我们要感谢 Kellie Breen Church 博士、Michael Kreisman 先生和 Jessica Jang 女士为收集代表性结果中显示的数据所做的贡献。这项工作得到了美国国立卫生研究院（NIH）R00 HD104994（R.B.M.）的支持。

所有程序均由科罗拉多州立大学 （#3960） 和加州大学圣地亚哥分校机构动物护理和使用委员会批准，并收集了代表性数据（S13235，PI Kellie Breen Church）。来自5只成年雌性和2只成年雄性C57 / BL6小鼠（9-16周龄）的数据在代表性数据部分中描述。如前所述，雌性小鼠在ICV注射和采血前3-4周进行卵巢切除术7。在实验之前，这些小鼠被饲养在12小时光照/ 12小时暗光循环中，并根据《实验动物护理和使用指南》自由获取饲料和水。
1. 进行开颅手术
注意:开颅手术可以在实际注射前一天或多天进行，这使得实验当天的注射过程更快。
<ol><li>如下所述准备开颅手术的材料。<ol><li>在工作台面上放置干净的台垫或悬垂材料。将异氟烷蒸发器的鼻锥粘在工作表面上（靠近实验者，但背对实验者;见 图1A）。</li>
		<li>将硅橡胶管放在无菌锋利的 18 G 针头的末端，使针尖突出 ~1 mm。将硅橡胶管放在无菌钝针的末端，使针尖突出~1毫米。 注意:每只小鼠应使用单独且无菌的预包装针头（锋利和钝）。</li>
		<li>收集电动剃须刀、碘磨砂膏、无菌纱布和酒精棉片，用于准备注射部位。</li>
	</ol></li>
	<li>如下所述，练习移动针头 2 毫米的横向和 1 毫米的尾部。<ol><li>用尺子和笔在一张纸上标记一个起点（这将是bregma），一个标记向左或向右2毫米，另一个标记在最后一个上方1毫米处（这将是注射部位;见 图1B）。</li>
		<li>练习将针头从起点移动到标记点 1 再到标记点 2（前囟到注射部位），直到确信该运动在没有导轨的情况下是可重复的。 注:此处描述的坐标在成年 C57/BL6 小鼠中有效，但其他品系或年龄组可能需要调整。</li>
	</ol></li>
	<li>如下所述准备小鼠进行开颅手术。<ol><li>将小鼠置于诱导室中，并在医用级氧气中用3%-4%异氟烷麻醉小鼠。通过练习和熟悉程序，将开颅手术的异氟烷暴露总持续时间减少到10分钟以内。 注意:异氟醚暴露对人体健康有害;在通风良好的地方使用经批准和检查的异氟烷汽化器，并清除废气。</li>
		<li>一旦脚趾反射消失，剃掉动物的头部。涂抹眼部润滑剂。</li>
		<li>将鼠标放在工作台面上，头部在鼻锥中以保持麻醉。</li>
		<li>按照供应商的指示施用镇痛剂，例如丁丙诺啡（0.6-0.8 mg/kg 小鼠体重，皮下注射）。</li>
		<li>用蘸有碘溶液的无菌纱布擦拭头部清洁注射部位（进行三次擦洗），然后用酒精擦洗垫擦拭（进行三次）。 注意:按照美国兽医协会的建议，去除毛发并清洁注射部位周围的皮肤，并使用无菌器械和针头，以防止感染。</li>
	</ol></li>
	<li>用非惯用手牢牢握住鼠标头部。将头部尽可能平放在工作台面上。</li>
	<li>找到注射部位。<ol><li>使用准备好的锋利的 18 G 针首先识别前盂;为此，将针沿着中线拖过头部皮肤，在喙尾平面上移动。想象一个等边三角形，其中两个顶点是眼睛，第三个顶点是囟的大致位置（见 图 1C）。</li>
		<li>从前乳开始，将针头向外侧移动2毫米，尾部向1毫米移动至注射部位。</li>
	</ol></li>
	<li>垂直握住针头的同时，用力将针头穿过皮肤和骨骼，直到管子与皮肤齐平。</li>
