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>

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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), 从而扩大的感官方式, 可以解决。
关键步骤:
对于获取健壮的形态学和行为数据以及避免人工制品, 有几个关键步骤。病毒载体的设计和启动子和血清型的选择对转基因区域的转导效率和范围有很强的影响, 因此对行为结果 (关于病毒传播和转导效率的细节), 请参见12). 需要高质量的病毒制剂, 以尽量减少病毒注射的副作用, 如果病毒溶液中存在污染物或细胞碎片, 就会发生这种情况。注射脊髓所需的手术必须非常小心地进行, 以避免手术引入的形态学和行为制品。特别是在椎板切除术和硬脑膜穿孔时, 需要特别注意处理脊髓。脊髓损伤会损害小鼠的躯体感觉和运动协调, 从而损害任何计划中的行为实验。此外, 仔细组装注射器是很重要的。微不正确的装配或堵塞会阻碍实验的进行。注射部位的脊柱组织必须在实验结束时进行检查, 以验证注射区的成功注射和转导, 并排除手术后组织损伤过多的原因。该协议使用病毒滴度高达 1x1013 GC/mL 的浓度, 没有明显的毒性, 但更高的病毒滴度可能会在某个时间点中毒。此外, 毒性也很可能取决于注射 AAV 编码的蛋白质。
修改:
该协议中的几个参数可以适应神经元种群和研究问题。注射 300 nL 病毒溶液在深度300µm 导致神经元转导的整个, 主要在背角。如果目标是对神经元进一步的腹侧, 深度可以调整。注射大量的病毒 (我们使用了多达 500 nL 每注射) 将导致更大的传播 dorsoventral 和 rostrocaudal 方向。这可能有助于在 rostrocaudal 范围内实现对额外细胞的靶向, 但会增加对侧侧的溢出, 这可能会妨碍对侧侧的使用作为内部控制。此外, 转基因细胞的 dorsoventral 程度会增加, 这可能是预期的效果, 或者应该避免。注射 300 nl 在深度300µm 避免副作用对马达控制在 GlyT2::Cre 小鼠, 而注射 500 nL 或注射在更大的深度偶尔地产生马达表型, 例如痉挛的后肢延长 (没有显示的数据)。注入较小的卷可用于将目标限制到更受限制的区域5。病毒滴度是另一个可以修改的参数, 实际上应该为每种病毒确定。对于细菌毒素, 如高效的 DTA, 低表达水平可能足以诱发预期效果, 而对于荧光记者和 DREADDs, 更高的滴度可能是必要的可检测或有效的表达。
方法对于现有/替代方法的意义:
