Name: optical control of a neuronal protein using a genetically encoded unnatural amino acid in neurons
Uploaded: 2016-03-28T14:00:00.000+00:00
Duration: 8 min 20 s
Description: Watch this Scientific Journal Video about Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons at JoVE.com

We thank Dr. S. Szobota for helpful discussion on neuron culture and Ca-P transfection. L.W. acknowledges support from The Salk Innovation Grant, the California Institute for Regenerative Medicine (RN1-00577-1), and the National Institutes of Health (1DP2OD004744).

使用机构动物护理和使用委员会（IACUC）批准的索尔克研究所，拉霍亚，加州的动物处理方案进行了研究中的所有程序 1. UAA纳入的Kir2.1，将所得PIRK在原代神经元的文化表情<ol><li> DNA建设<ol><li>选择的Kir2.1的用于UAA掺入目标部位。利用有关的Kir2.1的结构和功能的先验知识和信息，使之与光敏UAA团选定的部位实现的Kir2.1功能21的光调制。 </li><li>构建重组DNA编码的Kir2.1基因突变为TAG琥珀终止密码子的选择位点。使用标准克隆技术25向的Kir2.1-TAG的DNA克隆到哺乳动物表达质粒。 </li><li>克隆的tRNA /合成酶的基因是特异于UAA和响应于纳入UAATAG终止密码子到另一个哺乳动物表达质粒21。 注意:对于最佳UAA掺入不同的启动子和基因盒的组合（对于酰tRNA，合成酶，和的Kir2.1）可以在不同的质粒进行测试。 </li><li>使用小量获得高质量的质粒DNA或根据制造商的协议大量制备试剂盒。周围的260/280比例为1.9是最佳的纯化的DNA。当需要时，执行一个琼脂糖凝胶电泳以检查坦然的DNA 25的纯度。具有高纯度的超螺旋质粒DNA是最适合的神经元的转染。 </li></ol></li><li>文化大鼠海马原代神经元<ol><li>地方盖玻片（圆圈，直径12毫米）在24孔板上每一个盖玻片井，和外套每孔250微升的0.5毫克/毫升聚D-赖氨酸（在100mM硼酸盐缓冲液:1.24克硼酸在400ml去离子蒸馏水酸1.90克次硫酸钠四硼酸H 2 O或的DDH 2 </suB&gt;在pH8.5 O），在室温下超过10小时，密封。每次准备小狗10-12涂盖玻片。 </li><li>在夹层的日子，异氟醚麻醉他们，并斩首后收集出生后的幼鼠（1-4日龄）新生儿大脑。麻醉幼仔含异氟醚（2-4％）的麻醉室。等到他们失去意识，无法对触觉刺激和爪子的捏回应。 </li><li>使用标准的技术26，在温暖的从脑解剖海马（〜37℃）的盐水（10毫米的HEPES和20mM D-葡萄糖的Hank氏平衡盐溶液），并收集海马在一个锥形管用4.5ml温生理盐水。 </li><li>加入500微升2.5％的胰蛋白酶到海马（0.25％终胰蛋白酶浓度），并孵育在37℃的水浴中10分钟。 </li><li>彻底冲洗用盐水（3×10ml）和磨碎的组织。 </li><li>回收分离的神经元在1ml瓦特含有最低必需培养基（MEM）中补充有5％胎牛血清（FBS）的臂的生长培养基，21.2毫D-葡萄糖，2mM的L-谷氨酰胺，2％的B-27，和0.1％血清剂。 注意:在解剖当天添加L-谷氨酰胺和B-27的制备新鲜的生长培养基。 </li><li>计数与血球的神经元，并且在1.0-1.5×10 5个细胞板他们到盖玻片在24孔板/孔用500μl生长培养基密度通过40微米尼龙网过滤后。 </li><li>孵育神经元培养物在35℃在一个5％CO 2:95％空气的加湿培养箱中培养2-3周。为了达到最佳效果，避免孵化过程中尽可能扰乱文化。 </li></ol></li><li>原发性神经文化磷酸钙（Ca-P）转染<ol><li>使储备溶液并储存在4℃:0.5M的BES（N，N-双[2-羟基-乙基] -2-氨基乙磺酸）缓冲液（10倍）; 150毫米的 Na 2 HPO 4（100倍）; 2.8 M氯化钠（10X）;无菌DDH 2 O;无菌的1N NaOH。 </li><li>在转染的当天，使在DDH 2 O的新鲜的2.5M的CaCl 2溶液并无菌过滤以0.22微米过滤器。使含有50mM BES，1.5毫的 Na 2 HPO 4，和280 mM氯化钠在pH 7.00（调节pH值用NaOH）和无菌过滤用0.22微米过滤器的新鲜2倍的BES缓冲盐水（BBS）的缓冲器。 注:计算待转染的氯化钙溶液，并根据盖玻片的数量的BBS缓冲器的量。另外，也要看看1.3.4。 </li><li>用500μl新鲜预热的转染培养基更换生长的培养介质。 （转染介质可以用MEM加21.2毫D-葡萄糖进行）。 注意:不要丢弃旧媒体。 </li><li>计算DDH 2 O的量，并准备以使含1.65微升的CaCl 2，0.7微克的DNA，和16.5的转染溶液（每个盖玻片33微升）添加＃181; l 2个BBS。 </li><li>立即将其加入到培养物之前制备的转染溶液。要准备，首先结合氯化钙和DDH 2 O的同时缓慢摇动管。继续搅拌并缓慢加入DNA引入该溶液中。最后，加2个缓冲BBS滴边搅拌。 </li><li>立即加入30微升转染溶液中神经元文化的每个盖玻片。 </li><li>摇动培养皿几次以混合溶液，并在5％CO 2在35℃培养:95％空气的加湿培养箱中45分钟-1小时。神经元的转染培养液后，很细的Ca-P沉淀就会形成覆盖层的神经元。 </li><li>用500μl预热的洗涤缓冲液（135毫摩尔NaCl，20毫米的HEPES，4mM的氯化钾，2mM的氯化钙 ，1mM的MgCl 2的，1毫米的 Na 2 HPO 4，10mM葡萄糖，在pH 7.3，无菌替换转染培养基用0.22微米过滤器过滤），并在35孵育6;下在5％CO 2:95％空气加湿15-20分钟孵化器。洗步骤后钙磷沉淀物就会消失。 </li><li>替换用500μl新鲜的生长培养基的清洗缓冲液。再次，替换为保存原始媒体的生长培养基。 </li><li>添加UAA CMN（预混合在50μl温暖生长培养基）的培养，以达到1mM的终浓度。 </li><li>孵育转染的培养物在35℃在一个5％CO 2:95％空气的加湿培养箱中测定前12-48小时。 </li></ol></li><li>全细胞记录与光激活<ol><li>准备额外的/细胞内的解决方案。细胞内溶液包含135毫葡萄糖酸钾，10mM的氯化钠，2mM的MgCl 2的，10毫米的HEPES，1mM的EGTA，2.56毫K 2 ATP和0.3在pH 7.4毫的 Li 2 GTP。细胞外记录溶液含有150 mM氯化钠，3毫米氯化钾，5mM的MgCl 2的，0.5毫米氯化钙2，5mM葡萄糖和10mM HEPES，在pH 7.4。 </li><li&gt;设置的电钻机全细胞膜片钳:显微镜配备了4X和20X的目标，微分干涉对比（DIC）和mCitrine过滤器（激发:十分之四百九十五纳米，发射:二十五分之五百二十五纳米）;手;膜片钳放大器;数字转换器;数据采集​​和分析软件。 <ol><li>通过显微镜在钻机安装一个发光二极管（LED）与385 nm发射光1厘米的距离以45°角交付的焦点。检查LED的功率和光功率计。 </li></ol></li><li>从使用商业微量拉马有3-6MΩ吸液管电阻玻璃电极拉补丁移液器。按照制造商的说明来设置微量拉马。 <ol><li>为了测试吸液管电阻，先填充细胞内液的吸管，并在电极支架上的位置。浸在充满细胞外溶液在35毫米培养皿中的吸移管，放置在显微镜platform。浸入接地电极放入盘中以完成一个电路。 </li><li>打开放大器/数字转换器和启动数据采集软件。显示器用薄膜测试协议吸液管的阻力。 </li></ol></li><li>取出从孵化器神经元文化的盖玻片，并在细胞外液冲洗一次。采用真空硅脂，按住盖玻片在充满新鲜的细胞外液35毫米的培养皿的中间。 </li><li>放置在显微镜电平台上的盖玻片/皿。 </li><li>使用标准的膜片钳技术，修补与mCitrine荧光27神经元。使用电流钳（I-钳）方法记录的神经元活动。首先，通过注射小电流调节静息电位左右-72毫伏。然后，注入了一步电流（10-200 PA）诱导动作电位的连续发射（5-15赫兹）。 </li><li>手动，或使用数据采集软件，闪光脉冲（一种罪过同时记录LED灯的100毫秒-1秒持续时间），对神经元的GLE脉搏，看看动作电位的影响。 </li><li>添加0.5mM的氯化钡2浴和验证，如果动作电位被回收。 </li></ol></li></ol> 2. UAA纳入的Kir2.1，将所得PIRK在小鼠胚胎脑体内的表达<ol><li> DNA建设<ol><li>类似设计的质粒DNA作为在步骤1.1。对于体内表达，使用强启动子如CAG（巨细胞病毒增强鸡β-肌动蛋白启动子）。 </li><li>根据制造商的方案用不含内毒素的大量制备商业试剂盒纯化DNA。执行酚-氯仿提取，随后用乙醇沉淀25获得极高质量的DNA（缩合以2-5微克/微升）。 </li></ol></li><li> 在子宫内电的小鼠胚胎大脑皮层 注意:基本TECHNI在子宫内的电阙先前已描述28。 <ol><li>麻醉在胚胎14.5天（E14.5）用戊巴比妥钠（腹腔注射，每克体重50微克）或异氟醚（吸入，2-4％）一个定时怀孕小鼠。通过检查意识丧失和触觉刺激无反应和爪子捏监测麻醉深度。 </li><li>使在腹部正中一个小切口。轻轻地揭露钳和指尖子宫角。 </li><li>注入约1微升DNA溶液（每质粒2-5微克/微升，这取决于构建体）与通过子宫壁插入一玻璃吸管每窝的侧脑室。在大多数情况下，总胚胎约60-80％被注入。 </li><li>与电穿孔（33-35伏，50毫秒持续时间，950毫秒的间隔，4-8个脉冲）电穿孔胚胎。 </li><li>与返回子宫角向腹腔轻轻牛肝菌和指尖。缝合肌肉壁，然后用手术用缝合线的皮肤，以使胚胎继续发育。 </li></ol></li><li> UAA显微注射<ol><li>在E16.5再次使在腹部正中线一小切口，然后轻轻地揭露钳和指尖子宫角。 </li><li>注入约2-5微升CMN丙氨酸（500毫米）以电穿孔侧或通过子宫壁插入一玻璃吸管侧脑室的两侧。为了提高生物利用度CMN，使用二肽CMN丙氨酸（CMN丙氨酸） 体内 21提供CMN。 </li><li>再次，用钳子和指尖轻轻返回子宫角，以腹腔。缝合肌肉壁，然后用手术用缝合线的皮肤，以使胚胎继续发育。 </li></ol></li><li>获得急性脑片<ol><li>使含有119毫米氯化钠，2.5毫米氯化钾，1.3毫米氯化镁2，2.5毫米氯化钙1升人工脑脊液（学联） <sUB&gt; 2，1mM的的NaH 2 PO 4，26.2毫碳酸氢钠 ，和11毫米的葡萄糖在pH 7.3。 </li><li>取200毫升ACSF和快速冷冻在-80℃下20-30分钟。气泡ACSF的其余部分用5％ 的 CO 2:95％O 2的气体在室温下。 </li><li>准备和消毒手术工具从收获胚胎大脑。还准备冰桶。 </li><li>使烧瓶在微波约100毫升4-％低熔点琼脂糖溶液加热。冷却液约5分钟就开始凝固之前。 </li><li>安乐死CMN丙氨酸注射二氧化碳过量后鼠标12-24小时。使大的切口在腹部区域，以及从与细剪刀和镊子子宫解剖出来电穿孔/显微注射的胚胎。 </li><li>从细剪刀和镊子胚胎收获的大脑。 </li><li>将每个脑上置于冰桶10cm的培养皿。使用锋利的刀片分成两个半球，并且将每个半球与矢状切面触摸盘底部的菜。很快倒在了大脑琼脂糖溶液（每左右半球500微升）。 </li><li>使用锋利的刀片，平砍琼脂糖周围大脑，使大脑包埋琼脂糖块。 </li><li>使用组织粘合剂，胶式琼脂糖块的矢状切面表面上安装了vibratome。填写预冷学联的vibratome室和削减200微米的矢状脑片。 </li><li>孵育在ACSF在33℃补充有3毫肌醇 ，0.4毫抗坏血酸，和2mM丙酮酸钠为42分钟，急性切片，同时用5％的CO 2鼓泡:95％O 2的气体。 </li><li> 42分钟后，关闭加热器，并继续鼓泡孵育在室温切片。关闭加热器之后开始全细胞膜片记录至少15分钟。 </li></ol></li><li>全细胞记录与光激活<ol><li>准备INTRAC含130毫葡糖酸钾，4毫氯化镁 ，5mM的HEPES，1.1毫EGTA，3.4毫的 Na 2 ATP，10mM的肌酸磷酸钠，和0.1在用KOH调节pH为7.3毫米的Na 3 GTP ellular溶液。 </li><li>从使用商业微量拉马有3-6MΩ吸液管电阻玻璃电极拉补丁移液器。 </li><li>设置为全细胞膜片夹紧片电钻机和以2毫升/分钟速率用灌注泵superfuse与ACSF录音室。调整室温度为约33℃。 注意:切片电生理钻机配用灌注腔室，水浸物镜和一个温度控制器。此外，GFP过滤器（激发:三十零分之四百八十零纳米，发射:四十零分之五百三十五纳米）和mCherry滤光片（激发:二十零分之五百八十○nm发射:一百三十零分之六百七十五纳米）是必需的。 <ol><li>安装在用钻机由显微镜385 nm发射LED光1厘米的距离以45°交付到焦点角度。检查LED的功率和光功率计。 </li></ol></li><li>仔细挑选了在灌注室玻璃吸管和地点的脑切片。按住与竖琴的切片。 </li><li>来自新皮层区27与补丁mCherry / GFP荧光的神经元。使用电压钳位（V-钳）方法记录PIRK活动。首先，保持在-60毫伏的膜电位。然后，在固定的负膜电位（-100毫伏）或电压斜坡（-100 mV至+ 40毫伏）记录电流。具体来说，监测的Kir2.1具体内向电流在-100 mV的。 </li><li>手动，或使用数据采集软件，闪光灯LED光脉冲（100毫秒-1秒时长的单脉冲）的神经元，同时记录，看看是否PIRK蛋白质被激活。一旦PIRK被激活，在-100 mV的内向电流会显著增加。 </li><li>添加0.5mM的氯化钡2浴并且如果PIRK再次灭活验证。 </li></ol></li></ol>

