我们感谢 Hanns Zeilhofer 慷慨地支持这项工作。亨德里克 Wildner 得到了奥尔加 Mayenfisch 基金会的支持。我们感谢卡门 Birchmeier 的 Lmx1b 抗体。

所有动物实验均经瑞士州兽医局 (苏黎世) 批准, 并符合所有相关的规章制度和机构准则。
注: 所有材料连同各自的制造商和/或供应商在材料表中列出。
1. 产生依赖于 AAV 的向量
注意: 可以购买各种与不同启动子的依赖关系的向量 (请参见材料表), 或者, 如果所需的表达式构造不可用, 则可以通过修改现有的 AAV 构造来生成它。注意, 启动子和血清型可能对病毒转导的传播产生影响 (请参见12)。本协议的第一部分简要描述了两种不同的 AAV 向量的生成, 分别适用于函数实验的增益和损耗。
<ol><li>
遗传药理学激活 (功能增益)
	<ol>
<li>命令 pAAV. hSyn. hM3D (Gq)-mCherry (参见材料表), 专门由由设计药物 (DREADD) 介导的激活的设计器受体激活。</li>
		<li>在接受细菌刺培养后, 在 LB 板上切出细菌 (细菌级琼脂在 Luria-Bertani 液介质中溶解) 补充适当的抗生素。在晚上孵化37摄氏度, 并选择一个单一的殖民地在第二天。 注: 含有 AAV 向量基因组的质粒可能不稳定, 病毒基因组的重组, 特别是在病毒性倒置终端重复 (ITR) 中, 可能发生在大肠杆菌的放大过程中。我们建议使用 recA 缺乏/抑制的菌株, 如 MDS42 或 Stbl3, 以避免在含有 AAV 基因组的质粒内重组。</li>
		<li>根据所选择的 dna 最大试剂盒供应商给出的指示, 放大细菌, 并准备高质量的质粒 dna, 并提供商用的 dna 最大套件 (例如, 请参阅材料表)。通过对质粒 dna 整除的限制消化, 验证质粒 dna 的完整性和同一性, 对质粒 dna 的制备进行质量控制。 注: 在 ITRs 中有限制限制性 SmaI 的识别站点。在质量控制中包括 SmaI 摘要, 以验证 ITRs 的完整性。</li>
		<li>发送 DNA 到一个病毒载体的核心设施, 以产生高质量的 rAAV。 注: 高滴度, 高品质的病毒眼影是至关重要的, 以避免混淆表型造成的污染物在病毒的准备。因此, 我们建议 rAAV 在一个已建立的矢量核心设施中所产生的选择, 除非 AAV 在实验室中建立良好的生产。</li>
	</ol>
</li>
	<li>
消融 (丧失功能) 注: 细菌毒素或毒素受体可用于调解细胞消融或神经沉默。我们已经产生了 pAAV. EF1α. dta, 以以一种与生相关的方式表达白喉毒素片段 a (DTA)。<ol>
<li>要生成一个与之相关的病毒载体, 请选择一个合适的启动子 (例如, pAAV. EF1α hChR2 (H134R)-eYFP) 的一个与此相关的向量。通过聚合酶链反应放大选择的编码序列 (例如, DTA), 其中包括 AscI 和 NheI 或兼容限制限制性识别站点在它们的悬垂中 (参见福斯特et 。7)</li>
		<li>使用标准分子生物学技术, 执行对载体 DNA (pAAV. EF1α. hChR2 (H134R) eYFP) 和 PCR 扩增插入 (NheI-DTA-asci) 的限制性摘要, 并限制内切酶 AscI 和 NheI。结扎纯化的碎片。</li>
		<li>将结扎反应转化为合适的细菌, 根据供应商提供的可选择的细菌的协议, 将细菌印在 LB 板上。使用 recA 缺乏/抑制的菌株, 如 MDS42 或 Stbl3, 用于克隆和随后扩增的质粒 DNA。</li>
		<li>在改造后的一天, 选择克隆和接种在 LB 培养基中补充适当的抗生素过夜37摄氏度。第二天, 从培养细菌中提取质粒 DNA。通过对提取的质粒 DNA 进行限制文摘和测序, 验证成功的克隆。</li>
		<li>放大高质量, 细菌质粒 DNA (见步骤 1.1.3)。将扩增的 DNA 发送到病毒的载体核心设施中, 以用于生产计算机病毒。</li>
	</ol>
</li>
</ol>2. 脊髓细胞的转导
<ol><li>病毒溶液的制备 警告: 病毒是传染性试剂, 应根据相关指南进行处理。在大多数情况下, rAAVs 可以在生物安全级别 1 (BSL1) 中处理。<ol>
<li>在注射之日, 将所需纯化病毒的一股整除在冰上解冻, 并将其放在冰上直到注射前直接。避免重复冻融循环, 因为这些会降低病毒的有效效价。 注: 如有必要, 解冻病毒整除可保持在摄氏4摄氏度, 长达3天。</li>
		<li>稀释无菌0.9% 氯化钠或磷酸盐缓冲生理盐水 (PBS) 中的病毒微粒。 注: 适当的效价取决于实验目的, 并应在实验中确定。一个良好的总病毒负载开始是 3x109基因组拷贝 (3.33x1012 GC/mL, 3x300 nL 注入)。考虑到过量和一些损失, 当加载毛细管考虑到, 大约2.5 µL 的病毒解决方案将需要为每一个鼠标。</li>
	</ol></li>
	<li>椎管内注射 Micropipettes 的制备
	<ol>
<li>' 拉 ' 薄壁玻璃毛细血管 (外径1毫米) 上的微拉出创建一个 ~ 5.5 毫米长, 浅柄。使用 3.0 mm 加热器灯丝和程序00的设置 (P (a) 适应于表 1。 注: 拉拔可以提前完成。被拉扯的毛细血管应该被存放在一个闭合的容器在建模黏土避免尘土, 以后可能堵塞微。热处理的 micropipettes 是不必要的。</li>
		<li>用椎板钳夹紧所拉毛细管的小腿, 长度约为4.5 至5毫米, 以形成直径为25-35 微米的尖端开口。使用微米刻度的显微镜, 测量几个 micropipettes 的内径, 以了解开口的大小, 或每微的测量值。 注: 小提示可能导致堵塞;一个较大的提示可能会导致组织损伤和困难穿透脊髓。</li>
	</ol></li>
	<li>外科手术设置和工具的准备
	<ol>
<li>确保设备清洁, 准备使用。通过热处理或消毒, 通过擦拭70% 乙醇和消毒工具消毒工作区域。不要留下任何消毒剂的微升注射器, 这将损害病毒载体的使用。</li>