	<li>缩回针头，旋转针头，然后再次按压皮肤和骨骼。重复该过程，直到在骨头上形成一个小孔。</li>
	<li>使用钝针检查骨头上是否产生了足够的孔。目标是在骨头上开一个足够大的开口，以便钝针穿过。</li>
	<li>将鼠标从鼻锥中取出，用无菌纱布清除注射部位的任何血液，然后将鼠标放在笼子加热器上的笼子中，直到醒来。由于开颅手术的开口很小（18 G 针头），因此不需要用缝合线或伤口夹闭合该部位。 注意:这里产生的开口很小（18 G），因此许多机构认为这种手术是注射而不是手术。此外，虽然通过皮肤和骨骼向大脑开了一个口，但保持皮肤绷紧会导致孔不对齐，这可能有助于防止皮肤菌群或笼子垫料接触大脑。</li>
</ol>2. 进行注射
<ol><li>如下所述准备注射材料。<ol><li>在工作台面上放置干净的台垫或悬垂材料。将异氟烷蒸发器的鼻锥粘在工作表面上（靠近实验者，但背对实验者;见 图1A）。</li>
		<li>将一根 10 mm 长的 27 G 针头（斜角为 45°）连接到 5 μL 玻璃注射器上。将硅橡胶管放在针头上，使针尖突出 3.5 毫米;使用实验室胶带将管子固定在注射器主体上（见 图1D）。</li>
		<li>在试管中制备注射介质（药物、病毒或其他注射液）。对于本研究的代表性结果，注射无菌等渗盐水。 注意: 选择允许玻璃注射器和针头进入的试管，例如 2 mL 微量离心管。</li>
		<li>将 3 μL 注射介质吸入玻璃注射器中。首先，抽取超过所需体积的体积，然后弹出，直到注射器中残留 3 μL。</li>
		<li>收集碘磨砂膏、无菌纱布和酒精垫，用于准备注射部位。</li>
		<li>在计数模式下启动实验室计时器，并放置计时器，以便实验者在进样时可以看到它。</li>
		<li>练习像前一天一样移动针头 2 毫米的横向和 1 毫米的尾部。</li>
	</ol></li>
	<li>如下所述准备注射鼠标。<ol><li>将小鼠置于诱导室中，并用异氟醚麻醉小鼠。一旦脚趾反射消失，涂抹眼部润滑剂，然后将鼠标放在工作台面上，头部在鼻锥内。</li>
		<li>用碘和酒精各擦三片擦拭清洁注射部位。如步骤1.8所述，用18 G针（或钝针）识别注射部位。开颅手术期间形成的孔洞应该是可检测到的。 注意:如果没有提前进行开颅手术，或者如果无法检测到颅骨上的孔，则可以在此处进行开颅手术。</li>
	</ol></li>
	<li>如下所述用玻璃注射器进行注射。<ol><li>用非惯用手牢牢握住鼠标头部。将头部尽可能平放在工作台面上。</li>
		<li>将玻璃注射器的针头穿过头骨上的孔，直到放置在步骤2.1.2中制备的针头上的管子停留在小鼠的皮肤上。</li>
		<li>尽可能垂直握住注射器，注意冠状面和矢状面。在1分钟内缓慢注入培养基。</li>
		<li>注射完成后，将注射器和针头固定到位一分钟，以尽量减少回流。慢慢地将针头从鼠标头上缩回。</li>
		<li>将鼠标从鼻锥中取出，用无菌纱布（如果存在）清除注射部位的任何血液，然后将鼠标放在笼子加热器上的笼子中，直到醒来。</li>
	</ol></li>
</ol>3. 确认注射位置
<ol><li>如前所述，用盐水和4%多聚甲醛在磷酸盐缓冲盐水（PBS）中深度麻醉并灌注小鼠8。将大脑储存在4°C的PBS中的30%蔗糖中，直到大脑下沉（通常为2天）。</li>
	<li>如前所述，在低温恒温器上切割 20-50 μm 冠状切片9.切片时，观察组织块中的注射道。记录注射道是否与侧脑室明显相交。</li>
</ol>