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The authors have nothing to disclose.

实现有效的光调制中，重要的初步步骤是决定，其中掺入光响应UAA中靶蛋白。目标蛋白的结构和功能的信息是非常有帮助，指导候选场址的选择。与此同时，光调节的目的将确定哪些位点是最合适的。选择候补的站点之后，建议在进行初级神经元和在体内之前测试在哺乳动物细胞系，如人胚肾（HEK）细胞更容易培养和操作，该位点。要确定的Kir2.1光激活站点，沿线的Kir2.1的孔隙多个站点已初步在HEK细胞进行测试。的Kir2.1的C169，发现最佳的，因为在那里C169所在的Kir2.1孔径大到足以容纳CMN侧链但小的足以让CMN侧链阻断离子通道。当CMN在C169中的Kir2.1并入在HEK细胞中表达，叔他得到的突变的Kir2.1处于非活动状态，但一旦成了光的短普乐激活，确认成功PIRK发展21。 的正交tRNA的有效表达和合成是关键的细胞和在体内得到高UAA掺入效率。正交合成酶基因是使用聚合酶II启动子和多聚A信号通常在哺乳动物细胞和神经元中使用高效表达。为正交tRNA，一种特殊类型-3-聚合酶III启动子，表达如这里使用的H1启动子，具有3&#39;-侧翼序列一起如先前18,20描述是必要的。 对于体内研究，我们发现，在tRNA的表达盒的三个拷贝相比一个拷贝（ 图5A）增加CMN掺入效率。在哺乳动物细胞中的另一项研究，增加的tRNA表达盒的数目也增加UAA掺入效率30。疗法安伏，我们建议优化的tRNA表达盒的数量不同Uaas，细胞和靶蛋白时UAA整合效率需要改进。在细胞和体内 Uaas生物利用度是UAA整合的另一个关键因素。以前的研究表明Uaas的该酯化增加了它们的摄取在哺乳动物细胞31，和Uaas在二肽形式制剂增加UAA摄取到细胞中线虫 32。我们发现，直接馈送CMN到神经细胞的培养基是足够用于的Kir2.1 CMN掺入培养的原代神经元中表达，但不足以用于体内 21并入在小鼠大脑。因此，二肽CMN丙氨酸合成并注射到小鼠脑。寡肽转运PEPT2在啮齿动物的脑中33，其可促进二肽的运输成神经元中高度表达。该醒酒内在ptide将由蜂窝肽酶水解，得到UAA CMN掺入。事实上，这种优化，我们在胚胎小鼠大脑的多个区域，包括大脑皮层，丘脑和下丘脑21取得高效CMN纳入神经 ​​元蛋白。 在神经元成功PIRK表达对健康的神经元文化的编制很大程度上依赖。在协议中的每一个步骤应该在精确细致地进行。文化准备后，最好保持在孵化器中培养无干扰。打开和关闭门孵化器，以及进出孵化移动培养皿中，最多可以添加破坏文化的品质。下孵化温度（35℃而不是37℃）有助于减少兴奋性中毒。与此类似，钙磷转应格外小心来完成。 2X BBS缓冲的制备是关键的一步。这是卡利一个好主意诺贝特用于pH 6.90-7.15之间2倍的BBS缓冲器的最佳pH，因为新的DNA构建体或用制剂可以改变转染条件。该缓冲液pH可以精确到0.01位数，如果它有助于获得一致的结果来调整。在转染前，神经元培养物的生长培养基替换为新鲜的。关键的是要保存原始生长介质，并在转染后回加至培养恢复原始状态。在新鲜培养基维持神经培养转染后会与神经元的干扰，从回收的，因为它缺乏对细胞增殖的关键分子，如从神经元释放的生长因子。 在培养细胞和组织切片PIRK的光激活，适当的光源和交付方法需要确定。 CMN吸收长期和中波紫外线灯（280-400纳米）。然而，长期暴露于UV光，尤其是较短波长的UV光，会有害细胞。在SAMe时代，远红光能升温试样扰动细胞。因此，与单波长发射的LED是最佳PIRK活化。被选中;对于CMN光解，一个具有385纳米（Prizmatix〜40毫瓦）发射LED。 LED被外部安装在显微镜附近，以及光纤被用来精确地将光传送到在焦点的神经元。为可再现的数据采集，该光纤被设置距离在从焦点的神经元以45°角1厘米。在样品光功率测定40毫瓦/厘米2。此外，还建议定期检查使用光功率计LED性能。 我们表明， 在子宫内电是一种有效的技术，以在体内基因掺入Uaas。 在质粒DNA的子宫内电到小鼠胚胎皮层如前所述34，以小的修改被执行。为了表达neonata PIRK蛋白质11小鼠的大脑，需要（ 图5B）两种手术程序。第一步是注入基因构建为PIRK表达，随后通过电穿孔。两天后，进行第二大脑注入提供CMN-丙氨酸。这需要实践，而不强调动物太多熟练地进行手术。每个手术后，将动物后应搜索并在整个恢复定期检查。在大脑CMN充足及时供应是正常PIRK表达是至关重要的。 2-5微升CMN丙氨酸（500毫米）的通常注射到胚胎脑。有时，它有助于注入CMN丙氨酸在心室上都电穿孔和相对的半球中，由于CMN丙氨酸从相反侧将扩散到延长CMN供给电穿孔的一侧。考虑到在子宫内的电一般效用，这种技术不仅可以为新皮层的神经元也能神经元的应用在其他大脑区域，如纹状体，间脑，小脑，当电/注射部位调整为每个区域。胚胎/新生儿大脑比成人的大脑更加柔软，所以这将是很难得到急性切片电生理学实验。琼脂糖嵌入有助于稳定胚胎脑结构vibratome切割。尽管低熔点琼脂糖温度冲击最小化到脑组织，当浇在寒冷的大脑琼脂糖熔化的需要谨慎。全细胞记录期间温度冲击的从琼脂糖包埋的组织效果不明显。 遗传编码光敏Uaas在培养的神经元和在体内 ，由PIRK例举这里，将得到的光遗传学技术来控制在其天然环境中的光的各种神经蛋白活性。当前协议呈现一步一步的过程来执行PIRK表达和活化实验我ñ神经元文化在体外和体内的啮齿动物的大脑，占UAA系统的哺乳动物大脑的使用首先研制成功。它具有潜在的受益许多天然蛋白的研究，以及它们在疾病含义。例如，α - 氨基-3-羟基-5-甲基-4-异唑丙酸（AMPA）受体和N-甲基-D-天冬氨酸（NMDA）受体共享的Kir2.1通道相似的跨膜结构。因此这将是可行的PIRK方法适用于这些配体门控离子通道。此外，PIRK技术也可以被用来解决的Kir2.1相关的遗传性疾病，如安徒生综合症和心短QT综合征35,36的病理生理学。除了 ​​"块和释放"经由CMN的Cys，多氨基酸，例如酪氨酸，丝氨酸，赖氨酸，谷氨酸，天冬氨酸和甘氨酸已笼不同photoreleasable组37。因此，类似的策略可使用在体外和体内 d，来光调节神经元中这些氨基酸。此外，可逆光控制可以用偶氮含Uaas来实现，并且这样Uaas现在在大肠杆菌中被遗传编码的大肠杆菌和哺乳动物细胞38,39。 总之，本协议呈现光以控制在其原生设置的神经元蛋白的活性的一般方法。相比于涉及视蛋白家族和其他光敏感的蛋白质或结构域的其他光遗传学方法，这种方法使用遗传编码的光敏感Uaas和变化仅单个残基。因此，它应该有所研究的靶蛋白的功能，运输和定位的干扰最小。高现场选择性也可以与这些遗传编码的光响应Uaas实现，以提供详细的分子研究了更大的灵活性和特异性。该协议还提供了第一共mprehensive，易于遵循程序如何成功地掺入Uaas成蛋白质中的神经元在体外和体内 。通过基因编码Uaas 体外神经细胞和体内的蛋白质一般光学控制具有很大的潜力，造福于两国基础和转化科学，而该协议将有助于推动其应用。