	</ol></li>
	<li>外科动物的麻醉与准备 注: 手术总持续时间为60-90 分钟。<ol>
<li>选择 6-到10周的老老鼠, 以方便手术的过程。 注: 如果实验设计需要它, 注射年轻或年长的动物是可能的。任何应变和两性都可以原则上使用, 但使用相同的背景应变 (例如, C57BL/6J) 来比较不同基因的小鼠线之间的行为。如果性别差异是预期或有待调查, 分析男性和女性在不同的群体。</li>
		<li>用5% 异氟醚诱导麻醉。将动物放在一个热垫上的立体定向的框架, 并保持麻醉在 1-2. 5% 异氟醚 (气流速率900毫升/分钟)。监测整个手术的呼吸率。</li>
		<li>应用润滑剂眼膏预防手术中角膜干燥。</li>
		<li>剃掉动物的背部, 用湿纸巾仔细地去除毛发。用碘溶液对剃光皮肤进行消毒, 使皮肤干燥。</li>
		<li>给予镇痛治疗 (例如, 0.1-0.2 毫克/千克丁丙诺啡皮)。</li>
	</ol></li>
	<li>脊柱在腰椎脊髓水平的暴露 注: 由于脊髓和椎柱的差异性发育, 各自的水平没有对齐, 但腰椎椎线段 L4 与胸椎 T13 的水平相同。<ol>
<li>palpating 沿椎体柱定位最尾肋对。使用手术刀, 使 1.5-2.5 厘米纵向切割到皮肤开始从刚刚延髓到最尾肋对。</li>
		<li>用镊子将皮肤抬起, 用剪刀将其与底层肌肉分离。在整个手术中, 保持暴露的组织湿润与无菌0.9% 氯化钠。</li>
		<li>使用细钳和小剪刀, 使切口进入下一个, 薄膜层右侧的中线, 并切断它从棘突过程。它是分开的基础 paraspinous 肌肉, 但附加到棘突过程。</li>
	</ol></li>
	<li>腰椎脊髓水平的鉴别 注: 一旦椎柱暴露, 横突间韧带和背棘突的过程应该是可见的。几个解剖地标可以帮助确定正确的脊椎目标 (图 1B)。<ol>
<li>将皮肤向后拉向尾部, 露出髂嵴。在同一水平, 最尾对可见横突间韧带加入 L6 棘突过程。在尾部向后计数以延髓方向来识别感兴趣的椎骨。</li>
		<li>触及沿脊椎柱。最尾肋对位于 T13 椎延髓, 髋部的骨盆骨在 L6 椎骨的水平。</li>
		<li>定位脊柱一侧的肌腱是最白和最内侧的部位。T13 椎骨仅位于延髓。腰椎脊髓段 L4 位于椎体内。</li>
	</ol></li>
	<li>椎柱固定与腰髓外露
	<ol>
<li>把动物放到卷起的纸巾垫上, 把它抬高到立体定位框架的脊柱夹子上。</li>
		<li>固定靶椎以避免因呼吸而导致椎柱运动。为了达到这个目的, 对准目标脊椎相邻的夹钳, 固定一个钳位并在固定第二钳时用 Adson 钳握住椎柱。 注: 立柱应垂直于注射面, 并牢固固定, 使其不会在压力下移动。如有必要, 第二对钳可以固定在椎柱上。在这种情况下, 这是有用的两对钳固定在两个椎骨相邻的靶椎骨。</li>
		<li>把 paraspinous 的肌肉移除感兴趣的椎骨上。使用手术刀, 使平行切口只是内侧的肌腱平行的脊椎柱, 以及垂直切口延髓和骶管到靶椎骨。小心不要切得太深。用 rongeurs 把肌肉撕掉。使用镊子, 以清除椎体或硬脑膜以上的椎间隙的组织。</li>
		<li>在椎间隙, 背血管应可见于脊髓中线。对于单边注射, 执行部分椎板切除术, 钻入靶侧的脊椎中部的一个洞。在接近脊髓时, 使用一个0.5 毫米球形刀具和钻头的精细牙钻设备。用26G 斜面针取出残余的骨骼碎片, 以暴露脊髓。</li>
		<li>使用26G 斜面针, 穿孔的硬脑膜在钻孔, 并在椎间隙延髓和尾部到靶椎骨, 所有大约200µm 侧向血管。脑脊液应该从洞里逃出来, 脊髓应该有点肿胀。</li>
	</ol></li>
	<li>注射注射器的制备 注: 注射注射器应在注射开始前立即准备, 以减少粉尘颗粒堵塞微的危险。<ol>
<li>使用可拆卸针压缩配件套件将玻璃毛细管安装到微升注射器上。拧紧螺母, 确保紧固和安全配合。</li>
		<li>用无菌蒸馏水填充微升注射器, 然后用柱塞把它压出来。 注: 如果有太多的阻力, 使水不容易从尖端出来, 微可能被堵塞, 应该交换。</li>
		<li>将注射器安装到连接到电子控制 microinjector 的机器人上。小心不要碰任何与微。</li>
		<li>利用 microinjector, 绘制约1µL 的空气, 在水和病毒之间制造气泡。</li>
		<li>将2.5 µL 滴病毒溶液放到一片石蜡膜上, 小心地将微的尖端移动到液滴上, 然后使用立体定位框架将其绘制出来。然后仔细按下分配, 直到一点点病毒解决方案出现在尖端。</li>
		<li>收回注射器, 并用钢笔标记微, 并注意病毒溶液的水平;这将有助于监测输液是否按程序进行。</li>
	</ol></li>
	<li>椎管内注射
	<ol>
<li>将微的尖端移到硬脑膜中的一个孔上, 然后向下移动, 直到硬脑膜中有轻微的凹痕。为靶向脊髓背角注射, 向下移动500µm 100 µm 连续递增 (速度1毫米/秒), 然后200µm, 以允许机械稳定的组织。 注: 在这种情况下, 尖端的最后深度是300µm, 但可以根据目标区域进行调整。<ol>
<li>如果组织抗穿透力, 微可能会在硬脑膜上被抓住。在这种情况下, 收回和移动轻微的位于孔, 或使用针再次穿孔硬脑膜之前, 重复渗透尝试。</li>
		</ol>
</li>
		<li>在注射速度为 50 nl/分钟的情况下, 将泵按 300 nl 的目标注入量进行编程, 然后按 "开始" 按钮开始输液。</li>
		<li>注射完成后, 检查刻度, 以查看病毒水平是否下降, 并留在微3分钟, 让压力平衡之前, 慢慢缩回注射器。</li>
		<li>重复步骤 2.9. 1.-2.9. 3。其他两个注射点。</li>
	</ol></li>
	<li>缝合和恢复
	<ol>
<li>把脊椎钳和垫子放在老鼠下面。</li>
		<li>在针中断的层中闭合伤口, 用可吸收缝线缝合表面的组织层, 并用不可吸收的缝合缝进行皮肤。将碘消毒剂应用于缝合伤口。</li>
