我们非常感谢中国国家自然科学基金（第31800903项，授予郑伟）和温州科学技术计划（第Y2020426号，授予翁耀彦）对这项工作的支持。

本研究中描述的所有动物程序均按照美国国立卫生研究院《实验动物护理和使用指南》中概述的指南进行，并经温州医科大学机构动物护理和使用委员会批准。
1. 动物
<ol><li>购买年龄为8-12周，体重为20-22g的雄性C57BL / 6小鼠。</li>
	<li>获得转基因Rosa-LSL（Lox-StoP-Lox）-tdTomato（Ai9）小鼠系和雄性A2AR flox/ flox 小鼠。</li>
	<li>将小鼠随机分配到两组并饲养在笼子中，每个笼子最多5只小鼠，在标准的12小时光照/ 12小时黑暗循环下1周。</li>
	<li>为小鼠提供足够的食物和水，并将它们保持在25°C的恒定温度下。</li>
</ol>2. 用 AAV2/5-shRNA 特异性敲低 A2A R
<ol><li>称量每只鼠标的重量并记下值。</li>
	<li>用1%戊巴比妥钠腹膜内麻醉小鼠，剂量为6-8mL / kg，相当于60-80mg / kg戊巴比妥，并将小鼠放在加热垫上保温。 注意：根据小鼠的体重给予正确剂量的戊巴比妥钠以确保手术成功至关重要。麻醉不宜太深，因为它可能导致死亡，但不应太浅，因为小鼠可能会在手术过程中醒来，从而可能影响病毒注射的有效性。通过捏住脚趾并确保小鼠没有反应来检查适当的麻醉剂量。</li>
	<li>使用专门的剃须刀修剪麻醉小鼠的顶毛。</li>
	<li>在双眼上涂抹眼药膏以防止干燥，并将小鼠固定在立体定位装置上以固定大脑。用无菌窗帘盖住动物。</li>
	<li>用三轮碘伏消毒液和75%酒精对小鼠头颈部皮肤进行彻底消毒，以减少术后感染。然后局部将1%利多卡因局部施加在小鼠的头皮上，并做一个小切口，在显微镜下完全暴露颅骨。 注意：在整个过程中使用无菌器械。</li>
	<li>准备两个 10 μL 注射器：一个装有购买的 AAV2/5-A 2A R-shRNA（滴度：6.27 × 10 9 vg/μL），另一个装有购买的 AAV2/5-Scramble（滴度：6.21× 109 vg/μL）。 注：使用注射器时，前端需要连接玻璃毛细管，通过控制玻璃毛细管的长度可以获得10μL的范围。</li>
	<li>使用显微镜定位前囟和λ并设置坐标点（AP：-0.58;毫升：±1.10;DL：-2.20）在10μL注射器上（图1）。该坐标点基于小鼠大脑立体定位图谱15。</li>
	<li>在调整后的坐标点在头骨上钻一个小孔。 注意：在显微镜下，使用无菌微钻对头骨表面进行钻孔。钻孔后，通过去除覆盖大脑的脑膜并暴露下面的脑实质，可以防止血管损伤。这一步可以防止玻璃毛细管的尖端被脑膜折断。钻孔的直径应足以使针尖进入脑组织。</li>
	<li>通过侧向注射以 100 nL/min 的稳定速率将 2 μL AAV2/5-A2AR-shRNA 病毒注入脑室。在拔出之前将针头保持在原位 10 分钟。请注意，该病毒是通过单侧注射给药的。</li>
	<li>立即使用医用生物纤维蛋白胶水密封受伤的皮肤，以防止病毒流失，如果不快速涂抹胶水，可以在取下针头后用脑脊液冲洗干净。此外，不要使用棉签擦拭脑脊液，因为它也可能导致病毒丢失。</li>
	<li>为了促进恢复，将鼠标放在加热垫上，将体温保持在37.0±0.5°C，并将1%利多卡因局部涂抹在小鼠的头皮上。在诱导EAE模型之前，让小鼠休养2周，因为这段时间是AAV2 / 5-A 2A R-shRNA病毒表达和随后的A2AR敲低所必需的。</li>
</ol>3. 具有 X-overP1 Cre/位点 （Cre/LoxP） 系统的 A 2A R 的 ChP 特异性敲低
注意：可以使用前面描述的方法实现以下过程。有关详细的注射方法，请参阅步骤 2.1-2.11。
<ol><li>将 2 μL CRE-TAT 重组酶注射到 Rosa-LSL （Lox-StoP-Lox）-tdTomato 小鼠的每个侧脑室中作为实验组，将 2 μL 无菌磷酸盐缓冲盐水 （PBS） 作为对照组。使用与上述相同的方案（第2节），将2μL的CRE-TAT重组酶注射到A2AR flox/ flox 小鼠的每个侧脑室中，以及2μL无菌PBS作为对照组。</li>
	<li>准备冷冻组织切片并在注射 CRE-TAT 重组酶后 2 周进行核染色。有关详细信息，请参阅步骤 4.1-4.3。</li>
</ol>4.小鼠心内灌注