相对于传统的电刺激，光刺激，提供带到标本生理系统最小干扰较大的时间和空间分辨率。由于使用激光来刺激神经 ​​元于1971年1示范，许多创造性的方式被发明来控制外源性神经活动的光。 photocaged激动剂的光释放长期以来被用于研究神经元网络到配位体2,3,4的生理反应。这种技术的特异性有限，原因是笼激动剂的扩散。遗传特异性由异位表达光敏视蛋白渠道取得和泵5,6,7，并且已成功地应用到调制在不同的模式生物中选择的神经元网络。然而，这将是困难的应用此方法到光学控制各种其他神经元蛋白，由于从视蛋白的蛋白质与其它蛋白质接枝光响应竟被ð要求激烈工程可能改变研究中的蛋白质的自然特性。化学圈养的外源感光配体与蛋白质已经证明另一种方式来控制通道蛋白8,9,10的功能。该配体呈现或通过偶氮苯部分的光致异构化从蛋白质的结合位点抽出。束缚化学限制了应用主要是膜蛋白的胞外侧，不含细胞内侧面和细胞内蛋白质。 光敏Uaas，被纳入的蛋白质后，提供操纵蛋白质与光的一般策略。在早期的努力，tRNA的化学酰化photocaged Uaas显微注射入爪蟾卵母 ，以纳入Uaas到膜受体和离子通道11，它有先进的它们的结构-功能关系12,13,14的理解。这种注射方法主要限于大的卵母细胞。一个UAA的遗传掺入通过正交tRNA /合成酶对，通过内源性蛋白的翻译融合了UAA活细胞15,16,17,18绕过了技术上的挑战酰化tRNA的显微注射和。 UAA掺入神经 ​​元蛋白已被证明在初级神经元和神经干细胞19,20。最近，光响应UAA已经遗传掺入神经 ​​蛋白在哺乳动物脑中体内首次21。这些进步使得有可能研究与Uaas神经元蛋白在其天然细胞环境。 内向整流钾离子通道的Kir2.1是更容易通过K +电流成比出细胞的一个强有力的整流器，并且在调节生理过程，包括细胞的兴奋，血管张力，心脏速率，重是必不可少最终盐流和胰岛素释放22。的Kir2.1的过表达超极化的目标神经元，它变得不太可激发23,24的膜电位。以光学控制的Kir2.1在其天然细胞，Kang等人的基因掺入一个光响应UAA成的Kir2.1在哺乳动物细胞，神经元和胚胎鼠大脑21表示。闪光短暂的脉冲能够将UAA转化成天然氨基酸半胱氨酸，从而激活靶蛋白的Kir2.1。当这种光诱导内向整流钾（PIRK）通道蛋白在大鼠海马原代神经元中表达，它响应于光激活抑制神经元放电。此外，PIRK通道中的胚胎鼠新皮质中表达，并测定在皮层神经元的光激活PIRK电流。成功实现了UAA技术在哺乳动物大脑体内敞开了大门光学控制神经细胞的蛋白质在其天然环境中，这将使在分子水平上的神经元过程和机制的光学夹层。 在这个协议中，我们描述了Uaas遗传纳入文化和体内胚胎小鼠大脑初级神经元的程序。光响应UAA CMN和的Kir2.1用来说明该过程。方法评估成功的UAA整合并提供神经蛋白活性的光学控制。这个协议提供了一个明确的指导中的神经元和在体内基因编码Uaas，并以光学调节经由光敏UAA神经蛋白的功能。我们希望这个协议推动通过体内 UAA技术，神经科学和光遗传学生物学研究。

<table><tbody><tr><td>Cover Glasses, Circles, 12 mm, Thickness 0.13-0.17 mm</td><td>Carolina Biologicals</td><td>633029</td><td></td></tr><tr><td>Corning BioCoat Poly-D-Lysine</td><td>Corning Discovery Labware</td><td>354210</td><td></td></tr><tr><td>D-(+)-Glucose solution</td><td>Sigma-Aldrich</td><td>G8769</td><td></td></tr><tr><td>Iris Spatula-curved</td><td>Fine Science Tool</td><td>10092-12</td><td></td></tr><tr><td>Dissecting Knife - Fine Angled Tip</td><td>Fine Science Tool</td><td>10056-12</td><td></td></tr><tr><td>GlutaMAX-I Supplement</td><td>Life Technologies</td><td>35050-061</td><td></td></tr><tr><td>MITO+ Serum Extender</td><td>BD Biosciences</td><td>355006</td><td></td></tr><tr><td>Falcon 40 &amp;micro;m Cell Strainer</td><td>Corning Life Sciences</td><td>352340</td><td></td></tr><tr><td>BES (&lt;em&gt;N,N&lt;/em&gt;-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid, &lt;em&gt;N,N&lt;/em&gt;-Bis(2-hydroxyethyl)taurine)</td><td>Sigma-Aldrich</td><td>B6420</td><td></td></tr><tr><td>LED LIGHT SOURCE &amp;ndash; Black LED 385</td><td>Prizmatix Ltd.</td><td></td><td></td></tr><tr><td>Agarose, Low Melting Point, Analytical Grade</td><td>Promega</td><td>V2111</td><td></td></tr></tbody></table>

optical control of a neuronal protein using a genetically encoded unnatural amino acid in neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