		<li>终止麻醉, 将动物放在热垫上, 直到它恢复到它的主笼为止。</li>
	</ol></li>
	<li>术后护理
	<ol>
<li>在手术当天, 第二天, 然后每2-3 天监测动物的健康状况。确保动物有正常的步态, 健康的外观 (体重, 眼睛, 皮毛和行为), 伤口愈合。</li>
		<li>持续镇痛治疗 (例如, 0.1-0.2 毫克/千克丁丙诺啡皮) 必要时, 每天三次。</li>
	</ol></li>
</ol>3. 行为和形态学分析
<ol><li>
行为分析 注: 允许动物在开始测量前至少30分钟适应各自的设置, 让它们适应新的实验环境。<ol>
<li>
冯·弗雷测试
		<ol>
<li>将动物单独放置在金属格子地板上, 在大约10厘米 x 10 厘米的单独隔间里。</li>
			<li>刺激 hindpaw 的足底表面与注射部位的动态冯弗雷灯丝。注意动物从刺激中提取爪子的力。</li>
			<li>重复五次并计算每种动物的六测量的平均提取阈值。</li>
		</ol>
</li>
		<li>
针刺试验
		<ol>
<li>将动物放置在金属格子地板上, 在大约10厘米 x 10 厘米的单独隔间里。</li>
			<li>用钝的26克针, 不经皮肤穿透, 刺激 hindpaw 与注射部位的足底表面。评分行为为零 (无反应) 或一 (反应)。</li>
			<li>重复9次间隔3分钟刺激和计算百分比响应从10测量为每种动物。</li>
		</ol>
</li>
		<li>
DREADD 配体注射氯氮平-n-氧化物 (CNO)
		<ol>
<li>溶解 CNO 二甲基亚砜 (亚砜), 以创建一个0.2 毫克/µL 的股票溶液, 可以储存在室温下。</li>
			<li>在实验当天, 稀释的库存溶液在无菌0.9% 氯化钠到0.2 µg/µL 和注射10µL/克体重腹腔。注射控制动物与车 (0.2% 亚砜在0.9% 氯化钠)。 注: 根据经验, 注射后对行为的峰值影响可预期为1-3 小时, 效果应在24小时后完全消退。在激活 glycinergic 神经元的情况下, 观察到的行为包括减少疼痛和瘙痒反应。由 DREADD 介导的神经元激活所诱发的行为将取决于脊髓神经元的靶向子集。</li>
		</ol>
</li>
		<li>
Pruritogens 注射液诱导瘙痒
		<ol>
<li>将 pruritogens 溶于0.9% 氯化钠, 溶液为8毫克/毫升 (氯喹) 或10毫克/毫升 (组胺)。2小时后, CNO 管理, 注入10µL pruritogen 或车辆的解决方案皮下的足底表面的 hindpaw 同侧的脊柱注射部位。或者, 将 pruritogen intradermally 注射到小牛中, 这是前一天剃掉的, 以减少剃须本身造成的厌恶行为。</li>
		</ol>
</li>
		<li>
录像分析自发性 Nocifensive 或 Pruritogen 引起的瘙痒行为
		<ol>
<li>将动物单独放到透明的隔间 (大约10厘米直径), 从他们各自的笼子在地板上的一些床上用品。</li>
			<li>录像动物5分钟, 以记录自发和30分钟记录 pruritogen 诱导的行为。在5分钟的垃圾桶中分析视频离线。</li>
		</ol>
</li>
		<li>
疼痛与瘙痒行为
		<ol>
<li>观察并注意到退缩, 舔, 和咬的行为。 注意: 受影响的部位的退缩和舔被认为是对疼痛的反应, 而咬伤则与瘙痒有关。</li>
			<li>以正常速度分析视频, 测量鼠标舔或咬受影响站点的时间, 或者在慢动作中测量舔和咬反射之间更精确的区别。或者, 数一下舔/咬/退缩的次数。</li>
		</ol>
</li>
	</ol>
</li>
	<li>
形态学分析
	<ol>
<li>在注射病毒或行为实验结束后三周进行形态学分析。 注: 我们在注射后48小时内检测到 eGFP 表达, 但也发现在接下来的几天甚至几周内表达明显增加。对于有较强报告表达的细胞来说, 一周的潜伏期 (从椎管内注射到灌注) 可能就足够了。对于基因表达弱的细胞, 具有弱启动子或弱显影的病毒, 可能需要较长的表达以确保可检测的水平。</li>
		<li>
组织固定
		<ol>
<li>灌注每只老鼠 transcardially, 先用20毫升的冰冷人工脑脊液 (ACSF) 溶液 (氯化钠125毫米, NaHCO3 25 毫米, NaH2PO4 1.25 毫米, d-葡萄糖20毫米, 氯化钾2.5 毫米, CaCl2 2 毫米 MgSO4 1 毫米), 然后与100毫升4% 冰冷多聚甲醛 (在0.1 米磷酸钠缓冲液中, pH 值为 7.4)。 注意: 多聚甲醛是有毒的, 必须小心处理。</li>
			<li>立即解剖腰椎脊髓, 并后固定的组织为2小时与4% 多聚甲醛冰。用0.1 米磷酸钠缓冲液 (ph 7.4) 简要清洗后固定组织, 然后用25% 蔗糖溶液 (在0.1 米磷酸钠缓冲液中, pH 7.4) 在4°c 过夜。</li>
			<li>在恒温器上切下30微米的 cryoprotected 组织, 然后在显微镜上贴上切片。</li>
		</ol>
</li>
		<li>
免疫荧光染色
		<ol>
<li>在 PBS 洗净后, 应用 300 ul 的阻断溶液 (10% 正常驴血清0.3% 海卫 PBS) 的部分1小时的室温。</li>
			<li>孵化的幻灯片与 300 ul 的各自组合的主要抗体在阻塞解决方案 (见材料表) 过夜, 在4摄氏度。在 PBS 中每3次洗5分钟。</li>
			<li>在室温下, 在1小时的阻断溶液中, 用相应的二次抗体组合孵化出切片。在 PBS 中每3次洗5分钟。</li>
			<li>简要冲洗 ddH2O 中的幻灯片, 然后使用荧光安装介质装入盖玻片。</li>
			<li>使用配备20x 和40x 目标的共焦显微镜获取荧光图像。</li>
		</ol>
</li>
	</ol>
</li>
</ol>