使用徒手方法对小鼠进行脑室内注射

<ol>
	<li>Roseweir, A. 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=Discovery+of+potent+kisspeptin+antagonists+delineate+physiological+mechanisms+of+gonadotropin+regulation.">Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation.</a> Journal of Neuroscience. 29 (12), 3920-3929 (2009).</li><li>Kim, J. Y., Grunke, S. D., Levites, Y., Golde, T. E., Jankowsky, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+viral+injection+of+the+neonatal+mouse+brain+for+persistent+and+widespread+neuronal+transduction.">Intracerebroventricular viral injection of the neonatal mouse brain for persistent and widespread neuronal transduction.</a> Journal of Visualized Experiments. (91), e51863 (2014).</li><li>Taylor, Z. V., Khand, B., Porgador, A., Monsonego, A., Eremenko, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optimized+intracerebroventricular+injection+of+CD4(+)+T+cells+into+mice.">An optimized intracerebroventricular injection of CD4(+) T cells into mice.</a> STAR Protocols. 2 (3), 100725 (2021).</li><li>Russo, K. 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=Circadian+control+of+the+female+reproductive+axis+through+gated+responsiveness+of+the+RFRP-3+system+to+VIP+signaling.">Circadian control of the female reproductive axis through gated responsiveness of the RFRP-3 system to VIP signaling.</a> Endocrinology. 156 (7), 2608-2618 (2015).</li><li>Laursen, S. E., Belknap, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracerebroventricular+injections+in+mice.+Some+methodological+refinements.">Intracerebroventricular injections in mice. Some methodological refinements.</a> Journal of Pharmacological Methods. 16 (4), 355-357 (1986).</li><li>Haley, T. J., McCormick, W. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+effects+produced+by+intracerebral+injection+of+drugs+in+the+conscious+mouse.">Pharmacological effects produced by intracerebral injection of drugs in the conscious mouse.</a> British Journal of Pharmacology and Chemotherapy. 12 (1), 12-15 (1957).</li><li>McCosh, R. 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=Insulin-induced+hypoglycaemia+suppresses+pulsatile+luteinising+hormone+secretion+and+arcuate+Kiss1+cell+activation+in+female+mice.">Insulin-induced hypoglycaemia suppresses pulsatile luteinising hormone secretion and arcuate Kiss1 cell activation in female mice.</a> Journal of Neuroendocrinology. 31 (12), e12813 (2019).</li><li>Wu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcardiac+perfusion+of+the+mouse+for+brain+tissue+dissection+and+fixation.">Transcardiac perfusion of the mouse for brain tissue dissection and fixation.</a> Bio-Protocol. 11 (5), e3988 (2021).</li><li>Comba, 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=Laser+capture+microdissection+of+glioma+subregions+for+spatial+and+molecular+characterization+of+intratumoral+heterogeneity,+oncostreams,+and+invasion.">Laser capture microdissection of glioma subregions for spatial and molecular characterization of intratumoral heterogeneity, oncostreams, and invasion.</a> Journal of Visual Experiments. (158), e60939 (2020).</li><li>Porteous, R., 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=Reformulation+of+PULSAR+for+analysis+of+pulsatile+LH+secretion+and+a+revised+model+of+estrogen-negative+feedback+in+mice.">Reformulation of PULSAR for analysis of pulsatile LH secretion and a revised model of estrogen-negative feedback in mice.</a> Endocrinology. 162 (11), (2021).</li><li>Hohmann, J. G., 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=Differential+role+of+melanocortins+in+mediating+leptin's+central+effects+on+feeding+and+reproduction.">Differential role of melanocortins in mediating leptin's central effects on feeding and reproduction.</a> American Journal of Physiology: Regulatory, Integrative, and Comparative Physiology. 278 (1), R50-R59 (2000).</li><li>Gottsch, M. 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作者没有什么可透露的。

在这里，描述了一种在小鼠中进行ICV注射的简单有效的方法。由于该技术不需要立体定向框架，因此这种用于药物和实验剂集中递送的方法可供更多研究人员使用。此外，这种方法的通量相对较高，因为制备和注射程序可以快速执行。
由于此过程需要使用近似距离手动操作针头和玻璃注射器，因此建议新从业者在与活体动物一起工作之前花一些时间练习这些动作。此外，小?...