要结合基因UAA一成神经细胞的蛋白质，重要的第一步是设计合适的基因结构来实现，并在神经元高效表达基因。有用于UAA掺入3种成因组成部分:（1）靶基因与为UAA掺入（2）正交tRNA识别突变TAG终止密码子，和（3）正交氨选定的部位引入TAG琥珀终止密码子-tRNA合成酶的UAA充电到正交tRNA。每个部件需要由适当的启动子来驱动。这三个基因盒可包含在一个质粒或在几个质粒分离。为了表达在神经元PIRK，光反应UAA CMN使用正交tRNA 亮氨酸 CUA / CmnRS（CMN -tRNA合成酶）对演变为特异于CMN 21,29并入的Kir2.1。的Kir2.1基因残留Cys169突变为豪琥珀终止密码子（Kir2。1-TAG），和荧光蛋白mCitrine融合到的Kir2.1的C末端来可视化在神经元培养PIRK表达（ 图1）。 获得高质量的神经元的文化是成功PIRK实验的先决条件。日龄1-4只SD大鼠幼仔一般用于神经元培养的准备，但大鼠胚胎也经常使用。当在1.0-1.5×10 5个细胞/孔的密度在24孔板中铺板于盖玻片，应该看到不太多的小胶质细胞或其他碎片（ 图2A）以及分隔健康的神经元。健康的神经元有广泛的进程，以及细胞体（SOMA）是丰满（ 图2B）。独特可见suborganelle结构通常是不健康文化的标志。神经元培养物可以在35℃在5％CO 2维持2-3周:95％空气加湿培养箱。 Calcium磷酸盐（CA-P）转染适合敏感神经文化一个非常温和的转染方法。但是，它需要极高的精度和谨慎的神经元，以实现高转染效率。储备溶液需要被储存在4℃，混合以2×BBS缓冲器上转染当天。精确的pH值调节是关键，可重复获得成功的结果。在2.5M的氯化钙溶液还应该在转染当天新鲜。所有缓冲区需要过滤。此外，具有高纯度和质量新鲜DNA质粒改善的结果。为了达到最佳效果，可以得到新鲜的DNA迷你坦然从不杂草丛生大肠杆菌培养物（〜37℃在振荡培养箱12小时）。毕竟缓冲区准备好了，把解决方案在组织培养罩和转染准备。在低速用旋涡，搅拌转染溶液中，同时混合。紧接在混合溶液添加到该神经元的铜lture，并把培养皿回孵化器。停止转染后45分钟，1小时，并添加原始生长培养基回培养的连续培养。将转染的神经元可以从6小时转染终止（ 图3）后进行可视化。在CMN的在培养基中的存在，PIRK表达并PIRK表达神经元是绿色荧光由于融合到PIRK的C末端（ 图3D）的mCitrine蛋白。 PIRK表达神经元有正常基础生理和它们能够响应于注射的电流（ 图4）焙烧动作电位。然而，这一系列的动作电位与照到细胞（ 图4A）的简要光脉冲立即抑制。当0.5毫氯化钡2，一个的Kir2.1特异性抑制剂加入到浴中，神经元放电然后恢复时，指示该先前抑制由l至的Kir2.1的活化是由于飞行（ 图4B）。未转染对照神经元不给光或Ba 2+（ 图4C，D）响应。 在体内表达PIRK，高纯度的浓缩的DNA质粒（2-5微克/微升），应制备。在这些实验中使用的具体的质粒示于图5A。除了 ​​正交tRNA 亮氨酸 CUA / CmnRS对和目标的Kir2.1-TAG基因，基因编码绿色荧光蛋白与TAG突变（GFP-TAG）也是共同电穿孔作为荧光报道。 GFP荧光检测将表明所有三个质粒的成功交付，因为在GFP TAG抑制将需要两个tRNA的亮氨酸 CUA和CmnRS;在GFP-TAG CMN掺入建议中的Kir2.1-TAGas CMN掺入好，因为两个基因存在于同一细胞中。这三个基因构造都仁济反恐执行到小鼠胚胎的上E14.5侧脑室（ 图5B，左图）。电穿孔后，返回胚胎至腹腔，以允许胚胎继续发育。两天后，注入约2-5微升CMN丙氨酸（500毫米）以电穿孔侧或侧脑室的两侧，以类似的方式作为DNA注射（ 图5B，中图）。再次，返回胚胎连续发展腹腔。胚胎可以在E17.5收获成像或电试验。正如预期的，仅与CMN丙氨酸注射，绿色荧光的细胞在小鼠大脑皮层观察。与红色和绿色荧光的细胞应该CMN掺入的Kir2.1-TAG使PIRK通道（ 图6A）。因为它是对神经元培养完成从胚胎新皮层切片全细胞记录同样进行。绿色和红色荧光的神经元必须在NE没有内向电流gative持有的潜力，但光脉冲短暂迅速激活内向电流（ 图6B）。电流完全被加入的Ba 2+，确认它是由PIRK产生阻塞。 <img alt="图1" src="/files/ftp_upload/53818/53818fig1.jpg" /> 图1:对神经元培养方案示出示例性的质粒设计用于在神经元培养PIRK表达PIRK表达质粒集 。顶部质粒编码的Kir2.1基因（灰色）与C169站点下巨细胞病毒（CMV）启动子的单个氨基酸突变。 C169被突变为TAG琥珀终止密码子，其中所述UAA CMN将并入。的Kir2.1基因后跟mCitrine基因（绿色）。 mCitrine融合到的Kir2.1基因的C-末端形象化PIRK表达细胞。底部质粒编码CmnRS（粉红色），该CMN具体的合成，通过鼠标磷酸甘油酸激酶1（mPGK）启动子驱动。酰tRNA <sup<html> &gt;亮氨酸CUA（蓝色）时，正交tRNA 21，也表示在由H1启动子驱动的同一质粒，但在相反的方向。 <img alt="图2" src="/files/ftp_upload/53818/53818fig2.jpg" /> 图2:大鼠海马原代神经元文化示范图片显示健康大鼠海马神经细胞。 （A）神经元文化对10天的DIC图像在体外 （DIV）。成熟的神经，因为他们分支树突和轴突。小胶质细胞（小而圆细胞，而不进程）共存，但不要压倒文化。 （ 二 ）健康培养的神经元的放大图像DIC。一个健康的神经细胞呈现饱满细胞体（胞体），并宣布树突/轴突。 <a href="https://www.jove.com/files/ftp_upload/53818/53818fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <html> _content"FO: - together.within页保留="1"&gt; <img alt="图3" src="/files/ftp_upload/53818/53818fig3.jpg" /> 图3:在培养的原代神经元PIRK表达 DIC和体外培养，并用在图1中为PIRK表达描绘基因构建体转染大鼠海马神经元的荧光图像。当在培养未添加CMN转染（A和B）后，没有绿色荧光是从神经元检测。在CMN（1毫米）的生长介质的存在，转染的神经元能够表达全长PIRK-mCitrine蛋白，从而呈现出绿色荧光（C和D）。 <a href="https://www.jove.com/files/ftp_upload/53818/53818fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> .JPG"/&gt; 图4:PIRK的光活化抑制了海马神经元发射 （A）单光脉冲抑制海马神经元表达PIRK活动。代表电压痕迹在电流钳连续记录。动作电位发放是由20 pA的电流注入（Ⅰ-工序）诱发。光曝光（385纳米，40毫瓦/厘米2，1秒;用箭头表示）迅速和完全抑制神经元放电。烧制恢复与细胞外的500mM氯化钡2，其选择性地抑制的Kir2.1通道。 （B）的紫外线（UV）照射（385纳米，40毫瓦/厘米2，6秒;与黄色框表示）不改变控制，未转染的神经元的兴奋性。动作电位发放是由50 PA电流注入（Ⅰ-工序）诱发。 （C和D）既不紫外光照射，也不氯化钡2（500毫米）影响控制神经元的兴奋性。有力的行动胶质由50 PA电流注入（Ⅰ-工序）诱发。 <img alt="图5" src="/files/ftp_upload/53818/53818fig5.jpg" /> 图5:为在体内小鼠大脑皮层PIRK表达程序 。 （A）PIRK表达质粒设定。顶部的质粒:用于通过经由内部核糖体进入位点（IRES）的CAG启动子和mCherry从动CmnRS质粒。 mCherry荧光将核实CmnRS的成功表达。中东质粒:为配合亮氨酸的tRNA CUA，为CMN掺入21正交tRNA的三个副本的Kir2.1基因的质粒。底部质粒:为GFP_Y182 TAG质粒。对GFP的基因在允许Tyr182网站琥珀终止密码子（GFP_Y182 TAG）被设计为可视化CMN成功掺入代码的网站，21。 （B）卡通笑翼用于体内 PIRK表达的实验过程。为PIRK表达基因构建体注射到小鼠大脑皮层（E14.5）和子宫内 （左面板）电穿孔。两天后，CMN丙氨酸被注入到在所述电穿孔侧脑室或脑半球的两面（中图）。切片成像和电测定可以在E17.5-E18.5（右图）进行。 <img alt="图6" src="/files/ftp_upload/53818/53818fig6.jpg" /> 图6:在小鼠大脑皮层和光活化PIRK表达。 小鼠胚胎皮质神经元显示CMN纳入GFP和蛋白质的Kir2.1 在体内的（A）荧光图像。 图5A中的三个基因构建体电穿孔在子宫内 。 mCherry荧光演示CMN具体SYNTHE成功的表达TASE基因，CmnRS。 GFP荧光仅与CMN丙氨酸注射液（底部）检测，显示绿色荧光蛋白标记，并在TAG的Kir2.1可能CMN CMN纳入合并。因此，绿色和红色荧光指示所有三种质粒和CMN掺入成功表达。 （B）中从小鼠记录新皮层神经元表示PIRK的光依赖性激活的电流的第四曲线图。两天后图5A基因构建了电，CMN-阿拉宫内注射; CMN丙氨酸注射后12-48小时，从该胚制备新皮层急性切片。 PIRK表达在切片的神经元，由红色和绿色荧光检测，被前（黑色）记录和之后（蓝色）的光曝光（385纳米，8毫瓦/平方厘米，10秒为饱和曝光）。加入氯化钡2（500毫米）光活化后（橙色），以验证特定PIRK-电流。