<ol>
	<li>Goulding, M., Bourane, S., Garcia-Campmany, L., Dalet, A., Koch, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+downunder:+an+update+from+the+spinal+cord.">Inhibition downunder: an update from the spinal cord.</a> Curr Opin Neurobiol. 26, 161-166 (2014).</li><li>Todd, 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=Neuronal+circuitry+for+pain+processing+in+the+dorsal+horn.">Neuronal circuitry for pain processing in the dorsal horn.</a> Nat Rev Neurosci. 11 (12), 823-836 (2010).</li><li>Sandkuhler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Models+and+mechanisms+of+hyperalgesia+and+allodynia.">Models and mechanisms of hyperalgesia and allodynia.</a> Physiol Rev. 89 (2), 707-758 (2009).</li><li>Zeilhofer, H. U., Wildner, H., Yevenes, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+synaptic+inhibition+in+spinal+sensory+processing+and+pain+control.">Fast synaptic inhibition in spinal sensory processing and pain control.</a> Physiol Rev. 92 (1), 193-235 (2012).</li><li>Azim, E., Jiang, J., Alstermark, B., Jessell, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skilled+reaching+relies+on+a+V2a+propriospinal+internal+copy+circuit.">Skilled reaching relies on a V2a propriospinal internal copy circuit.</a> Nature. 508 (7496), 357-363 (2014).</li><li>Cui, L., 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=Identification+of+Early+RET++Deep+Dorsal+Spinal+Cord+Interneurons+in+Gating+Pain.">Identification of Early RET+ Deep Dorsal Spinal Cord Interneurons in Gating Pain.</a> Neuron. 91 (6), 1413 (2016).</li><li>Foster, 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=Targeted+ablation,+silencing,+and+activation+establish+glycinergic+dorsal+horn+neurons+as+key+components+of+a+spinal+gate+for+pain+and+itch.">Targeted ablation, silencing, and activation establish glycinergic dorsal horn neurons as key components of a spinal gate for pain and itch.</a> Neuron. 85 (6), 1289-1304 (2015).</li><li>Francois, 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=A+Brainstem-Spinal+Cord+Inhibitory+Circuit+for+Mechanical+Pain+Modulation+by+GABA+and+Enkephalins.">A Brainstem-Spinal Cord Inhibitory Circuit for Mechanical Pain Modulation by GABA and Enkephalins.</a> Neuron. 93 (4), 822-839 (2017).</li><li>Peirs, 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=Dorsal+Horn+Circuits+for+Persistent+Mechanical+Pain.">Dorsal Horn Circuits for Persistent Mechanical Pain.</a> Neuron. 87 (4), 797-812 (2015).</li><li>Petitjean, 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=Dorsal+Horn+Parvalbumin+Neurons+Are+Gate-Keepers+of+Touch-Evoked+Pain+after+Nerve+Injury.">Dorsal Horn Parvalbumin Neurons Are Gate-Keepers of Touch-Evoked Pain after Nerve Injury.</a> Cell Rep. 13 (6), 1246-1257 (2015).</li><li>Zhang, Y., 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=Identifying+local+and+descending+inputs+for+primary+sensory+neurons.">Identifying local and descending inputs for primary sensory neurons.</a> J Clin Invest. 125 (10), 3782-3794 (2015).</li><li>Haenraets, K., 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=Spinal+nociceptive+circuit+analysis+with+recombinant+adeno-associated+viruses:+the+impact+of+serotypes+and+promoters.">Spinal nociceptive circuit analysis with recombinant adeno-associated viruses: the impact of serotypes and promoters.</a> J Neurochem. , (2017).</li><li>Abraira, V. 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=The+Cellular+and+Synaptic+Architecture+of+the+Mechanosensory+Dorsal+Horn.">The Cellular and Synaptic Architecture of the Mechanosensory Dorsal Horn.</a> Cell. 168 (1-2), 295-310 (2017).</li><li>Wildner, 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=Genome-wide+expression+analysis+of+Ptf1a-+and+Ascl1-deficient+mice+reveals+new+markers+for+distinct+dorsal+horn+interneuron+populations+contributing+to+nociceptive+reflex+plasticity.">Genome-wide expression analysis of Ptf1a- and Ascl1-deficient mice reveals new markers for distinct dorsal horn interneuron populations contributing to nociceptive reflex plasticity.</a> J Neurosci. 33 (17), 7299-7307 (2013).</li><li>Inquimbert, P., Moll, M., Kohno, T., Scholz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereotaxic+injection+of+a+viral+vector+for+conditional+gene+manipulation+in+the+mouse+spinal+cord.">Stereotaxic injection of a viral vector for conditional gene manipulation in the mouse spinal cord.</a> J Vis Exp. (73), e50313 (2013).</li><li>Kohro, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+minimally-invasive+method+for+microinjection+into+the+mouse+spinal+dorsal+horn.">A new minimally-invasive method for microinjection into the mouse spinal dorsal horn.</a> Sci Rep. 5, 14306 (2015).</li><li>Bourane, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gate+control+of+mechanical+itch+by+a+subpopulation+of+spinal+cord+interneurons.">Gate control of mechanical itch by a subpopulation of spinal cord interneurons.</a> Science. 350 (6260), 550-554 (2015).</li><li>Bourane, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+spinal+circuit+for+light+touch+and+fine+motor+control.">Identification of a spinal circuit for light touch and fine motor control.</a> Cell. 160 (3), 503-515 (2015).</li><li>Duan, B., 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=Identification+of+spinal+circuits+transmitting+and+gating+mechanical+pain.">Identification of spinal circuits transmitting and gating mechanical pain.</a> Cell. 159 (6), 1417-1432 (2014).</li><li>Gutierrez-Mecinas, 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=Preprotachykinin+A+is+expressed+by+a+distinct+population+of+excitatory+neurons+in+the+mouse+superficial+spinal+dorsal+horn+including+cells+that+respond+to+noxious+and+pruritic+stimuli.">Preprotachykinin A is expressed by a distinct population of excitatory neurons in the mouse superficial spinal dorsal horn including cells that respond to noxious and pruritic stimuli.</a> Pain. 158 (3), 440-456 (2017).</li><li>Awatramani, R., Soriano, P., Rodriguez, C., Mai, J. J., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryptic+boundaries+in+roof+plate+and+choroid+plexus+identified+by+intersectional+gene+activation.">Cryptic boundaries in roof plate and choroid plexus identified by intersectional gene activation.</a> Nat Genet. 35 (1), 70-75 (2003).</li><li>Kim, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linking+genetically+defined+neurons+to+behavior+through+a+broadly+applicable+silencing+allele.">Linking genetically defined neurons to behavior through a broadly applicable silencing allele.</a> Neuron. 63 (3), 305-315 (2009).</li><li>Kim, J. C., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+fate-mapping+approaches:+new+means+to+explore+the+embryonic+origins+of+the+cochlear+nucleus.">Genetic fate-mapping approaches: new means to explore the embryonic origins of the cochlear nucleus.</a> Methods Mol Biol. 493, 65-85 (2009).</li></ol>