到目前为止, 主要有两种技术用于功能性地审问感知信号传输所需的神经回路。许多研究人员用椎管内注射 rAAV 编码器和效应蛋白来 recombinase 表达小鼠, 以标记和操作脊髓神经元亚群。椎管内注射作为一种操作脊髓细胞的技术, 以前被描述为15,16。这里概述的协议是专门设计, 以传感器基因标记的神经元在三个连续段的腰椎脊髓, 同时尽量减少手术操作。这是通过将三种注射中的两个注入到 T13 椎骨的椎间隙延髓和尾部, 然后通过钻孔进入 T13 进行第三次注射而不是切除椎骨来实现的。靶向 L3-L5 段是支配后肢的感觉神经元的主要终止区。我们证明, 这种传导方式足以在 glycinergic 神经元的操作后产生强健的行为变化, 并表明它适合于分析各种不同基因标记的脊髓神经元。
用于控制脊髓神经元亚群功能的第二种技术是以杂交转基因动物为基础的。在这里, 表达 recombinase 在脊髓神经元的特定子集必须交叉到记者老鼠, 以实现所需的标记或效应蛋白的表达在各自的神经元子集17,18,19. 为了说明在比较使用记者小鼠或椎管内病毒注射获得的结果时可能发生的一些差异, 我们要么越过 RORβ创能的动物到一项依赖于一份报告的记者鼠标或注入 RORβ 与 rAAV 的报告员一起进行的小鼠。本文比较了 RORβ rAAV 中获得的报告基因表达与报告基因表达的对比;R26Tom鼠标。报告基因表达从椎管内注射的一项依赖 rAAV 被限制到脊髓神经元, 而 R26汤姆记者老鼠诱发 tdTomato 表达也发现在星形胶质细胞。这表明 RORβ是瞬态表达的星形胶质细胞。类似地, Mecinas et在病毒注射后发现了一个更受限制的报告表达式 (空间和细胞类型特定), 与 Tac1 小鼠20中的小鼠诱发的记者表达相比. 通过使用 rAAV 记者椎管内注射入成年小鼠脊髓, 记者和/或效应基因表达可以有效地限制在成人中表达基因的细胞, 从而避免重组/表达在那些有早期短暂基因活动的人群。
椎管内 rAAV 注射液的第二个主要优点是能够限制 rAAV 编码基因在注射部位的表达。在转基因小鼠中, 以驱动介导的表达通常不仅在感兴趣的神经元亚群中发现, 而且在神经系统中的其他人群中也有。Dymecki 和同事, 以及 Ma 和古尔丁的小组, 已经开发了优雅的方式使用交叉记者鼠标的方法, 避免重组的部分神经系统, 应保持不变的17, 18,19,21,22,23。使用敲鼠来获得标记或效应蛋白的表达也可以假设是较少变量, 因为只有一个基因组副本每个细胞的各自表达式磁带和表达式卡带是存在的所有细胞在感兴趣的区域.相比之下, 在病毒转导后, 各自表达式卡带的存在以及表达盒的拷贝数取决于转导效率, 这可能因细胞、细胞、注射和注射而异, 而且取决于病毒很多。最后, 病毒介导的基因表达对传统遗传方法的主要缺点是手术的必要性。手术介导的损伤/炎症可能直接影响神经元和电路。因此, 在接受相同手术的情况下, 控制小鼠必须加入。
未来应用:
椎管内注射可用于分析和操作任何基因鉴定的脊柱亚群通过过度表达的标记和效应蛋白。因此, 将来可能会有许多人采用这种技术来研究神经元的特定焦点。此外, 除了使用椎管内注射 rAAVs, 以实现外源效应蛋白的过度表达, 该技术也可用于沉默或过度表达 tshr 内源性蛋白, 因此, 研究任何特定基因的功能, 高空间和时间分辨率。