将实验药物（如药物1、病毒2 或细胞3）输送到大脑通常是神经内分泌研究所必需的。如果该药剂不容易穿过血脑屏障，或者实验目标是专门测试药剂的中枢作用，那么有一种可靠的方法将注射剂输送到大脑中是很重要的。此外，注射到脑室内 （ICV） 空间提供了将药物广泛分布在大脑中的机会，并提供了较大的靶区，从而增加了成功注射的可能性2. 
进行 ICV 注射的常用方法是放置永久性留置插管。在这种方法中，需要立体定位框架来定位市售或定制的套管，因为套管被粘合或胶合到位。通常，在康复后，通过套管给予超生理剂量的血管紧张素 II，如果立即观察到饮酒行为，则认为套管放置正确4.这种方法具有许多优点，包括能够进行长期输注和多次注射同一只动物的能力;此外，如果使用血管紧张素II，则可以在施用实验化合物之前确认正确的放置。然而，放置永久性插管存在一些局限性，包括对昂贵设备（立体定位框架）的要求、放置后插管损坏的可能性（例如，小鼠可以咀嚼笼子伴侣的插管）以及永久性插管周围感染的可能性。可以使用立体定位框架3 进行单次 ICV 注射，这虽然有效，但需要大量暴露于麻醉，因此可能会掩盖治疗的一些急性生理和行为影响。此外，将小鼠放置在立体定位框架中需要大量训练才能实现稳定放置并防止耳道破裂。
在这里，描述了一种在小鼠中进行徒手注射的既定方法。这种方法基于以前的报告5,6。这种技术的优点是简单、快速，不需要立体定位框架等专用设备。如下所述，该过程涉及操纵玻璃注射器相对于小鼠头部的标志进行注射，这可以快速完成，因此在实验当天只需要几分钟的气体麻醉。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>18-gauge blunt needles</td>
			<td>SAI Infusion</td>
			<td>B18-150</td>
			<td></td>
		</tr>
		<tr>
			<td>18-gauge needles</td>
			<td>BD Medical</td>
			<td>305195</td>
			<td></td>
		</tr>
		<tr>
			<td>Alcohol pads</td>
			<td>Fisher Scientific</td>
			<td>22-363-750</td>
			<td></td>
		</tr>
		<tr>
			<td>Bench pad</td>
			<td>Fisher Scientific</td>
			<td>14-206-62AC22</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine solution</td>
			<td>Fisher Scientific</td>
			<td>NC1696484</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>Patterson Vet Supply</td>
			<td>07-892-5235</td>
			<td>Controlled substance</td>
		</tr>
		<tr>
			<td>Eyelube</td>
			<td>Fisher Scientific</td>
			<td>50-218-8442</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass syringe</td>
			<td>Hamilton</td>
			<td>7634-01</td>
			<td></td>
		</tr>
		<tr>
			<td>Injection needle</td>
			<td>Hamilton</td>
			<td>7803-01</td>
			<td>27 gauge, Small Hub RN needle, point style: 4, Needle length: 10cm, Angle: 45</td>
		</tr>
		<tr>
			<td>Isoflurane&#160;&#160;</td>
			<td>Patterson Vet Supply</td>
			<td>07-893-8441</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane vaporizer</td>
			<td>Vet Equip</td>
			<td>V-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Laboratory Tape</td>
			<td>VWR</td>
			<td>89098-128</td>
			<td></td>
		</tr>
		<tr>
			<td>Medical grade oxygen</td>
			<td>Airgas</td>
			<td>OX USPEA</td>
			<td></td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Millipore-Sigma</td>
			<td>8.18715.1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffered Saline</td>
			<td>Fisher Scientific</td>
			<td>J67802.K2</td>
			<td></td>
		</tr>
		<tr>
			<td>PulsaR Software</td>
			<td>Open source, University of Otago</td>
			<td></td>
			<td>See ref 9</td>
		</tr>
		<tr>
			<td>Ruler</td>
			<td>Fisher Scientific</td>
			<td>12-00-152</td>
			<td></td>
		</tr>
		<tr>
			<td>Silastic tubing (0.040&#34; I.D.)</td>
			<td>DOW</td>
			<td>508-005</td>
			<td></td>
		</tr>
		<tr>
			<td>Silastic tubing (0.078&#34; I.D.)</td>
			<td>DOW</td>
			<td>508-009</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile saline</td>
			<td>VWR</td>
			<td>101320-574</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose&#160;</td>
			<td>Fisher Scientific</td>
			<td>S5-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