作者没有什么可透露的。

椎管内注射自动增值服务可能成为一个强大的技术在一个研究实验室, 使分析脊髓细胞具有较高的时间和空间的解决方案。该协议使三主要脊柱段支配由感觉神经元扩展其外围轴突到后肢的传导。传感三段产生健壮和可重复的行为数据。它还能在单椎管内注射后检测出更大的感官区域。例如, 相同的注射机制允许测试爪子 (冯弗雷, 哈格里夫斯,等) 和大腿 (皮下注射, 机械受体刺激), 从而扩大?...

背脊髓是人体和大脑周围信息交换的必要条件。感官刺激, 如热, 冷, 接触, 或有害刺激检测的专门的外围神经元, 这将信息传递给脊髓背角神经元。在这里, 一个复杂的抑制和兴奋中间神经元网络调节并最终通过脊髓投射神经元传递感官信息到 supraspinal 站点 1, 2.由脊柱间和投射神经元之间进行的计算, 以此来确定哪些信息被抑制或中继到何种强度。感官刺激的整合的变化, 如抑制和刺激之间的平衡改变, 可能导致感官功能障碍, 如过敏性或痛觉 (通常不痛刺激后的痛苦感觉)。这些更改被认为是各种慢性疼痛状态的根本原因3,4。因此, 脊柱电路在感官加工中具有高度的重要性, 因此在感知有机体的环境和自我方面是非常重要的。随着分子、遗传和外科技术的最近出现和结合, 使基因识别的脊髓神经元亚群精确操作, 科学家们现在开始了解底层的脊柱电路负责不同感官方式的加工。
椎管内注射 rAAV 为野生型或转基因小鼠, 极大地促进了对脊髓神经元特定子集功能的操作、分析和理解5,6,7,8,9,10,11. 此技术允许在空间上提供标记蛋白 (如 gfp/gfp 融合蛋白)、报告蛋白 (如 GCaMP) 或效应蛋白 (如细菌毒素、channelrhodopsin 或遗传药理学受体)。脊髓神经元受限的方式。局部注射 rAAVs 依赖于转基因小鼠, 表达 recombinase 在脊髓神经元的特定子集, 允许对各自神经元种群的具体分析。我们使用这种技术来标记、消融、抑制或激活脊髓 glycinergic 神经元, 证明它们是控制疼痛和瘙痒传输的脊髓门的重要部分7。在这些实验中, 椎管内注射 rAAV 为 GlyT2::Cre 小鼠, 使脊髓 glycinergic 神经元有选择性的操作。因此, 可以避免同时操作包含对动物生存至关重要的 glycinergic 神经元的 supraspinal 电路。
虽然椎管内注射 rAAVs 限制了对注射部位的感染, 但病毒转导不仅可以发生在局部神经元中, 而且也可能出现在通过轴突投射连接到注射部位的神经元中。后者经常被用来追踪中枢神经系统的区域, 为大脑中特定的细胞核提供神经元输入。然而, 轴突投射的感染也可能是一个混淆的因素, 当一个定义的神经元群体应在特定的地点进行研究。为了解决这些问题, 我们最近对 AAV 血清型和表达盒进行了全面分析, 以确定血清型和促进剂, 可用于最小化或最大限度地逆行转导。本文在脊柱电路研究的背景下, 分析了不同血清型和促进剂对背根神经节、延髓丘脑髓质 (RVM) retrogradely 传感器神经元的能力, 以及体感皮层12. 因此, 本议定书中概述的技术可以用于分析注射部位的脊髓神经元, 也可用于分析为脊髓注入部位提供输入的投射神经元。在这里描述的协议中, 三注射 rAAV 到腰脊髓左侧, 使三腰椎段 (L3-L5) 神经元转导。L3-L5 段接受大部分的感觉输入从后肢同侧到注射部位。我们证明, L3-L5 基因标记神经元的功能操作足以唤起强健的行为变化, 从而为基因标记的神经元亚型的电路功能提供功能性证据。