<ol><li>施用60-80mg / kg戊巴比妥钠对小鼠进行深度麻醉。通过捏脚趾确认麻醉平面，并使用 40 mL 无菌 PBS 溶液和 20 mL 4% 多聚甲醛 （PFA） 进行经心灌注。 注意：肝脏中的白色表明灌注成功，而灌注过程中肺肿大或PBS从口腔流出表明手术失败。</li>
	<li>快速提取小鼠大脑，确保最小的蛋白质降解。</li>
	<li>将小鼠脑浸没在4%PFA / PBS中过夜用于固定后，然后用30%蔗糖PB溶液替换72小时。 注意：避免脑组织过度脱水至关重要，因此大脑不应在蔗糖PB溶液中停留太久。</li>
</ol>5. 冷冻组织切片和染色
<ol><li>将先前脱水的小鼠大脑嵌入最佳切割温度（OCT）胶中，冷冻嵌入的大脑，并使用滑动切片机切割厚度为20μm的冠状切片。</li>
	<li>将大脑部分放在载玻片上。一旦脑切片干燥，将它们储存在-20°C冰箱中用于后续实验。</li>
	<li>将每个载玻片放入框架盒中，然后将框架浸入装有 PBS 的容器中以彻底冲洗载玻片。用PBS溶液轻轻冲洗脑载玻片三次，每次10分钟。 注意： 应小心，以防止大脑部分从载玻片上掉下来。</li>
	<li>用4′，6-二脒基-2-苯基吲哚（DAPI）溶液染色脑载玻片10分钟。</li>
	<li>用PBS溶液冲洗脑载玻片5分钟。</li>
	<li>将一滴抗淬灭封固剂涂在大脑部分。</li>
	<li>将盖玻片放在脑切片上，用指甲油密封盖玻片，并使用常规荧光显微镜分析切片。</li>
</ol>6. EAE诱导
注意：在 shRNA 或 CRE-TAT 重组酶注射 2 周后进行 EAE 诱导11.
<ol><li>通过将 2.5 mg 髓鞘少突胶质细胞糖蛋白 （MOG35-55） 与 2 mL PBS 混合来制备水溶液。通过将 结核分枝杆菌 （H37Ra） 与不完全弗氏佐剂 （IFA） 混合来制备完整的弗氏佐剂 （CFA） 油溶液。</li>
	<li>将水溶液和油溶液以 1：1 的比例混合。使用三通管将混合物搅打到水包油状态。 注意： 三通管也称为三通管。它是一种塑料管，可以控制溶液的流动方向，使MOG35-55 和CFA充分混合。</li>
	<li>使用高速均质机在冰浴条件下为EAE模型制作MOG抗原乳液。</li>
	<li>用1%戊巴比妥钠腹膜内麻醉小鼠，剂量为6-8mL / kg，相当于60-80mg / kg戊巴比妥。</li>
	<li>皮下注射MOG抗原乳剂，体积为10mL / kg，每次注射四次。 注意：仔细选择注射部位很重要，因为不同的部位可能对小鼠的发病率和死亡率产生不同的影响。此外，重复注射MOG会导致免疫耐受，因此研究小组选择了一种单一注射方法来防止这种潜在问题。</li>
	<li>注射 MOG 后立即腹膜内注射 500 ng/mL 百日咳毒素 （PT），剂量为 5 mg/kg。 注意：PT增加血脑屏障（BBB）的通透性，并促进T细胞浸润到大脑中。</li>
	<li>48小时后，腹膜内以相同体积注射PT溶液。</li>
</ol>7. 神经功能缺损评分
<ol><li>每天使用从0到15的评定量表16 对小鼠进行评估和分级，以根据以下神经功能缺损评估EAE的发生率和严重程度： 尾巴：0 表示没有迹象，1 表示半瘫痪的尾巴，2 表示完全瘫痪的尾巴。 四肢：0 表示没有体征，1 表示步态无力或改变，2 表示瘫痪，3 表示肢体完全瘫痪。</li>
	<li>给完全瘫痪的四肢瘫痪动物打 12 分，给死亡率打 15 分。</li>
</ol>8. 苏木精-伊红（H&E）染色
<ol><li>取脱水的小鼠大脑并将其嵌入熔融石蜡中。让石蜡块冷却并固化以备后用。 注意：石蜡块应完全干燥和冷却，以避免组织损伤。</li>
	<li>将小鼠脑石蜡块切成5μm厚的载玻片。将石蜡小鼠脑切片放在载玻片上，并在60°C烘箱中干燥3小时。</li>
	<li>将载玻片依次浸入二甲苯溶液I中10分钟，二甲苯溶液II浸泡10分钟，100%酒精I浸泡3分钟，100%酒精II浸泡3分钟，95%酒精浸泡3分钟，90%酒精浸泡3分钟，80%酒精浸泡3分钟，浸泡在蒸馏水中1分钟。</li>
</ol>9. 定量聚合酶链反应（qPCR）分析
<ol><li>用PBS灌注小鼠后，从心室中取出ChP并用Trizol提取RNA。使用第一个链 cDNA 合成试剂盒合成 cDNA。</li>
	<li>使用Ex Taq SYBR-green预混液和实时荧光定量PCR系统进行qPCR分析。使用以下 A2AR 引物：forward - GCCATCCCATTCGCCATCA;反向 - GCAATAGCCAAGAGGCTGAAGA。</li>
</ol>