free-hand intracerebroventricular injections in mice

神经内分泌系统的研究通常需要将药物、病毒或其他实验剂直接输送到小鼠的大脑中。脑室内 （ICV） 注射允许实验药物在整个大脑中广泛递送（特别是在心室附近的结构中）。在这里，描述了在成年小鼠中进行徒手ICV注射的方法。通过在小鼠头部使用视觉和触觉标志，可以快速可靠地向侧脑室注射。注射是用实验者手中的玻璃注射器进行的，并放置在与地标大致距离的地方。因此，该技术不需要立体定位框架。此外，该技术只需要短暂的异氟醚麻醉，这允许随后评估清醒、行为自由的小鼠的小鼠行为和/或生理学。徒手ICV注射是将实验剂有效输送到活体小鼠大脑中的强大工具，可以与其他技术相结合，如频繁的血液采样、神经回路操作或 体内 记录，以研究神经内分泌过程。

成功执行后，该技术允许将实验剂快速输送到心室系统中。 图2A显示了接受ICV注射3μL无菌等渗盐水（许多药理学化合物的载体）的卵巢切除小鼠的促黄体激素（LH）脉冲曲线。该示例表明，短暂暴露于气体麻醉和单独向心室系统注射 3 μL 液体不会改变搏动性 LH 分泌。ICV注射后3 h，对动物实施安乐死，收集新鲜冷冻的神经组织，在低温恒温器上切割;注射道与侧脑室明显相交?...

Watch this Scientific Journal Video about Insights, Innovations, and Future Avenues in Reproductive Neurocontrol at JoVE.com

作者聚焦:生殖神经控制的见解、创新和未来途径 

在这里，描述了一种使用徒手方法（即没有立体定位装置）在小鼠中进行脑室内注射的简单快速的方法。

小鼠徒手脑室内注射

The investigation of neuroendocrine systems often requires the delivery of drugs, viruses, or other experimental agents directly into the brains of mice. ...

我们对生殖功能的神经控制感兴趣，特别是大脑如何调节控制性腺的激素的释放，无论是在健康条件下还是在应对压力时。这种技术的主要优点是相对简单和快速。将药物输送到侧脑室是在测试信号通路以支持更详细的后续实验时广撒网的好方法。关于生殖的神经内分泌控制还有很多东西需要了解，包括促黄体生成素脉冲的分子机制、应激期间调节脉冲频率的机制以及排卵前 LH 激增的产生。未来，我们对将不同的发现整合到模型中特别感兴趣。

free-hand-intracerebroventricular-injections-in-mice

Research

JoVE Journal

Neuroscience

Free-Hand Intracerebroventricular Injections in Mice

9.5K 次观看.  Colorado State University. 我们对生殖功能的神经控制感兴趣，特别是大脑如何调节控制性腺的激素的释放，无论是在健康条件下还是在应对压力时。这种技术的主要优点是相对简单和快速。将药物输送到侧脑室是在测试信号通路以支持更详细的后续实验时广撒网的好方法。关于生殖的神经内分泌控制还有很多东西需要了解，包括促黄体生成素脉冲的分子机制、应激期间调节脉冲频率的机制以及?...

视频: 作者聚焦:生殖神经控制的见解、创新和未来途径 

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 ...

科研

神经科学

Simultaneous Electrophysiological Recording and Micro-injections of Inhibitory Agents in the Rodent Brain

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing system was developed that allows for the injection of a drug while recording electrophysiological signals from the affected neuronal population. This method provides a simple and economical alternative to commercial solutions. A glass pipette was modified by combining it with a hypodermic needle and a silver filament. The injectrode is attached to commercial microsyringe pump for drug delivery. This results in a technique that provides real-time pharmacodynamics feedback through multi-unit extracellular signals originating from the site of drug delivery. As a proof of concept, we recorded neuronal activity from the superior colliculus elicited by flashes of light in rats, concomitantly with delivery of drugs through the injectrode. The injectrode recording capacity permits the functional characterization of the injection site favoring precise control over the localization of drug delivery. Application of this method also extends far beyond what is demonstrated here, as the choice of chemical substance loaded into the injectrode is vast, including tracing markers for anatomic experiments.