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>Equipment</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 &#956;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 &#215; 20 Plan-apochromat objective</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>1.3 NA &#215; 40 EC Plan-Neofluar oil-immersion objective</td>
			<td>Zeiss</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Surgical Tools</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Consumables and Chemicals</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&#160; (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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Mice</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6J mice&#160; (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&#946;Cre)</td>
			<td>The Jackson Laboratory</td>
			<td>RRID:IMSR_JAX:023526</td>
			<td></td>
		</tr>
		<tr>
			<td>&#160;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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Viral vectors</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.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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Plasmids</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&#945;.flex.hChR2(H134R)-eYFP</td>
			<td>Addgene</td>
			<td>20298</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Bacteria</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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Reagents</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 &#38; 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>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Antibodies (dilution)</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 &#38; 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, 目的是说明在 rAAV 介导的表达的差异, 当注射一项依赖于 rAAV 的转基因小鼠。因此, 我们注射了与 AAV1 相关的。CAG.flex.eGFP 载体进入 GlyT2::Cre 转基因小鼠脊髓。与以前一样, eGFP 表达在背和腹角观察到。然而, 正如预期的那样, eGFP 的表达变得更受限制, 反映了 GlyT2+ 神经元的分布,即在深背角的浅背角和致密表达式中相对稀疏的表达 (图 1E)。
其次, 为了证明解决脊髓神经元亚群电路功能的可行性, 对不同效应蛋白 (DTA 和 hM3Dq) 两种不同的自动增值服务编码进行了 GlyT2::Cre 转基因小鼠的注射。类似于三注射 AAV1 后观察到的病毒表达。eGFP 为野生型小鼠, 在 AAV1 注射后, 腰椎段 L3-L5 有抑制神经元 (Pax2+) 的强力消融。EF1a.flex.DTA 到 Glyt2::Cre 鼠标 (图 2A, C)。这些片段中 glycinergic 抑制神经元的丢失诱发了明显的机械超敏 (图 2D) 和自发厌恶行为, 指向同侧后肢 (图 2E)。厌恶行为导致自我造成的损害, 可以观察到爪子, 小腿和大腿 (数据, 见福斯特et 等。7) 在激活 glycinergic 神经元时, 通过注射 AAV1.hSyn.flex.hM3Dq 和随后腹腔注射 CNO (图 2B), 观察到相反的效果。老鼠对有害的机械刺激 (图 2F) 和其他有害刺激变得麻木 (见福斯特et 。7) 此外, 当使用 pruritogens 组胺或氯喹处理时, glycinergic 神经元的 hM3Dq-mediated 激活有效抑制 prurifensive 响应 (图 2G)。这些结果表明, 三注射 rAAV 到腰椎脊髓能够传感器一个区域的腰椎脊髓足以观察到强健的行为变化诱发的相应后肢的刺激。
在最后的一组实验中, 我们想证明在使用该报告的小鼠与创报道病毒相比有潜在的差异。因此, 选择了一个在小鼠脊髓中显示受限表达模式的创能驱动基因。基因 RORβ已被建议主要表现在抑制中间神经元的深背角 13, 14.本研究使用 RORβ "创" 敲鼠, 并越过他们 Rosa26 液 氧-停止-液-tdTomato (R26汤姆) 的记者小鼠, 这导致 tdTomato 在所有的细胞显示的表达在任何时间点在分析之前 (图 3A)。RORβ中 tdTomato + 单元格的特征;R26Tom小鼠揭示了背角神经元和星形胶质细胞的表达 (图 3B, C)。事实上, 定量的 tdTomato + 细胞表明, 大多数细胞的重组 (58%) 是非神经元。然后, 我们注入了一个依赖于 tdTomato 的记者 rAAV (AAV1。CAG.flex.tdTomato) RORβ P40 小鼠的脊髓 ( 图 3D)。与 R26Tom介导的记者表达式相反, 我们发现所有 tdTomato + 细胞被报告病毒标记为神经元 (图 3E, F)。