脉络丛中的选择性基因沉默

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提交人表示，他们没有相互竞争的经济利益或其他披露要申报。

该研究提出了两种不同的方法来靶向敲低ChP基因。第一种方法涉及将含有Cre重组酶的CRE-TAT注射到A2AR flox/ flox小鼠中。第二种方法需要ICV注射携带A2AR的shRNA的AAV2/5。通过利用这两种策略，本工作实现了 ChP 中 A 2A R 的选择性敲低，并能够证明抑制 ChP 中 A2AR 信号传导对 EAE 病理学的保护作用。值得注意的是，该协议包括两个关键步骤。首先，立体定位操作是必...

脉络丛 （ChP） 通常被认为通过分泌脑脊液 （CSF） 和脑源性神经营养因子 （BDNF） 来帮助维持脑功能稳态1,2。在过去三十年中，越来越多的研究表明，ChP 代表了免疫细胞浸润到中枢神经系统 （CNS） 的独特途径。
ChP 的紧密连接 （TJ） 由单层 ChP 上皮细胞组成，通过阻止大分子和免疫细胞进入大脑来维持免疫稳态3。然而，在某些病理条件下，ChP 组织会检测脑脊液和血液中的危险相关分子模式 （DAMP） 并做出反应，导致异常免疫浸润和脑功能障碍 4,5。尽管ChP起着关键作用，但它的小尺寸和在大脑中的独特位置使得很难在不影响其他大脑区域的情况下研究其功能。因此，在ChP中特异性地操纵基因表达是了解其功能的理想方法。
最初，环化重组酶 （Cre） 转基因系在 ChP 中表达的基因特异性启动子的控制下表达 Cre，通常用于通过与 flox 候选基因杂交来删除靶基因 6,7,8。例如，转录因子叉头盒J1（FoxJ1）仅在产前小鼠脑的ChP上皮细胞中表达7。因此，FoxJ1-Cre 系通常用于删除位于 ChP 6,9 中的基因。然而，这种策略的成功很大程度上取决于启动子的特殊性。逐渐发现 FoxJ1 的表达模式不够独特，因为 FoxJ1 也存在于大脑和外周系统其他部位的纤毛上皮细胞中7。为了克服这一局限性，进行了脑室内 （ICV） 注射 Cre 重组酶，以将重组酶输送到絮状转基因系的脑室中。该策略显示出高度特异性，仅在 ChP 组织中存在 tdTomato 荧光就证明了这一点 10,11。然而，这种方法仍然受到氟化转基因小鼠品系可用性的限制。为了解决这个问题，研究人员采用了腺相关病毒 （AAV） 的 ICV 注射来实现 ChP 特异性敲低或靶基因的过表达12,13。对不同 AAV 血清型的 ChP 感染的综合评估显示，AAV2/5 和 AAV2/8 在 ChP 中表现出很强的感染能力，而不感染其他大脑区域。然而，发现 AAV2/8 感染心室周围的室管膜，而 AAV2/5 组未显示感染14。该方法的优点是克服了获得floxed转基因动物的局限性。
本文描述了使用两种方法在 ChP 中敲低基因的分步方案：携带腺苷 A 2A 受体 （A 2A R） 的 shRNA 的 AAV2/5 的 ICV 和由 TAT 序列 （CRE-TAT） 重组酶组成的 Cre 重组酶蛋白，以实现 A2A R 的 ChP 特异性敲低。研究结果表明，敲低 ChP 中的 A2AR 可以缓解实验性自身免疫性脑脊髓炎 （EAE）。该详细方案为 ChP 功能研究和 ChP 中基因的特异性敲低提供了有用的指导。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>A2ARflox/flox mice</td>
			<td>State Key Laboratory of Ophthalmology, Optometry and Visual Science, Wenzhou Medical University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>AAV2/5-A2AR-ShRNA virus</td>
			<td>Shanghai Heyuan Biotechnology Co. LTD</td>
			<td>pt-4828</td>
			<td></td>
		</tr>
		<tr>
			<td>antifade mounting medium</td>
			<td>Beyotime Biotechnology</td>
			<td>0100-01</td>
			<td></td>
		</tr>
		<tr>
			<td>borosilicate glass capillary</td>
			<td>Beijing Meiyaxian Technology Co. Ltd</td>
			<td>B100-50-10</td>
			<td></td>
		</tr>
		<tr>
			<td>brain stereotaxic apparatus</td>
			<td>RWD, Shenzhen</td>
			<td>69100</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 mice</td>