<table><tbody><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>micropipette puller: DMZ-Universal-Electrode-Puller</td><td>Zeitz</td><td>NA</td><td></td></tr><tr><td>anesthesia unit: Oxymat3 oxygen concentrator</td><td>Weinmann</td><td>NA</td><td></td></tr><tr><td>anesthesia unit: VIP 3000 Veterinary Vaporizer</td><td>Midmark</td><td>NA</td><td></td></tr><tr><td>Heat mat: Mio Star Thermocare 100</td><td>Migros</td><td>717614700000</td><td></td></tr><tr><td>Electric shaver</td><td>Philips</td><td>BT9290</td><td></td></tr><tr><td>surgical microscope (OPMI pico)</td><td>Zeiss</td><td>NA</td><td></td></tr><tr><td>Small animal stereotaxic apparatus</td><td>Kopf</td><td>NA</td><td></td></tr><tr><td>Neurostar StereoDrive (optional)</td><td>Neurostar</td><td>NA</td><td></td></tr><tr><td>Model 51690 Cunningham mouse spinal adaptor</td><td>Harvard Apparatus</td><td>72-4811</td><td></td></tr><tr><td>PHD Ultra syringe pump with nanomite</td><td>Harvard Apparatus</td><td>70-3601</td><td></td></tr><tr><td>Hamilton 701 RN 10 &amp;mu;l glass microliter syringe</td><td>Hamilton</td><td>7635-01</td><td></td></tr><tr><td>Hamilton Removable needle (RN) compression fitting 1 mm</td><td>Hamilton</td><td>55750-01</td><td></td></tr><tr><td>fine dentistry drilling apparatus: Osada success 40</td><td>Osada</td><td>OS-40</td><td></td></tr><tr><td>spherical cutter, 0.5mm</td><td>Busch</td><td>12001005B</td><td></td></tr><tr><td>electronic von Frey anesthesiometer</td><td>IITC</td><td>23905</td><td></td></tr><tr><td>flexible von Frey hairs</td><td>IITC</td><td>#7</td><td></td></tr><tr><td>LSM710 Pascal confocal microscope</td><td>Zeiss</td><td>NA</td><td></td></tr><tr><td>0.8 NA &amp;times; 20 Plan-apochromat objective</td><td>Zeiss</td><td>NA</td><td></td></tr><tr><td>1.3 NA &amp;times; 40 EC Plan-Neofluar oil-immersion objective</td><td>Zeiss</td><td>NA</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Surgical Tools&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Scalpel Handle #4, 13cm</td><td>Fine Science Tools</td><td>10004-13</td><td></td></tr><tr><td>Extra Fine Bonn Scissors</td><td>Fine Science Tools</td><td>14084-08</td><td></td></tr><tr><td>Adson forceps, 1 x 2 teeth, 12 cm</td><td>Fine Science Tools</td><td>11027-12</td><td></td></tr><tr><td>Friedman-Pearson rongeurs, curved, 0.7 mm cup</td><td>Fine Science Tools</td><td>16121-14</td><td></td></tr><tr><td>Dumont #2 laminectomy forceps</td><td>Fine Science Tools</td><td>11223-20</td><td></td></tr><tr><td>Olsen-Hegar needle holders, serrated, 8.5 mm clamp length</td><td>Fine Science Tools</td><td>12002-12</td><td></td></tr><tr><td>Fine forceps #5</td><td>Fine Science Tools</td><td>11254-20</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Consumables and Chemicals&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Thin-wall glass capillary, 1mm outside diameter</td><td>World Precision Instruments</td><td>TW 100-3</td><td></td></tr><tr><td>Syringes (1, 5 and 20 ml)</td><td>B. Braun</td><td>(9166917V, 4606051V, 4606205V)</td><td></td></tr><tr><td>26G beveled needle</td><td>B. Braun</td><td>4665457</td><td></td></tr><tr><td>Sterile scalpel blades</td><td>B. Braun</td><td>BB523</td><td></td></tr><tr><td>Surgical sutures Safil Quick+ 4/0, absorbable</td><td>B. Braun</td><td>C1046220</td><td></td></tr><tr><td>Surgical sutures Premilene 5/0, non-absorbable</td><td>B. Braun</td><td>C0932191</td><td></td></tr><tr><td>Sterile PBS or saline (0.9%)</td><td></td><td>NA</td><td></td></tr><tr><td>Ethanol, 70% (disinfectant)</td><td></td><td>NA</td><td></td></tr><tr><td>Iodine solution (e.g. Braunol)</td><td>B. Braun</td><td>18380</td><td></td></tr><tr><td>Anaesthetics (e.g. Attane isoflurane)</td><td>Provet</td><td>2222</td><td></td></tr><tr><td>Aldasorber</td><td>Provet</td><td>333526</td><td></td></tr><tr><td>analgesics (e.g. buprenorphine: temgesic)</td><td>Indivior</td><td>GTIN: 7680419310018</td><td></td></tr><tr><td>Ophthalmic ointment (e.g. vita-pos)</td><td>Pharma medica</td><td>GTIN: 4031626710635</td><td></td></tr><tr><td>Cotton swabs (e.g. from)</td><td>IVF Hartmann</td><td>1628100</td><td></td></tr><tr><td>Facial tissues (e.g. from)</td><td>Uehlinger AG</td><td>2015.10018</td><td></td></tr><tr><td>Superfrost plus microscope slides</td><td>ThermoScientific</td><td>J1800AMNZ</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Mice&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>C57BL/6J mice (wildtype)</td><td>The Jackson Laboratory</td><td>RRID:IMSR_JAX:000664</td><td></td></tr><tr><td>Rorbtm1.1(cre)Hze/J mice (ROR&amp;beta;Cre)</td><td>The Jackson Laboratory</td><td>RRID:IMSR_JAX:023526</td><td></td></tr><tr><td>Gt(ROSA)26Sortm14(CAG-tdTomato)Hze/J mice (R26Tom)</td><td>The Jackson Laboratory</td><td>RRID: IMSR_JAX:007914</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Viral vectors&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>AAV1.CB7.CI.eGFP.WPRE.rBG (AAV1.CAG.eGFP)</td><td>Penn Vector Core</td><td>AV-1-PV1963</td><td></td></tr><tr><td>AAV1.CAG.flex.eGFP.WPRE.bGH (AAV1.CAG.flex.eGFP)</td><td>Penn Vector Core</td><td>AV-1-ALL854</td><td></td></tr><tr><td>AAV1.CAG.flex.tdTomato.WPRE&lt;br /&gt;.bGH (AAV1.CAG.flex.tdTomato)</td><td>Penn Vector Core</td><td>AV-1-ALL864</td><td></td></tr><tr><td>AAV1.EF1a.flex.DTA.hGH (AAV1.EF1a.flex.DTA)</td><td>Penn Vector Core</td><td>Custom production</td><td></td></tr><tr><td>AAV1.hSyn.DIO.hM3D(Gq)-mCherry.hGH (AAV.flex.hM3D(Gi))</td><td>Penn Vector Core</td><td>Custom production</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pAAV.hSyn.flex.hM3D(Gq)-mCherry</td><td>Addgene</td><td>44361</td><td></td></tr><tr><td>pAAV.EF1&amp;alpha;.flex.hChR2(H134R)-eYFP</td><td>Addgene</td><td>20298</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Bacteria&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MDS42</td><td>ScarabGenomics</td><td></td><td></td></tr><tr><td>Stbl3</td><td>ThermoScientific</td><td>C737303</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>EndoFree Plasmid Maxi Kit</td><td>Quiagen</td><td>12362</td><td></td></tr><tr><td>NucleoBond PC 500</td><td>Machery &amp;amp; Nagel</td><td>740574</td><td></td></tr><tr><td>clozapine-N-oxide (CNO)</td><td>Enzo Life Sciences</td><td>BBL-NS105-0025</td><td></td></tr><tr><td>chloroquine diphosphate salt</td><td>Sigma</td><td>C6628</td><td></td></tr><tr><td>histamine</td><td>Sigma</td><td>H7125</td><td></td></tr><tr><td>Dapi</td><td>Invitrogen</td><td>D3571</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies (dilution)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rabbit anti-GFP (1:1000)</td><td>Molecular Probes</td><td>RRID:AB_221570</td><td></td></tr><tr><td>Rabbit anti-NeuN (1:3000)</td><td>Abcam</td><td>RRID:AB_10711153</td><td></td></tr><tr><td>Goat anti-Pax2 (1 : 200)</td><td>R &amp;amp; D Systems</td><td>RRID:AB_10889828</td><td></td></tr><tr><td>Guinea pig anti-Lmx1b (1 : 10 000)</td><td>Dr Carmen Birchmeier</td><td>Muller et al. 2002</td><td></td></tr><tr><td>Rabbit anti-GFAP (1 : 1000)</td><td>DakoCytomation</td><td>RRID:AB_10013382</td><td></td></tr><tr><td>Secondary antibodies raised in donkey (1:800)</td><td>Jackson ImmunoResearch Laboratories</td><td>NA</td><td></td></tr></tbody></table>

adeno-associated virus-mediated transgene expression in genetically defined neurons of the spinal cord