Here, we craft a glass pipette with dual functions: inhibition of deep brain structures by microinjections of drugs and real-time monitoring of their effects through simultaneous electrophysiological recordings.

同时电生理记录和抑制剂在啮齿动物大脑微注射

Here we describe a method for the construction of a single-use &#8220;injectrode&#8221; using commercially accessible and affordable parts. A probing ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

测定冷灵敏度和适应小鼠一种简单廉价的方法

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

Intracerebroventricular Injection of Amyloid-&#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, progressive accumulation of A&#946; oligomers and plaques is observed. Such A&#946; abnormalities are believed to block long-term potentiation, impair synaptic function, and induce cognitive deficits. Clinical and experimental evidences have revealed that the acute increase of A&#946; levels in the brain allows development of Alzheimer-like phenotypes. Hence, a detailed protocol describing how to acutely generate an AD mouse model via the intracerebroventricular (ICV) injection of A&#946; is necessary in many cases. In this protocol, the steps of the experiment with an A&#946;-injected mouse are included, from the preparation of peptides to the testing of behavioral abnormalities. The process of preparing the tools and animal subjects before the injection, of injecting the A&#946; into the mouse brain via ICV injection, and of assessing the degree of cognitive impairment are easily explained throughout the protocol, with an emphasis on tips for effective ICV injection of A&#946;. By mimicking certain aspects of AD with a designated injection of A&#946;, researchers can bypass the aging process and focus on the downstream pathology of A&#946; abnormalities.

Intracerebroventricular Injection of Amyloid-&amp;#946; Peptides in Normal Mice to Acutely Induce Alzheimer-like Cognitive Deficits

The amyloid-&#946; (A&#946;)-injected animal model enables the administration of a defined quantity and species of A&#946; fragments and reduces individual differences within each study group. This protocol describes the intracerebroventricular (ICV) injection of A&#946; without stereotactic instruments, enabling the production of Alzheimer-like behavioral abnormalities in normal mice.

正常小鼠β淀粉样肽侧脑室注射敏锐地诱导老年痴呆样认知缺陷

Amyloid-&#946; (A&#946;) is a major pathological mediator of both familial and sporadic Alzheimer's disease (AD). In the brains of AD patients, ...

Intracerebroventricular and Intravascular Injection of Viral Particles and Fluorescent Microbeads into the Neonatal Brain

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the hematogenous route, which involves infection of the endothelial cells and pericytes of the brain. The second is the intracerebroventricular (ICV) route. Once within the central nervous system (CNS), viruses may spread to the subarachnoid space, meninges, and choroid plexus via the cerebrospinal fluid. In experimental models, the earliest stages of CNS viral distribution are not well characterized, and it is unclear whether only certain cells are initially infected. Here, we have analyzed the distribution of cytomegalovirus (CMV) particles during the acute phase of infection, termed primary viremia, following ICV or intravascular (IV) injection into the neonatal mouse brain. In the ICV injection model, 5 &#181;l of murine CMV (MCMV) or fluorescent microbeads were injected into the lateral ventricle at the midpoint between the ear and eye using a 10-&#181;l syringe with a 27 G needle. In the IV injection model, a 1-ml syringe with a 35 G needle was used. A transilluminator was used to visualize the superficial temporal (facial) vein of the neonatal mouse. We infused 50 &#181;l of MCMV or fluorescent microbeads into the superficial temporal vein. Brains were harvested at different time points post-injection. MCMV genomes were detected using the in situ hybridization method. Fluorescent microbeads or green fluorescent protein expressing recombinant MCMV particles were observed by fluorescent microscopy. These techniques can be applied to many other pathogens to investigate the pathogenesis of encephalitis.

Here, we describe a simple method of intracerebroventricular and intravascular injection of viral particles or fluorescent microbeads into the neonatal mouse brain. The localization pattern of the virus and nanoparticles could be detected by microscopic evaluation or by in situ hybridization.

病毒颗粒和荧光微球侧脑室和血管内注入新生儿脑

In the study on the pathogenesis of viral encephalitis, the infection method is critical. The first of the two main infectious routes to the brain is the ...