Watch this Scientific Journal Video about Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons at JoVE.com

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

optical-control-neuronal-protein-using-genetically-encoded-unnatural

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.9K 次观看.  Tufts University. Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

视频: Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Scalable Fluidic Injector Arrays for Viral Targeting of Intact 3-D Brain Circuits

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted in neurological and psychiatric disorders--would be greatly facilitated by a technology for rapidly targeting genes to complex 3-dimensional neural circuits, enabling fast creation of "circuit-level transgenics." We have recently developed methods in which viruses encoding for light-sensitive proteins can sensitize specific cell types to millisecond-timescale activation and silencing in the intact brain. We here present the design and implementation of an injector array capable of delivering viruses (or other fluids) to dozens of defined points within the 3-dimensional structure of the brain (Figure. 1A, 1B). The injector array comprises one or more displacement pumps that each drive a set of syringes, each of which feeds into a polyimide/fused-silica capillary via a high-pressure-tolerant connector. The capillaries are sized, and then inserted into, desired locations specified by custom-milling a stereotactic positioning board, thus allowing viruses or other reagents to be delivered to the desired set of brain regions. To use the device, the surgeon first fills the fluidic subsystem entirely with oil, backfills the capillaries with the virus, inserts the device into the brain, and infuses reagents slowly (&lt;0.1 microliters/min). The parallel nature of the injector array facilitates rapid, accurate, and robust labeling of entire neural circuits with viral payloads such as optical sensitizers to enable light-activation and silencing of defined brain circuits. Along with other technologies, such as optical fiber arrays for light delivery to desired sets of brain regions, we hope to create a toolbox that enables the systematic probing of causal neural functions in the intact brain. This technology may not only open up such systematic approaches to circuit-focused neuroscience in mammals, and facilitate labeling of brain regions in large animals such as non-human primates, but may also open up a clinical translational path for cell-specific optical control prosthetics, whose precision may enable improved treatment of intractable brain disorders. Finally, such devices as described here may facilitate precisely-timed fluidic delivery of other payloads, such as stem cells and pharmacological agents, to 3-dimensional structures, in an easily user-customizable fashion.

Controlling and analyzing neural circuits in vivo would be facilitated by a technology for delivery of viruses and other reagents to desired 3-dimensional sets of brain regions. We demonstrate customized fluidic injector array fabrication, and delivery of virally-encoded optical sensitizers, enabling optical manipulation of complex brain circuits.

可伸缩流控喷油器阵列病毒完整的3 - D的大脑回路为目标

Our understanding of neural circuits--how they mediate the computations that subserve sensation, thought, emotion, and action, and how they are corrupted ...