			<td>Beijing Vital Charles River Laboratory Animal Technology Company</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CRE-TAT recombinase</td>
			<td>Millipore</td>
			<td>SCR508</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Absin</td>
			<td>B25A031</td>
			<td></td>
		</tr>
		<tr>
			<td>frozen slicing machine</td>
			<td>Leica</td>
			<td>CM1950</td>
			<td></td>
		</tr>
		<tr>
			<td>H37Ra</td>
			<td>Becton Dickinson and company</td>
			<td>231141</td>
			<td></td>
		</tr>
		<tr>
			<td>Hamilton syringe</td>
			<td>Hamilton, American</td>
			<td>P/N:&#160;86259</td>
			<td></td>
		</tr>
		<tr>
			<td>Incomplete Freunds adjuvant</td>
			<td>Sigma</td>
			<td>F5506</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser confocal microscope</td>
			<td>Zeiss</td>
			<td>LSM900</td>
			<td></td>
		</tr>
		<tr>
			<td>MOG35-55</td>
			<td>Suzhou Qiangyao Biotechnology Co., LTD</td>
			<td>4010006243</td>
			<td></td>
		</tr>
		<tr>
			<td>OCT glue</td>
			<td>Epredia</td>
			<td>6502p</td>
			<td></td>
		</tr>
		<tr>
			<td>paraformaldehyde</td>
			<td>Chengdu Kelong Chemical Reagent Company</td>
			<td>30525-89-4</td>
			<td></td>
		</tr>
		<tr>
			<td>pentobarbital sodium</td>
			<td>Boyun Biotech</td>
			<td>PC13003</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette gun</td>
			<td>Eppendorf</td>
			<td>N45014F</td>
			<td></td>
		</tr>
		<tr>
			<td>PrimeScript 1st Strand cDNA Synthesis Kit</td>
			<td>Takara</td>
			<td>&#160;6110A</td>
			<td></td>
		</tr>
		<tr>
			<td>Real- Time PCR System</td>
			<td>BioRad</td>
			<td>CFX96</td>
			<td></td>
		</tr>
		<tr>
			<td>Rosa-LSL (Lox-StoP-Lox)-tdTomato mice</td>
			<td>Jackson Laboratory</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sucrose</td>
			<td>Sangon Biotech</td>
			<td>A502792-0500</td>
			<td></td>
		</tr>
		<tr>
			<td>super high speed homogenizer</td>
			<td>IKA</td>
			<td>3737025</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Invitrogen</td>
			<td>15596026</td>
			<td></td>
		</tr>
		<tr>
			<td>xylene solution</td>
			<td>Chengdu Kelong Chemical Reagent Company</td>
			<td>1330-20-7</td>
			<td></td>
		</tr>
	</tbody>
</table>