Intracerebroventricular Treatment with Resiniferatoxin and Pain Tests in Mice

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In addition to its peripheral function, its involvement in brain functions has also been suggested. Resiniferatoxin (RTX), an ultrapotent TRPV1 agonist, has been known to induce long-term desensitization of TRPV1, and this desensitization has been an alternative approach for investigating the physiological relevance of TRPV1-expressing cells. Here we describe a protocol for intracerebroventricular (i.c.v.) treatment with RTX in mice. Procedures are described for testing nociception to peripheral TRPV1 stimulation (RTX test) and mechanical stimulation (tail pressure test) then follow. Although the nociceptive responses of mice that had been administered RTX i.c.v. were comparable to those of the control groups, RTX-i.c.v.-administered mice were insensitive to the analgesic effect of acetaminophen, suggesting that i.c.v. RTX treatment can induce supraspinal-selective TRPV1 desensitization. This mouse model can be used as a convenient experimental system for studying the role of TRPV1 in brain/supraspinal function. These techniques can also be applied to studies of the central actions of other drugs.

The transient receptor potential vanilloid type 1 (TRPV1) in the supraspinal region has been suggested to play some roles in the brain function. Described here is a protocol for intracerebroventricular injection of resiniferatoxin for supraspinal TRPV1 desensitization in mice. Procedures for some pain tests are also presented.

小鼠用树脂叶毒素和疼痛测试进行脑血管内治疗

The transient receptor potential vanilloid type 1 (TRPV1), a thermosensitive cation channel, is known to trigger pain in the peripheral nerves. In ...

In Vivo Intracerebral Stereotaxic Injections for Optogenetic Stimulation of Long-Range Inputs in Mouse Brain Slices

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional investigation of long-range connections requires targeted recordings of single neurons combined with the specific stimulation of identified distant inputs. This is often difficult to achieve with conventional and electrical stimulation techniques, because axons from converging upstream brain areas may intermingle in the target region. The stereotaxic targeting of a specific brain region for virus-mediated expression of light-sensitive ion channels allows selective stimulation of axons originating from that region with light. Intracerebral stereotaxic injections can be used in well-delimited structures, such as the anterior thalamic nuclei, in addition to other subcortical or cortical areas throughout the brain.
Described here is a set of techniques for precise stereotaxic injection of viral vectors expressing channelrhodopsin in the mouse brain, followed by photostimulation of axon terminals in the brain slice preparation. These protocols are simple and widely applicable. In combination with whole-cell patch clamp recording from a postsynaptically connected neuron, photostimulation of axons allows the detection of functional synaptic connections, pharmacological characterization, and evaluation of their strength. In addition, biocytin filling of the recorded neuron can be used for post-hoc morphological identification of the postsynaptic neuron.

This protocol describes a set of methods to identify the cell-type specific functional connectivity of long-range inputs from distant brain regions using optogenetic stimulations in ex vivo brain slices.

在Vivo脑内立体注射中，用于小鼠脑切片中远距离输入的光遗传学刺激

Knowledge of cell-type specific synaptic connectivity is a crucial prerequisite for understanding brain-wide neuronal circuits. The functional ...

Injections of AAV Vectors for Optogenetics in Anesthetized and Awake Behaving Non-Human Primate Brain

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of optogenetics, however, requires that safety and efficacy be demonstrated in animal models, ideally in non-human primates (NHPs), because of their neurological similarity to humans. The number of candidate vectors that are potentially useful for neuroscience and medicine is vast, and no high-throughput means to test these vectors yet exists. Thus, there is a need for techniques to make multiple spatially and volumetrically accurate injections of viral vectors into NHP brain that can be identified unambiguously through postmortem histology. Described herein is such a method. Injection cannulas are constructed from coupled polytetrafluoroethylene and stainless-steel tubes. These cannulas are autoclavable, disposable, and have low minimal-loading volumes, making them ideal for the injection of expensive, highly concentrated viral vector solutions. An inert, red-dyed mineral oil fills the dead space and forms a visible meniscus with the vector solution, allowing instantaneous and accurate measurement of injection rates and volumes. The oil is loaded into the rear of the cannula, reducing the risk of co-injection with the vector. Cannulas can be loaded in 10 min, and injections can be made in 20 min. This procedure is well suited for injections into awake or anesthetized animals. When used to deliver high-quality viral vectors, this procedure can produce robust expression of optogenetic proteins, allowing optical control of neural activity and behavior in NHPs.