最后, 对两组小鼠的 tdTomato + 神经元的身份进行了分析 (RORβ);R26Tom和 RORβ注塑注入 AAV1.CAG.flex.tdTomato). 在这两种情况下, 大多数神经元是抑制性的 (> 85%) 和神经元兴奋的少数 (< 20%)(图 3 G I), 它与以前对 RORβ + 神经元13、14的标识的评估一致。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57382/57382fig1.jpg"> 图 1: AAV1-eGFP/AAV1-flex-eGFP 椎管内注射 (A) AAV1 椎管内注射的示意图图示。eGFP (AAV1-eGFP) 进入腰椎脊髓, 从后肢支配。(B)腰椎椎线段 L3-L4 的解剖位置可从鼠标背部的上往下看。皮肤被打开以暴露脊椎柱。脊椎 T13-L6 的脊柱过程被染色为解剖参照, 箭头表示髂嵴。(C)一个完整的腰椎脊髓的代表图像。绿色荧光表示脊髓的病毒转基因区域。(D)通过从野鼠 AAV1-eGFP-transduced 的腰椎脊髓中的横断面的代表图像。(E) AAV1 的剖面的代表图像。eGFP-转基因的 GlyT2::Cre 鼠脊髓。虚线代表脊髓的灰质和浅背角的轮廓。刻度条 C = 1 毫米, D = 100 µm.<a href="/files/ftp_upload/57382/57382fig1large.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/57382/57382fig2.jpg"> 图 2: 脊髓表达 Glycinergic 神经元的功能操纵 (A + B)AAV1 椎管内注射的示意图。EF1a.flex.DTA (A)或 AAV1。EF1a.flex.hM3Dq (B)进入腰椎脊髓。疫苗依赖于白喉毒素片段 A (DTA) 的表达将导致 glycinergic 神经元 (GlyT2+) 的消融, 而遗传药理学设计器受体 hM3Dq 的依赖表达则会使 GlyT2 神经元激活氯氮平-n-氧化物 (CNO) (B)。(C)三注射 AAV1。EF1a.flex.DTA 进入 GlyT2::Cre 小鼠的 L3-L5 段, 导致在同侧的各段, 而不是对侧的抑制神经元明显丧失。(D) GlyT2 神经元的丢失诱发了对 AAV1 注射的 GlyT2::Cre 小鼠同侧足爪的机械性弗雷刺激的长期敏感反应。EF1a.flex.DTA, 但如果注射的是阴性小鼠, 则没有观察到任何变化。(E) GlyT2 神经元的丢失诱发自发厌恶行为, 使人联想到慢性瘙痒。(F) hM3Dq-mediated 激活 GlyT2 神经元, 减轻针刺刺激诱发的有害机械疼痛。(G) hM3Dq-mediated 激活 GlyT2 神经元减少 pruritogen (氯喹或组胺) 诱发厌恶行为。数据被表示为卑鄙的电子扫描电镜. ** p < 0.001;** p < 0.01。刻度条 C = 1 毫米克 = 克。图像被重新使用和修改, 从寄养等。7<a href="/files/ftp_upload/57382/57382fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57382/57382fig3.jpg"> 图 3: 基因和病毒介导的 RORβ表达细胞的标记。(A)图中显示了 RORβ中荧光 tdTomato 报告器的相关表达式的策略;Rosa26 液氧-停止-tdTomato (R26Tom) 小鼠。(B)染色 RORβ的脊髓部分;R26tom小鼠发现, NeuN + RORβ-Tom 神经元可以在 I. iv。(C)在 RORβ tdTomato 中的 GFAP + 细胞中也观察到了与此相关的基因表达.R26Tom小鼠, 提示在发育过程中 RORβ在星形胶质细胞中表达。箭头表示 iii. 层中的双标记星形胶质细胞。(D)图中显示椎管内注射 AAV (rAAV1.CAG.flex.tdTomato) 病毒进入 RORβ的创局小鼠, 以推动当地的与此相关的 tdTomato 表达.(E)染色 RORβ注射 AAV-汤姆病毒的小鼠脊髓部分.RORβ神经元被定位于脊髓背角的浅层, 星形胶质细胞 tdTomato 的表达不存在。(F) RORβ NeuN 在 RORβ脊髓段中表达神经元标记物的百分比;R26tom小鼠和 RORβ创能小鼠注射 AAV-弯曲-汤姆病毒.(G H)染色在(G) RORβ a. 的脊髓部分.R26Tom小鼠和(H) RORβ注射 AAV--tom 病毒的小鼠, 显示 RORβ-汤姆神经元与抑制标记 Pax2 或兴奋标记 Lmx1b 之间定位. (I)百分比 RORβ-汤姆表达 Lmx1b 和 Pax2 的神经元在 RORβ中;R26tom小鼠和 RORβ创能小鼠注射 AAV-Tom 病毒.数据被表示为平均值的电子扫描电镜. 数据来自2-3 只老鼠, 每只老鼠1-3 节。缩放条形图表示100µm (B、e) 和20µm (在高放大图像中为C、e ), G和H)。<a href="/files/ftp_upload/57382/57382fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>变量</td>
			<td>设置值</td>
			<td>单位</td>
		</tr>
<tr>
<td>热 (H)</td>
			<td>450</td>
			<td>-(与辐射热功率成正比的值)</td>
		</tr>
<tr>
<td>力初拉力 (F (日))</td>
			<td>20</td>
			<td>-(与施加于力线圈的电压成正比的值)</td>
		</tr>
<tr>
<td>距离阈值 (s (日))</td>
			<td>25</td>
			<td>0.12 毫米</td>
		</tr>
<tr>
<td>延迟 heatstop (t (H))</td>
			<td>30</td>
			<td>0.5 毫秒</td>
		</tr>
<tr>
<td>距离 heatstop (s (H))</td>
			<td>0</td>
			<td>0.12 毫米</td>
		</tr>
<tr>
<td>延时拉 1 (t (F1))</td>
			<td>200</td>
			<td>0.5 毫秒</td>
		</tr>
<tr>
<td>强制拉 1 (F1)</td>
			<td>300</td>
			<td>-(与施加于力线圈的电压成正比的值)</td>
		</tr>
<tr>
<td>距离拉扯 2 (s (F2))</td>
			<td>30</td>
			<td>0.12 毫米</td>
		</tr>
<tr>
<td>强制拉 2 (F2)</td>
			<td>600</td>
			<td>-(与施加于力线圈的电压成正比的值)</td>
		</tr>
<tr>
<td>调整 (AD)</td>
			<td>0</td>
			<td>-</td>
		</tr>
</tbody></table>表 1: 拉拔器设置