targeted knockdown of genes in the choroid plexus

脉络丛 （ChP） 是免疫细胞在生理和病理条件下浸润到中枢神经系统 （CNS） 的关键通道。最近的研究表明，调节 ChP 活性可以预防中枢神经系统疾病。然而，由于其微妙的结构，在不影响其他大脑区域的情况下研究 ChP 的生物学功能具有挑战性。本研究提出了一种使用腺相关病毒 （AAV） 或由 TAT 序列组成的环化重组酶 （Cre） 重组酶 （CRE-TAT） 重组酶蛋白在 ChP 组织中敲低基因的新方法。结果表明，将AAV或CRE-TAT注射到侧脑室后，荧光完全集中在ChP中。 利用这种方法，该研究利用RNA干扰（RNAi）或X-overP1（Cre/LoxP）系统的Cre/位点成功敲低了ChP中的腺苷A 2A受体（A2A R），并表明这种敲低可以缓解实验性自身免疫性脑脊髓炎（EAE）的病理。该技术可能对未来研究 ChP 在中枢神经系统疾病中的作用具有重要意义。

通过 ICV 注射 AAV2/5-shRNA 或 CRE-TAT 敲低 ChP 特异性 A2AR A2AR 在 ChP 中作为 EAE 发病机制中神经信息的强大调节因子的作用尚不清楚。敲低 ChP 特异性 A 2A R 表达可以揭示 A2AR 对 EAE和其他神经系统炎症中枢免疫系统的调节作用。本研究使用 ICV 注射 CRE-TAT 来降低 A 2A R flox/flox 小鼠 ChP 中 A2AR 的表达。为了确保 ChP 特异性，我们首先将 ...

Watch this Scientific Journal Video about Insights and Innovations in Gene Expression Manipulation Techniques for Choroid Plexus Research at JoVE.com

作者聚焦：脉络丛研究中基因表达操作技术的见解和创新 

在这里，我们描述了一种选择性地改变脉络丛中基因表达的方法，同时避免对其他大脑区域的任何影响。

脊索丛中基因的靶向敲除

The choroid plexus (ChP) serves as a critical gateway for immune cell infiltration into the central nervous system (CNS) under both physiological and ...

本研究的目的是研究操纵脉络丛中基因表达的技术。我们的研究引入了两种方法，这些方法只允许在脉络丛中使用技术，而不会对其他区域产生任何明显的影响。我们的研究结果可能会增强神经科学中脉络丛的未来功能研究。我们的研究回顾了通过选择性锁定腺苷到受体来抑制 T 细胞浸润脉络丛足以保护小鼠免受 EAE 病理学的影响。这一发现支持了这样一种观点，即检测脉络丛中的门户活动可能为治疗多发性硬化症提供一种有前途的新策略。本研究提出了两种在脉络丛中进行基因表达操作的替代方法。ICV 将 Cre 重组酶注射到这些转基因小鼠中或 ICV 注射 AAV2/5，或者在 WT 小鼠中靶向 SHI。这些方法克服了促进特异性和转基因小鼠育种的问题。