科研

神经科学

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

电压钳荧光测定<em&gt;爪蟾</em&gt;使用荧光非天然氨基酸的卵母细胞

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

生物化学

Using Affordable LED Arrays for Photo-Stimulation of Neurons

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single cells in response to electrical or pharmacological manipulations1,2. With the invention of methods to optically control genetically targeted neurons (optogenetics), researchers now have an unprecedented level of control over specific groups of neurons in the standard slice preparation. In particular, photosensitive channelrhodopsin-2 (ChR2) allows researchers to activate neurons with light3,4. By combining careful calibration of LED-based photostimulation of ChR2 with standard slice electrophysiology, we are able to probe with greater detail the role of adult-born interneurons in the olfactory bulb, the first central relay of the olfactory system. Using viral expression of ChR2-YFP specifically in adult-born neurons, we can selectively control young adult-born neurons in a milieu of older and mature neurons. Our optical control uses a simple and inexpensive LED system, and we show how this system can be calibrated to understand how much light is needed to evoke spiking activity in single neurons. Hence, brief flashes of blue light can remotely control the firing pattern of ChR2-transduced newborn cells.

Adult-born neurons expressing ChR2 can be manipulated in slice electrophysiological preparations in order to examine their contribution towards the function of olfactory neural circuits.

使用光刺激神经元的负担得起的LED阵列

Standard slice electrophysiology has allowed researchers to probe individual components of neural circuitry by recording electrical responses of single ...

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical properties, the amino acid has multiple uses in biochemical applications. This report describes a protocol for the genetic incorporation of biosynthesized DOPA and its application to protein conjugation. DOPA is biosynthesized by a tyrosine phenol-lyase (TPL) from catechol, pyruvate, and ammonia, and the amino acid is directly incorporated into proteins by the genetic incorporation method using an evolved aminoacyl-tRNA and aminoacyl-tRNA synthetase pair. This direct incorporation system efficiently incorporates DOPA with little incorporation of other natural amino acids and with better protein yield than the previous genetic incorporation system for DOPA. Protein conjugation with DOPA-containing proteins is efficient and site-specific and shows its usefulness for various applications. This protocol provides protein scientists with detailed procedures for the efficient biosynthesis of mutant proteins containing DOPA at desired sites and their conjugation for industrial and pharmaceutical applications.

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation

Here, we present a protocol for the genetic incorporation of L-dihydroxyphenylalanine biosynthesized from simple starting materials and its application to protein conjugation.

Biosynthesized l-dihydroxyphenylalanine (多巴) 的遗传合并及其在蛋白质共轭中的应用

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical ...

Optimizing the Genetic Incorporation of Chemical Probes into GPCRs for Photo-crosslinking Mapping and Bioorthogonal Chemistry in Live Mammalian Cells

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and reactive moieties onto proteins directly in the live cell. Each ncAA is incorporated by a dedicated orthogonal suppressor-tRNA/amino-acyl-tRNA-synthetase (AARS) pair that is imported into the host organism. The incorporation efficiency of different ncAAs can greatly differ, and be unsatisfactory in some cases. Orthogonal pairs can be improved by manipulating either the AARS or the tRNA. However, directed evolution of tRNA or AARS using large libraries and dead/alive selection methods are not feasible in mammalian cells. Here, a facile and robust fluorescence-based assay to evaluate the efficiency of orthogonal pairs in mammalian cells is presented. The assay allows screening tens to hundreds of AARS/tRNA variants with a moderate effort and within a reasonable time. Use of this assay to generate new tRNAs that significantly improve the efficiency of the pyrrolysine orthogonal system is described, along with the application of ncAAs to the study of G-protein coupled receptors (GPCRs), which are challenging objects for ncAA mutagenesis. First, by systematically incorporating a photo-crosslinking ncAA throughout the extracellular surface of a receptor, binding sites of different ligands on the intact receptor are mapped directly in the live cell. Second, by incorporating last-generation ncAAs into a GPCR, ultrafast catalyst-free receptor labeling with a fluorescent dye is demonstrated, which exploits bioorthogonal strain-promoted inverse Diels Alder cycloaddition (SPIEDAC) on the live cell. As ncAAs can be generally applied to any protein independently on its size, the method is of general interest for a number of applications. In addition, ncAA incorporation does not require any special equipment and is easily performed in standard biochemistry labs.

A facile fluorescence assay is presented to evaluate the efficiency of amino-acyl-tRNA-synthetase/tRNA pairs incorporating non-canonical amino-acids (ncAAs) into proteins expressed in mammalian cells. The application of ncAAs to study G-protein coupled receptors (GPCRs) is described, including photo-crosslinking mapping of binding sites and bioorthogonal GPCR labeling on live cells.

生物哺乳动物细胞光交联图谱和 Bioorthogonal 化学中化学探针的遗传受体优化

The genetic incorporation of non-canonical amino acids (ncAAs) via amber stop codon suppression is a powerful technique to install artificial probes and ...

化学

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

Optogenetic methods have emerged as a powerful tool for elucidating neural circuit activity underlying a diverse set of behaviors across a broad range of species. Optogenetic tools of microbial origin consist of light-sensitive membrane proteins that are able to activate (e.g., channelrhodopsin-2, ChR2) or silence (e.g., halorhodopsin, NpHR) neural activity ingenetically-defined cell types over behaviorally-relevant timescales. We first demonstrate a simple approach for adeno-associated virus-mediated delivery of ChR2 and NpHR transgenes to the dorsal subiculum and prelimbic region of the prefrontal cortex in rat. Because ChR2 and NpHR are genetically targetable, we describe the use of this technology to control the electrical activity of specific populations of neurons (i.e., pyramidal neurons) embedded in heterogeneous tissue with high temporal precision. We describe herein the hardware, custom software user interface, and procedures that allow for simultaneous light delivery and electrical recording from transduced pyramidal neurons in an anesthetized in vivo preparation. These light-responsive tools provide the opportunity for identifying the causal contributions of different cell types to information processing and behavior.

A Method for High Fidelity Optogenetic Control of Individual Pyramidal Neurons In vivo

The recent development of neuroscience tools that combine genetics and optics, termed "optogenetics", enables control over neural circuit activity with an unprecedented level of spatial and temporal resolution. Here we provide a protocol for integrating in vivo recording with optogenetic manipulation of genetically-defined subsets of prefrontal cortical and subicular pyramidal neurons.

单个锥体神经元的高保真光遗传学控制方法<em&gt;在体内</em

Optogenetic methods  have emerged as a powerful tool for elucidating neural circuit activity  underlying a diverse set of behaviors across a broad range ...