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

Nanyang Technological University

Research

JoVE Journal

Neuroscience

17.9K 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

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

从胚胎脊髓​​连合神经的解剖和文化

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

科研

神经科学

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

脊髓电二:外吸电极的制作

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Viral Tracing of Genetically Defined Neural Circuitry

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus (RABV), and more recently vesicular stomatitis virus (VSV), for studying circuitry, is becoming increasingly popular. These higher throughput methods use viruses that transmit between neurons in either the anterograde or retrograde direction.
Recently, a modified RABV for monosynaptic retrograde tracing was developed. (Figure 1A). In this method, the glycoprotein (G) gene is deleted from the viral genome, and resupplied only in targeted neurons. Infection specificity is achieved by substituting a chimeric G, composed of the extracellular domain of the ASLV-A glycoprotein and the cytoplasmic domain of the RABV-G (A/RG), for the normal RABV-G1. This chimeric G specifically infects cells expressing the TVA receptor1. The gene encoding TVA can been delivered by various methods2-8. Following RABV-G infection of a TVA-expressing neuron, the RABV can transmit to other, synaptically connected neurons in a retrograde direction by nature of its own G which was co-delivered with the TVA receptor. This technique labels a relatively large number of inputs (5-10%)2 onto a defined cell type, providing a sampling of all of the inputs onto a defined starter cell type.
We recently modified this technique to use VSV as a transsynaptic tracer9. VSV has several advantages, including the rapidity of gene expression. Here we detail a new viral tracing system using VSV useful for probing microcircuitry with increased resolution. While the original published strategies by Wickersham et al.4 and Beier et al.9 permit labeling of any neurons that project onto initially-infected TVA-expressing-cells, here VSV was engineered to transmit only to TVA-expressing cells (Figure 1B). The virus is first pseudotyped with RABV-G to permit infection of neurons downstream of TVA-expressing neurons. After infecting this first population of cells, the virus released can only infect TVA-expressing cells. Because the transsynaptic viral spread is limited to TVA-expressing cells, presence of absence of connectivity from defined cell types can be explored with high resolution. An experimental flow chart of these experiments is shown in Figure 2. Here we show a model circuit, that of direction-selectivity in the mouse retina. We examine the connectivity of starburst amacrine cells (SACs) to retinal ganglion cells (RGCs).

A method of tracing synaptically connected neurons is described. We use TVA specificity of an upstream cell to probe whether a cell population of interest receives synaptic input from genetically defined cell types.

病毒遗传背景明确的神经回路跟踪

Classical methods for studying neuronal circuits are fairly low throughput. Transsynaptic viruses, particularly the pseudorabies (PRV) and rabies virus ...

Spinal Cord Transection in the Larval Zebrafish

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6&#160;weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2&#160;days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

After spinal transection, adult zebrafish have functional recovery by six weeks post-injury. To take advantage of larval transparency and faster recovery, we present a method for transecting the larval spinal cord. After transection, we observe sensory recovery beginning at 2&#160;days post-injury, and C-bend movement by 3 days post-injury.

在斑马鱼幼虫脊髓横断

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability ...

RNA-Seq Analysis of Differential Gene Expression in Electroporated Chick Embryonic Spinal Cord

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome wide analysis of differentially expressed transcripts after such an experimental manipulation has the potential to uncover an almost complete picture of the downstream effects caused by the transfected construct. This work describes a simple method for comparing transcriptomes from samples of transfected embryonic spinal cords comprising all steps between electroporation and identification of differentially expressed transcripts. The first stage consists of guidelines for electroporation and instructions for dissection of transfected spinal cord halves from HH23 embryos in ribonuclease-free environment and extraction of high-quality RNA samples suitable for transcriptome sequencing. The next stage is that of bioinformatic analysis with general guidelines for filtering and comparison of RNA-Seq datasets in the Galaxy public server, which eliminates the need of a local computational structure for small to medium scale experiments. The representative results show that the dissection methods generate high quality RNA samples and that the transcriptomes obtained from two control samples are essentially the same, an important requirement for detection of differential expression genes in experimental samples. Furthermore, one example is provided where experimental overexpression of a DNA construct can be visually verified after comparison with control samples. The application of this method may be a powerful tool to facilitate new discoveries on the function of neural factors involved in spinal cord early development.