targeted-knockdown-of-genes-in-the-choroid-plexus

Research

JoVE Journal

Neuroscience

Targeted Knockdown of Genes in the Choroid Plexus

805 次观看.  Wenzhou Medical University. 本研究的目的是研究操纵脉络丛中基因表达的技术。我们的研究引入了两种方法，这些方法只允许在脉络丛中使用技术，而不会对其他区域产生任何明显的影响。我们的研究结果可能会增强神经科学中脉络丛的未来功能研究。我们的研究回顾了通过选择性锁定腺苷到受体来抑制 T 细胞浸润脉络丛足以保护小鼠免受 EAE 病理学的影响。这一发现支持了这样一种观点，即检?...

视频: 作者聚焦：脉络丛研究中基因表达操作技术的见解和创新 

Targeted Training of Ultrasonic Vocalizations in Aged and Parkinsonian Rats

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting communication abilities. 1, 2 Targeted training (speech/voice therapy) can improve specific voice deficits,3, 4 although the underlying mechanisms of behavioral interventions are not well understood. Systematic investigation of voice deficits and therapy should consider many factors that are difficult to control in humans, such as age, home environment, age post-onset of disease, severity of disease, and medications. The method presented here uses an animal model of vocalization that allows for systematic study of how underlying sensorimotor mechanisms change with targeted voice training. The ultrasonic recording and analysis procedures outlined in this protocol are applicable to any investigation of rodent ultrasonic vocalizations.
The ultrasonic vocalizations of rodents are emerging as a valuable model to investigate the neural substrates of behavior.5-8 Both rodent and human vocalizations carry semiotic value and are produced by modifying an egressive airflow with a laryngeal constriction.9, 10 Thus, rodent vocalizations may be a useful model to study voice deficits in a sensorimotor context. Further, rat models allow us to study the neurobiological underpinnings of recovery from deficits with targeted training.
To model PD we use Long-Evans rats (Charles River Laboratories International, Inc.) and induce parkinsonism by a unilateral infusion of 7 &mu;g of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle which causes moderate to severe degeneration of presynaptic striatal neurons (for details see Ciucci, 2010).11, 12 For our aging model we use the Fischer 344/Brown Norway F1 (National Institute on Aging).
Our primary method for eliciting vocalizations is to expose sexually-experienced male rats to sexually receptive female rats. When the male becomes interested in the female, the female is removed and the male continues to vocalize. By rewarding complex vocalizations with food or water, both the number of complex vocalizations and the rate of vocalizations can be increased (Figure 1).
An ultrasonic microphone mounted above the male's home cage records the vocalizations. Recording begins after the female rat is removed to isolate the male calls. Vocalizations can be viewed in real time for training or recorded and analyzed offline. By recording and acoustically analyzing vocalizations before and after vocal training, the effects of disease and restoration of normal function with training can be assessed. This model also allows us to relate the observed behavioral (vocal) improvements to changes in the brain and neuromuscular system.

Voice disorders are debilitating in aging and Parkinson disease. The ultrasonic vocalizations of rats, also affected by these conditions, can be used to study these voice disorders, their neural substrates, and the nature of functional recovery with behavioral intervention.

老年帕金森病大鼠的超声波发声的有针对性的培训

Voice deficits are a common complication of both Parkinson disease (PD) and aging; they can significantly diminish quality of life by impacting ...