As currently implemented, optogenetics in non-human primates requires injection of viral vectors into the brain. An optimal injection method should be reliable and, for many applications, capable of targeting individual sites of arbitrary depth that are readily and unambiguously identified in postmortem histology. An injection method with these properties is presented.

在麻醉和清醒行为的非人灵长类动物大脑中注射用于光遗传学的AAV载体

Optogenetic techniques have revolutionized neuroscience research and are poised to do the same for neurological gene therapy. The clinical use of ...

Intracerebroventricular Delivery of Gut-Derived Microbial Metabolites in Freely Moving Mice

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have revealed that gut microbiota-derived metabolites modulate brain-mediated physiological functions through intricate gut-brain pathways in the host. Short-chain fatty acids (SCFAs) are the major bacteria-derived metabolites produced during dietary fiber fermentation by the gut microbiome. Secreted SCFAs from the gut can act at multiple sites in the periphery, affecting the immune, endocrine, and neural responses due to the vast distribution of SCFAs receptors. Therefore, it is challenging to differentiate the central and peripheral effects of SCFAs through oral and intraperitoneal administration of SCFAs. This paper presents a video-based method to interrogate the functional role of SCFAs in the brain via a guide cannula in freely moving mice. The amount and type of SCFAs in the brain can be adjusted by controlling the infusion volume and rate. This method can provide scientists with a way to appreciate the role of gut-derived metabolites in the brain.

Gut-derived microbial metabolites have multifaceted effects leading to complex behavior in animals. We aim to provide a step-by-step method to delineate the effects of gut-derived microbial metabolites in the brain via intracerebroventricular delivery via a guide cannula.

自由移动小鼠中肠道来源的微生物代谢物的脑室内递送

The impact of gut microbiota and their metabolites on host physiology and behavior has been extensively investigated in this decade. Numerous studies have ...

在新生小鼠中进行鞘内注射的综合方案

Intrathecal Injection of Newborn Mouse for Genome Editing and Drug Delivery

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and treatments. By directly delivering medications and treatments into the cerebrospinal fluid of the central nervous system, this method achieves higher localized drug concentrations while reducing systemic side-effects compared to other routes such as intravenous, subcutaneous, or intramuscular injections. Its importance extends beyond clinical settings, as intrathecal injection plays a vital role in preclinical studies focused on treating neurogenetic disorders in rodents and other large animals, including non-human primates. However, despite its widespread application, intrathecal injection in young, particularly neonatal pups, poses significant technical challenges due to their small size and fragile nature. Successful and reliable administration of intrathecal injections in newborn mice requires meticulous attention to detail and careful consideration of various factors. Thus, there is a crucial need for a standardized protocol that not only provides instructions but also highlights key technical considerations and good laboratory practices to ensure procedural consistency, as well as the safety and welfare of the animals.
To address this unmet need, we present a detailed and comprehensive protocol for performing intrathecal injections specifically in newborn pups on postnatal day 1 (P1). By following the step-by-step instructions, researchers can confidently perform intrathecal injections in neonatal pups, enabling the accurate delivery of drugs, antisense oligos, and viruses for gene replacement or genome editing-based treatments. Furthermore, the importance of adhering to good laboratory practices is emphasized to maintain the well-being of animals and ensure reliable experimental outcomes. This protocol aims to address the technical challenges associated with intrathecal injections in neonatal mice, ultimately facilitating advances in the field of neurogenetic research that aims to develop potential therapeutic interventions.

作者聚焦: 鞘内注射 – 一种高效可靠的递送方法，用于测试新生小鼠大脑中基因编辑的功效

The present protocol outlines step-by-step instructions for performing intrathecal injections in neonatal mice for gene editing and drug delivery.

新生小鼠鞘内注射用于基因组编辑和药物递送

Intrathecal injection is a commonly employed procedure in both pediatric and adult clinics, serving as an effective means to administer medications and ...