脊髓神经元亚群的选择性操纵主要是通过两种不同的方法实现的: 1) 交叉遗传学, 利用双或三种转基因小鼠来实现有选择性表达的记者或效应者。基因 (例如, 从 Rosa26 轨迹) 在期望脊柱人口。2) 椎管内注射重组腺相关病毒 (rAAV);本文对选择的报告者或效应基因进行了 AAV 矢量编码, 并将其用于表达 recombinase 在所期望的神经元亚群中的小鼠脊髓。该协议描述了如何产生 rAAV 载体和如何传感器脊髓背角 L3-L5 与 rAAVs 神经元。由于腰椎段 L3-L5 是由那些从后肢传递感官信息的外周感觉神经元支配的, 自发的行为和对向侧向一侧与注射侧的感觉测试的反应, 可以分析了操作神经元在感官处理中的作用。我们提供的例子, 这项技术可以用来分析基因定义的脊髓神经元子集。在转基因小鼠中, 病毒介导的转基因基因表达的主要优势与经典的小鼠转基因基因表达相比有以下特点: 1) 不同的 rAAVs 编码各种记者或效应蛋白可注入单一的转基因生产线, 从而克服了创建多个转基因小鼠线的需要。2) 椎管内注射限制了对注射部位和注射后的时间对细胞的操作。主要缺点有: 1) rAAVs 的报告基因表达更具变数。2) 手术是需要传感器脊髓神经元的兴趣。这两种方法中哪一种比较合适取决于神经元的数量和要处理的研究问题。

为了说明椎管内注射 rAAV 编码标记蛋白所能获得的表达水平, 我们首先注射了 AAV1。eGFP 进入野生型小鼠的腰椎脊髓。三个注射间隔约1毫米, 产生了几乎连续感染腰椎段 L3 到 L5 (图 1A-c)。病毒注射在深度300µm 从脊柱表面导致主要感染的细胞在脊髓背角。但是, 也可以在腹角 (图 1D) 中找到受感染的细胞。下一步, 目的?...

Watch this Scientific Journal Video about Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord at JoVE.com

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

腺相关病毒介导的脊髓基因神经元的转基因表达

椎管内注射 recombinase 依赖重组腺相关病毒 (rAAV) 可以用来操纵任何基因标记的细胞类型的脊髓。在这里, 我们描述如何传感器神经元的背角的腰椎脊髓。这种技术可以对纵的神经元亚型进行功能性审问。

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

adeno-associated-virus-mediated-transgene-expression-genetically

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

18.0K 次观看.  University of Zurich. 椎管内注射 recombinase 依赖重组腺相关病毒 (rAAV) 可以用来操纵任何基因标记的细胞类型的脊髓。在这里, 我们描述如何传感器神经元的背角的腰椎脊髓。这种技术可以对纵的神经元亚型进行功能性审问。

视频: Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Intracranial Injection of Adeno-associated Viral Vectors

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific subsets of cells in different brain regions both in vivo and in brain sections. Unlike the injection of fluorescent dyes, viral labeling offers targeting of individual cell types and is less expensive and time consuming than establishing transgenic mouse lines. In this technique, an adeno-associated viral (AAV) vector is injected intracranially using stereotaxic coordinates, a micropipette and an automated pump for precise delivery of AAV to the desired area with minimal damage to the surrounding tissue. Injection parameters can be tailored to individual experiments by adjusting the animal age at injection, injection location, volume of injection, rate of injection, AAV serotype and the promoter driving gene expression. Depending on the conditions chosen, virally-induced transgene expression can allow visualization of groups of cells, individual cells or fine cellular processes, down to the level of dendritic spines. The experiment shown here depicts an injection of double-stranded AAV expressing green fluorescent protein for the labeling of neurons and glia in the mouse primary visual cortex.

Here we present the intracranial injection of AAV vectors for fluorescent labeling of neurons and glia in the visual cortex.

颅内注射腺相关病毒载体

Intracranial injection of viral vectors engineered to express a fluorescent protein is a versatile labeling technique for visualization of specific ...

科研

神经科学

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

立体定向注射病毒载体的基因操作在小鼠脊髓条件

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

新生小鼠脑侧脑室注射病毒的持续和广泛的神经元传导

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Dorsal Root Ganglion Injection and Dorsal Root Crush Injury as a Model for Sensory Axon Regeneration

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each other anatomically, it is important to identify an appropriate model to use for the study of axon regeneration. By using a suitable model, we can formulate a specific treatment based on the severity of injury, the neuronal cell type of interest, and the desired spinal tract for assessing regeneration. Within the sensory pathway, DRG neurons are responsible for relaying sensory information from the periphery to the CNS. We present here a protocol that uses a DRG injection with a viral vector and a concurrent dorsal root crush injury in the lower cervical spinal cord of an adult rat as a model to study sensory axon regeneration. As demonstrated using a control virus, AAV5-GFP, we show the effectiveness of a direct DRG injection in transducing DRG neurons and tracing sensory axons into the spinal cord. We also show the effectiveness of the dorsal root crush injury in denervating the forepaw as an injury model for evaluating axon regeneration. Despite the requirement for specialized training to perform this invasive surgical procedure, the protocol is flexible, and potential users can modify many parts to accommodate their experimental requirements. Importantly, it can serve as a foundation for those in search of a suitable animal model for their studies. We believe that this article will help new users to learn the procedure in a very efficient and effective manner.

This protocol presents the use of a dorsal root ganglion (DRG) injection with a viral vector and a concurrent dorsal root crush injury in an adult rat as a model to study sensory axon regeneration. This model is suitable for investigating the use of gene therapy to promote sensory axon regeneration.

背根神经节内注射和背根挤压伤为感官轴突再生模型

Achieving axon regeneration after nervous system injury is a challenging task. As different parts of the central nervous system (CNS) differ from each ...