科研

神经科学

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

局部和全局的方法评估热痛觉<em&gt;果蝇</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

A Visual Description of the Dissection of the Cerebral Surface Vasculature and Associated Meninges and the Choroid Plexus from Rat Brain

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain proper, that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis. The tissue harvested is suitable for biochemical and physiological analysis, and the MAV has been shown to be sensitive to damage produced by amphetamine and hyperthermia 1,2. As well, the major and minor cerebral vasculatures harvested in MAV are of potentially high interest when investigating concussive types of head trauma. The MAV dissected in this presentation consists of the pial and some of the arachnoid membrane (less dura) of the meninges and the major and minor cerebral surface vasculature. The choroid plexus dissected is the structure that resides in the lateral ventricles as described by Oldfield and McKinley3,4,5,6. The methods used for harvesting these two tissues also facilitate the harvesting of regional cortical tissue devoid of meninges and larger cerebral surface vasculature, and is compatible with harvesting other brain tissues such as striatum, hypothalamus, hippocampus, etc. The dissection of the two tissues takes from 5 to 10 min total. The gene expression levels for the dissected MAV and choroid plexus, as shown and described in this presentation can be found at GSE23093 (MAV) and GSE29733 (choroid plexus) at the NCBI GEO repository. This data has been, and is being, used to help further understand the functioning of the MAV and choroid plexus and how neurotoxic events such as severe hyperthermia and AMPH adversely affect their function.

This video presentation shows a method of harvesting the two most important highly vascular structures that support forebrain function. They are the cerebral surface (superficial) vasculature along with associated meninges (MAV) and the choroid plexus which are necessary for cerebral blood flow and cerebrospinal fluid (CSF) homeostasis.

从大鼠脑的解剖脑表面血管及相关脑膜和脉络丛的可视描述，

This video presentation was created to show a method of harvesting the two most important highly vascular structures, not residing within the brain ...

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

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

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

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

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

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

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. Choroid plexus epithelial cells (CPECs) covering the surface of choroid plexus tissue harbor multiple unique cilia, but most of the functions of these cilia remain to be investigated. To uncover the function of CPEC cilia with particular reference to their motility, an ex vivo observation system was developed to monitor ciliary motility during embryonic, perinatal and postnatal periods. The choroid plexus was dissected out of the brain ventricle and observed under a video-enhanced contrast microscope equipped with differential interference contrast optics. Under this condition, a simple and quantitative method was developed to analyze the motile profiles of CPEC cilia for several hours ex vivo. Next, the morphological changes of cilia during development were observed by scanning electron microscopy to elucidate the relationship between the morphological maturity of cilia and motility. Interestingly, this method could delineate changes in the number and length of cilia, which peaked at postnatal day (P) 2, while the beating frequency reached a maximum at P10, followed by abrupt cessation at P14. These techniques will enable elucidation of the functions of cilia in various tissues. While related techniques have been published in a previous report1, the current study focuses on detailed techniques to observe the motility and morphology of CPEC cilia ex vivo.

Observation of the Ciliary Movement of Choroid Plexus Epithelial Cells Ex Vivo

In this study, a detailed light microscopic technique was optimized for real-time observation and analysis of the motion of CPEC cilia ex vivo together with an electron microscopic method for ultrastructural analysis.

脉络丛上皮细胞的纤毛运动的观察<em&gt;离体</em

The choroid plexus is located in the ventricular wall of the brain, the main function of which is believed to be production of cerebrospinal fluid. ...

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory Receptor Neurons (ORNs) housed in hair-like structures, called chemosensilla, of the antennae. On the ORNs&#39; membranes, Odorant Receptors (ORs) are believed to be involved in odor coding. Thus, being able to identify genes localized to the ORNs is necessary to recognize OR genes, and provides a fundamental basis for further functional in situ studies. The RNA expression levels of specific ORs in insect antennae are very low, and preserving insect tissue for histology is challenging. Thus, it is difficult to localize an OR to a specific type of sensilla using RNA in situ hybridization. In this paper, a detailed and highly effective RNA in situ hybridization protocol particularly for lowly expressed OR genes of insects, is introduced. In addition, a specific OR gene was identified by conducting double-color fluorescent in situ hybridization experiments using a co-expressing receptor gene, Orco, as a marker.

Localization of Odorant Receptor Genes in Locust Antennae by RNA In Situ Hybridization

This paper describes a detailed and highly effective RNA in situ hybridization protocol particularly for low-level expressed Odorant Receptor (OR) genes, as well as other genes, in insect antennae using digoxigenin (DIG)-labeled or biotin-labeled probes.