This video shows the basic steps for performing whole transcriptome analysis on dissected chick embryonic spinal cord samples after transfection with in ovo electroporation.

对基因差异表达的RNA序列中电穿孔鸡胚脊髓分析

In ovo electroporation of the chick neural tube is a fast and inexpensive method for identification of gene function during neural development. Genome ...

Diffusion Imaging in the Rat Cervical Spinal Cord

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a diagnostic and prognostic tool in cases of disease or injury. Diffusion weighted imaging (DWI), is sensitive to the thermal motion of water molecules and allows for inferences of tissue microstructure. This report describes a protocol to acquire and analyze DWI of the rat cervical spinal cord on a small-bore animal system. It demonstrates an imaging setup for the live anesthetized animal and recommends a DWI acquisition protocol for high-quality imaging, which includes stabilization of the cord and control of respiratory motion. Measurements with diffusion weighting along different directions and magnitudes (b-values) are used. Finally, several mathematical models of the resulting signal are used to derive maps of the diffusion processes within the spinal cord tissue that provide insight into the normal cord and can be used to monitor injury or disease processes noninvasively.

The goal of this protocol is to obtain high-quality diffusion weighted magnetic resonance imaging (DWI) of the rat spinal cord for noninvasive characterization of tissue microstructure. This protocol describes optimizations of the MRI sequence, radiofrequency coil, and analysis methods to enable DWI images free from artifacts.

在大鼠颈脊髓弥散成像

Magnetic resonance imaging (MRI) is the state of the art approach for assessing the status of the spinal cord noninvasively, and can be used as a ...

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model.
For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons

This manuscript describes methods for electrophysiological recordings from spinal neurons of zebrafish embryos and larvae. The preparation maintains neurons in situ and often involves minimum dissection. These methods allow for the electrophysiological study of a variety of spinal neurons, from the initial electrical excitability acquisition through the early larval stages.

斑马鱼<em&gt;原位</em&gt;脊髓制备用于脊髓感觉和运动神经元的电生理记录

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing ...

Spinal Cord Neurons Isolation and Culture from Neonatal Mice

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on postnatal day 1-3. A mouse litter, usually 4-10 pups born from one breeding pair, is gathered for one experiment, and spinal cords are collected individually from each mouse after euthanasia with isoflurane. The spinal column is dissected out and then the spinal cord is released from the column. The spinal cords are then minced to increase the surface area of delivery for an enzymatic protease that allows for the neurons and other cells to be released from the tissue. Trituration is then used to release the cells into solution. This solution is subsequently fractionated in a density gradient to separate the various cells in solution, allowing for neurons to be isolated. Approximately 1-2.5 x 106 neurons can be isolated from one litter group. The neurons are then seeded onto wells coated with adhesive factors that allow for proper growth and maturation. The neurons take approximately 7 days to reach maturity in the growth and culture medium and can be used thereafter for treatment and analysis.

This study presents a technique for the isolation of neurons from WT neonatal mice. It requires the careful dissection of the spinal cord from the neonatal mouse, followed by the separation of neurons from the spinal cord tissue through mechanical and enzymatic cleavage.

脊髓神经元从新生小鼠中分离和培养

We present a protocol for the isolation and culture of spinal cord neurons. The neurons are obtained from neonatal C57BL/6 mice and are isolated on ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Laminectomy and Spinal Cord Window Implantation in the Mouse

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated digital vaporizer is utilized to achieve a stable plane of anesthesia at a low-flow rate of isoflurane. A single vertebral spine is removed, and a commercially available cover-glass is overlaid on a thin agarose bed. A 3D-printed plastic backplate is then affixed to the adjacent vertebral spines using tissue adhesive and dental cement. A stabilization platform is used to reduce motion artifact from respiration and heartbeat. This rapid and clamp-free method is well-suited for acute multi-photon fluorescence microscopy. Representative data are included for an application of this technique to two-photon microscopy of the spinal cord vasculature in transgenic mice expressing eGFP:Claudin-5 &#8212; a tight junction protein.

This protocol describes implantation of a glass window onto the spinal cord of a mouse to facilitate visualization by intravital microscopy.

拉米内切手术和脊髓窗口植入小鼠

This protocol describes a method for spinal cord laminectomy and glass window implantation for in vivo imaging of the mouse spinal cord. An integrated ...