Subpial Adeno-associated Virus 9 (AAV9) Vector Delivery in Adult Mice

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. Using subpially-placed polyethylene catheters (PE-10 or PE-5) for AAV9 delivery, potent transgene expression through the spinal parenchyma (white and gray matter) in subpially-injected spinal segments has been demonstrated. Because of the wide range of transgenic mouse models of neurodegenerative diseases, there is a strong desire for the development of a potent central nervous system (CNS)-targeted vector delivery technique in adult mice. Accordingly, the present study describes the development of a spinal subpial vector delivery device and technique to permit safe and effective spinal AAV9 delivery in adult C57BL/6J mice. In spinally immobilized and anesthetized mice, the pia mater (cervical 1 and lumbar 1-2 spinal segmental level) was incised with a sharp 34 G needle using an XYZ manipulator. A second XYZ manipulator was then used to advance a blunt 36G needle into the lumbar and/or cervical subpial space. The AAV9 vector (3-5 &#181;L; 1.2 x 1013 genome copies (gc)) encoding green fluorescent protein (GFP) was then injected subpially. After injections, neurological function (motor and sensory) was assessed periodically, and animals were perfusion-fixed 14 days after AAV9 delivery with 4% paraformaldehyde. Analysis of horizontal or transverse spinal cord sections showed transgene expression throughout the entire spinal cord, in both gray and white matter. In addition, intense retrogradely-mediated GFP expression was seen in the descending motor axons and neurons in the motor cortex, nucleus ruber, and formatio reticularis. No neurological dysfunction was noted in any animals. These data show that the subpial vector delivery technique can successfully be used in adult mice, without causing procedure-related spinal cord injury, and is associated with highly potent transgene expression throughout the spinal neuraxis.

Subpial Adeno-associated Virus 9 AAV9 Vector Delivery in Adult Mice

The goal of the present study was to develop and validate the potency and safety of spinal adeno-associated virus 9 (AAV9)-mediated gene delivery by using a novel subpial gene delivery technique in adult mice.

在成年小鼠中的亚型腺相关病毒9（AAV9）载体递送

The successful development of a subpial adeno-associated virus 9 (AAV9) vector delivery technique in adult rats and pigs has been reported on previously. ...

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Direct Intrathecal Injection of Recombinant Adeno-associated Viruses in Adult Mice

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, and excellent transduction efficacy in the CNS.&#160;Previous studies have demonstrated the therapeutic potency of AAV-delivered gene therapy in neurodegenerative disorders by IT administration. However, high rates of unpredictable failure due to the technical limitation of IT administration in small animals have been reported. Here, we established a scoring system to indicate the success extent of lumbar puncture in small animals by adding 1% lidocaine hydrochloride into the injection solution. We further show that the extent of transient weakness following injection can predict the transduction efficiency of AAV. Thus, this IT injection method can be used to optimize therapeutic trials in mouse models of CNS diseases that afflict wide regions of the CNS.

Here we present a direct intrathecal injection technique using 1% lidocaine hydrochloride in a viral solution to ensure efficient adeno-associated virus delivery to small animals and establish a scoring system to predict transduction efficiency in the central nervous system according to the degree of transient weakness induced by lidocaine.

重组腺相关病毒在成年小鼠中的直接注射

Intrathecal (IT) injection of adeno-associated virus (AAV) has drawn considerable interest in CNS gene therapy by virtue of its safety, noninvasiveness, ...

Direct Injection of a Lentiviral Vector Highlights Multiple Motor Pathways in the Rat Spinal Cord

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. Injection directly into spinal cord tissue bypasses these barriers, providing access to cell bodies or synapses where molecules can be incorporated. Combining viral vector technology with this method allows for introduction of target genes into nervous tissue for the purpose of gene therapy or tract tracing. Here a virus engineered for highly efficient retrograde transport (HiRet) is introduced at the synapses of propriospinal interneurons (PNs) to encourage specific transport to neurons in the spinal cord and brainstem nuclei. Targeting PNs takes advantage of the numerous connections they receive from motor pathways such as the rubrospinal and reticulospinal tracts, as well as their interconnection with each other throughout spinal cord segments. Representative tracing using the HiRet vector with constitutively active green fluorescent protein (GFP) shows high fidelity details of cell bodies, axons and dendritic arbors in thoracic PNs and in reticulospinal neurons in the pontine reticular formation. HiRet incorporates well into brainstem pathways and PNs but shows age dependent integration into corticospinal tract neurons. In summary, spinal cord injection using viral vectors is a suitable method for introduction of proteins of interest into neurons of targeted tracts.

This protocol demonstrates injection of a retrogradely transportable viral vector into rat spinal cord tissue. The vector is taken up at the synapse and transported to the cell body of target neurons. This model is suitable for retrograde tracing of important spinal pathways or targeting cells for gene therapy applications.

直接注射慢病毒载体突出大鼠脊髓中的多种运动途径

Introducing proteins of interest into cells in the nervous system is challenging due to innate biological barriers that limit access to most molecules. ...

Targeting the Corticospinal Tract in Neonatal Rats with a Double-Viral Vector using Combined Brain and Spine Surgery

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric spinal cord injuries (SCIs) compared to adult SCIs. In addition, reliably introducing therapies into the target cells of the central nervous system (CNS) can be challenging, and inaccuracies can compromise the efficacy of the study or therapy. This protocol combines viral vector technology with a novel surgical technique&#160;to accurately introduce gene therapies into neonatal rats at postnatal day 5. Here, a virus engineered for retrograde transport (retroAAV2) of Cre is introduced at the axon terminals of corticospinal neurons in the spinal cord, where it is subsequently transported to the cell bodies. A double-floxed&#160;inverted orientation (DIO) designer receptor exclusively activated by designer drug(s) (DREADD) virus is then injected into the somatomotor cortex of the brain. This double-infection technique promotes the expression of the DREADDs only in the co-infected corticospinal tract (CST) neurons.&#160;Thus, the simultaneous co-injection of the somatomotor cortex and cervical CST terminals is a valid method for studying the chemogenetic modulation of recovery following cervical SCI models in neonatal rats.

This protocol demonstrates a novel method for applying gene therapies to subpopulations of cells in neonatal rats at postnatal ages 5-10 days by injecting an anterograde chemogenetic modifier into the somatomotor cortex and a retrogradely transportable Cre recombinase into the cervical spinal cord.

使用脑和脊柱联合手术用双病毒载体靶向新生大鼠的皮质脊髓束

Successfully tackling the obstacles that constrain research on neonatal rats is important for studying the differences in outcomes seen in pediatric ...