通过RNA定位蝗虫天线中的气味受体基因<em&gt;原位</em&gt;杂交

Insects have evolved sophisticated olfactory reception systems to sense exogenous chemical signals. These chemical signals are transduced by Olfactory ...

Cochlear Surface Preparation in the Adult Mouse

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous etiologies such as exposure to excessive noise, the use of ototoxic medications, bacterial or viral ear infections, head injuries, and the aging process. Loss of sensory hair cells is a common pathological feature of the varieties of acquired hearing loss. Additionally, the inner hair cell synapse can be damaged by mild insults. Therefore, surface preparations of cochlear epithelia, in combination with immunolabeling techniques and confocal imagery, are a very useful tool for the investigation of cochlear pathologies, including losses of ribbon synapses and sensory hair cells, changes in protein levels in hair cells and supporting cells, hair cell regeneration, and determination of report gene expression (i.e., GFP) for verification of successful transduction and identification of transduced cell types. The cochlea, a bony spiral-shaped structure in the inner ear, holds the auditory sensory end organ, the organ of Corti (OC). Sensory hair cells and surrounding supporting cells in the OC are contained in the cochlear duct and rest on the basilar membrane, organized in a tonotopic fashion with high-frequency detection occurring in the base and low-frequency in the apex. With the availability of molecular and genetic information and the ability to manipulate genes by knockout and knock-in techniques, mice have been widely used in biological research, including in hearing science. However, the adult mouse cochlea is miniscule, and the cochlear epithelium is encapsulated in a bony labyrinth, making microdissection difficult. Although dissection techniques have been developed and used in many laboratories, this modified microdissection method using cell and tissue adhesive is easier and more convenient. It can be used in all types of adult mouse cochleae following decalcification.

This article presents a modified cochlear surface preparation method that requires decalcification and use of a cell and tissue adhesive to adhere the pieces of cochlear epithelia to 10 mm round cover slips for immunohistochemistry in adult mouse cochleae.

成年小鼠中的科利耳表面制备

Auditory processing in the cochlea depends on the integrity of the mechanosensory hair cells. Over a lifetime, hearing loss can be acquired from numerous ...

Targeted Neuronal Injury for the Non-Invasive Disconnection of Brain Circuitry

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly useful for disorders in which identifiable neuronal circuitry plays a key role, such as epilepsy and movement disorders. Currently available surgical modalities, while effective, generally involve an invasive surgical procedure, which can result in surgical injury to non-target tissues. Consequently, it would be of value to expand the range of surgical approaches to include a technique that is both non-invasive and neurotoxic.
Here, a method is presented for producing focal, neuronal lesions in the brain in a non-invasive manner. This approach utilizes low-intensity focused ultrasound together with intravenous microbubbles to transiently and focally open the Blood Brain Barrier (BBB). The period of transient BBB opening is then exploited to focally deliver a systemically administered neurotoxin to a targeted brain area. The neurotoxin quinolinic acid (QA) is normally BBB-impermeable, and is well-tolerated when administered intraperitoneally or intravenously. However, when QA gains direct access to brain tissue, it is toxic to the neurons. This method has been used in rats and mice to target specific brain regions. Immediately after MRgFUS, successful opening of the BBB is confirmed using contrast enhanced T1-weighted imaging. After the procedure, T2 imaging shows injury restricted to the targeted area of the brain and the loss of neurons in the targeted area can be confirmed post-mortem utilizing histological techniques. Notably, animals injected with saline rather than QA do demonstrate opening of the BBB, but dot not exhibit injury or neuronal loss. This method, termed Precise Intracerebral Non-invasive Guided surgery (PING) could provide a non-invasive approach for treating neurological disorders associated with disturbances in neural circuitry.

The goal of the protocol is to provide a method for producing non-invasive neuronal lesions in the brain. The method utilizes Magnetic Resonance-guided Focused Ultrasound (MRgFUS) to open the Blood Brain Barrier in a transient and focal manner, in order to deliver a circulating neurotoxin to the brain parenchyma.

脑回路非侵入性断开的靶向神经元损伤

Surgical intervention can be quite effective for treating certain types of medically intractable neurological diseases. This approach is particularly ...