Optogenetic Manipulation of Neuronal Activity to Modulate Behavior in Freely Moving Mice

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of single neurons and region-specific transmitter release, up to whole neuronal circuitries in the central nervous system, and allows the direct measurement of behavioral outcomes. Neurons express optogenetic tools via an injection of viral vectors carrying the DNA of choice, such as Channelrhodopsin2 (ChR2). Light is brought into specific brain regions via chronic optical implants that terminate directly above the target region. After two weeks of recovery and proper tool-expression, mice can be repeatedly used for behavioral tests with optogenetic stimulation of the neurons of interest.
Optogenetic modulation has a high temporal and spatial resolution that can be accomplished with high cell specificity, compared to the commonly used methods such as chemical or electrical stimulation. The light does not harm neuronal tissue and can therefore be used for long-term experiments as well as for multiple behavioral experiments in one mouse. The possibilities of optogenetic tools are nearly unlimited and enable the activation or silencing of whole neurons, or even the manipulation of a specific receptor type by light.
The results of such behavioral experiments with integrated optogenetic stimulation directly visualizes changes in behavior caused by the manipulation. The behavior of the same animal without light stimulation as a baseline is a good control for induced changes. This allows a detailed overview of neuronal types or neurotransmitter systems involved in specific behaviors, such as anxiety. The plasticity of neuronal networks can also be investigated in great detail through long-term stimulation or behavioral observations after optical stimulation. Optogenetics will help to enlighten neuronal signaling in several kinds of neurological diseases.

With optogenetic manipulation of specific neuronal populations or brain regions, behavior can be modified with high temporal and spatial resolution in freely moving animals. By using different optogenetic tools in combination with chronically implanted optical fibers, a variety of neuronal modulations and behavioral testing can be performed.

神经活性的光遗传学操作，调节自由移动小鼠的行为

Optogenetic modulation of neuronal circuits in freely moving mice affects acute and long-term behavior. This method is able to perform manipulations of ...

行为学

Reliably Engineering and Controlling Stable Optogenetic Gene Circuits in Mammalian Cells

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, regardless of the method used. Toward this goal, optogenetic gene expression systems have gained much attention over the past decade for spatiotemporal control of protein levels in mammalian cells. However, most existing circuits controlling light-induced gene expression vary in architecture, are expressed from plasmids, and utilize variable optogenetic equipment, creating a need to explore characterization and standardization of optogenetic components in stable cell lines. Here, the study provides an experimental pipeline of reliable gene circuit construction, integration, and characterization for controlling light-inducible gene expression in mammalian cells, using a negative feedback optogenetic circuit as a case example. The protocols also illustrate how standardizing optogenetic equipment and light regimes can reliably reveal gene circuit features such as gene expression noise and protein expression magnitude. Lastly, this paper may be of use for laboratories unfamiliar with optogenetics who wish to adopt such technology. The pipeline described here should apply for other optogenetic circuits in mammalian cells, allowing for more reliable, detailed characterization and control of gene expression at the transcriptional, proteomic, and ultimately phenotypic level in mammalian cells.

Reliably controlling light-responsive mammalian cells requires the standardization of optogenetic methods. Toward this goal, this study outlines a pipeline of gene circuit construction, cell engineering, optogenetic equipment operation, and verification assays to standardize the study of light-induced gene expression using a negative-feedback optogenetic gene circuit as a case study.

可靠地工程和控制哺乳动物细胞中稳定的光遗传学基因回路

Reliable gene expression control in mammalian cells requires tools with high fold change, low noise, and determined input-to-output transfer functions, ...

生物工程

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

The purpose of this protocol is to characterize the effect of gene knockdown on presynaptic function within intact neuronal circuits. We describe a workflow on how to combine artificial microRNA (miR)-mediated RNA interference with optogenetics to achieve selective stimulation of manipulated presynaptic boutons in acute brain slices. The experimental approach involves the use of a single viral construct and a single neuron-specific promoter to drive the expression of both an optogenetic probe and artificial miR(s) against presynaptic gene(s). When stereotactically injected in the brain region of interest, the expressed construct makes it possible to stimulate with light exclusively the neurons with reduced expression of the gene(s) under investigation. This strategy does not require the development and maintenance of genetically modified mouse lines and can in principle be applied to other organisms and to any neuronal gene of choice. We have recently applied it to investigate how the knockdown of alternative splice isoforms of presynaptic P/Q-type voltage-gated calcium channels (VGCCs) regulates short-term synaptic plasticity at CA3 to CA1 excitatory synapses in acute hippocampal slices. A similar approach could also be used to manipulate and probe the neuronal circuitry in vivo.

This protocol provides a workflow on how to combine artificial microRNA-mediated RNA interference with optogenetics to stimulate specifically presynaptic boutons with reduced expression of selective gene(s) within intact neuronal circuits.

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 ...

Residue-Specific Exchange of Proline by Proline Analogs in Fluorescent Proteins: How "Molecular Surgery" of the Backbone Affects Folding and Stability

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often detrimental to protein folding and, in particular, chromophore maturation in green fluorescent proteins and related variants. A reasonable alternative is to manipulate the translation of the protein so that all Pro residues are replaced residue-specifically by analogs, a method known as selective pressure incorporation (SPI). The built-in chemical modifications can be used as a kind of &#34;molecular surgery&#34; to finely dissect measurable changes or even rationally manipulate different protein properties. Here, the study demonstrates the usefulness of the SPI method to study the role of prolines in the organization of the typical &#946;-barrel structure of spectral variants of the green fluorescent protein (GFP) with 10-15 prolines in their sequence: enhanced green fluorescent protein (EGFP), NowGFP, and KillerOrange. Pro residues are present in connecting sections between individual &#946;-strands and constitute the closing lids of the barrel scaffold, thus being responsible for insulation of the chromophore from water, i.e., fluorescence properties. Selective pressure incorporation experiments with (4R)-fluoroproline (R-Flp), (4S)-fluoroproline (S-Flp), 4,4-difluoroproline (Dfp), and 3,4-dehydroproline (Dhp) were performed using a proline-auxotrophic E. coli strain as expression host. We found that fluorescent proteins with S-Flp and Dhp are active (i.e., fluorescent), while the other two analogs (Dfp and R-Flp) produced dysfunctional, misfolded proteins. Inspection of UV-Vis absorption and fluorescence emission profiles showed few characteristic alterations in the proteins containing Pro analogs. Examination of the folding kinetic profiles in EGFP variants showed an accelerated refolding process in the presence of S-Flp, while the process was similar to wild-type in the protein containing Dhp. This study showcases the capacity of the SPI method to produce subtle modifications of protein residues at an atomic level (&#34;molecular surgery&#34;), which can be adopted for the study of other proteins of interest. It illustrates the outcomes of proline replacements with close chemical analogs on the folding and spectroscopic properties in the class of &#946;-barrel fluorescent proteins.

To overcome the limitations of classical site-directed mutagenesis, proline analogs with specific modifications were incorporated into several fluorescent proteins. We show how the replacement of hydrogen by fluorine or of the single by double bonds in proline residues ("molecular surgery") affects fundamental protein properties, including their folding and interaction with light.

荧光蛋白中脯氨酸类似物对脯氨酸的残基特异换:骨架的"分子手术"如何影响折叠和稳定性

Replacement of proline (Pro) residues in proteins by the traditional site-directed mutagenesis by any of the remaining 19 canonical amino acids is often ...

一个神经元蛋白在神经元使用遗传编码的非天然氨基酸的光控制

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