我们承认以下资金来源:NIH NINDS – K08NS101122 （JB）， R01NS040337 （JK）， R01NS044370 （JK）， 弗吉尼亚大学医学院 （JB）。

所有动物研究均获得弗吉尼亚大学机构动物护理和使用委员会（IACUC）的批准。
1. 电极建筑/电缆建筑
<ol><li>使用单极绝缘不锈钢丝（0.005"裸直径，0.008"涂层）制成与母插座连接器（母插座连接器0.079）连接的电极。</li>
	<li>使用特殊的定制电缆将动物连接到放大器。<ol>
<li>将一个 4 针公连接器（公连接器 0.079"）连接到 4 通道单位增益阻抗匹配运算放大器（运算放大器）。将10K电阻连接到连接到9 V电池的电线上。未连接到运算放大器的地线充当电池的中点。</li>
		<li>将电缆的一端（AWG，0.012" 外径）连接到运算放大器，并将电缆的另一端连接到放大器。</li>
	</ol>
</li>
</ol>2. 电极植入手术
<ol><li>在向动的罩中用 4 - 5% 异氟醚麻醉幼崽（出生后第 9 天）。在手术开始之前，向幼崽注射布比卡因（0.02-0.05 mL，0.25%皮下局部浸润）。</li>
	<li>一旦动物无法移动，就转移到带有鼻锥的立体定向阶段。使用耳杆的反面，因为它很软，可以保持头部稳定。在这个年龄，幼崽没有完全发育的耳朵来使用耳杆的尖头。</li>
	<li>降低异氟醚的流量，并将其保持在2.5-3%。在整个手术过程中，请密切注意幼犬的稳定呼吸。捏住尾巴以检查疼痛反应，然后进行切口。</li>
	<li>用β-丁和酒精（3个循环的交替碘和70%乙醇）对颅骨上的切口区域进行灭菌。覆盖周围的身体部位，使切口区域可见。</li>
	<li>从眼睛上方稍微打开头皮前后，缩回约0.5厘米的皮肤。将鼠标头部重新定位在立体定位阶段，以便皮肤向外拉出暴露的头骨。</li>
	<li>使用棉签将过氧化氢涂抹在颅骨上，然后用手术刀刀片将颅骨刮干净。头骨非常柔软;刮擦时要小心。</li>
	<li>涂抹一滴（约50μL）粘合剂，并使用其涂抹器将其涂抹在暴露的颅骨周围。暴露在紫外线下40秒以固定粘合剂。</li>
	<li>使用暴露的边角作为参考来测量坐标。种植电极在海马体的CA1区域[-3.5 mm背腹（DV），±2 mm内侧-侧（ML），-1.75 mm深（D）]和双侧在顶叶皮层[-1.22 mm DV，±0.5 mm ML，-1 mm D]和小脑中的参考电极17。使用32 G针在标记区域创建一个孔。</li>
	<li>清洁颅骨表面的血液。在立体定位臂的帮助下，连接到母插座连接器的下电极进入大脑，并用牙科丙烯酸固定到位。将电极植入大脑。插座连接器耳机位于颅骨顶部，由牙科丙烯酸粘合在一起。</li>
	<li>电极固定后，皮下注射酮洛芬（5mg / kg）在肩胛间区域。将幼崽放回母亲身边。 注意:一次将一半的带有耳机的垃圾介绍给母亲，而不是一次介绍一个。这将防止母亲损坏小狗的耳机。</li>
</ol>3. 脑电图设置和记录（基线/损伤前）
<ol><li>电极植入后恢复24小时后，将每只动物置于加热（37°C）定制的Plexiglas室中进行脑电图记录。该室还将用作缺氧室。</li>
	<li>通过柔性电缆（定制运算放大器电缆）将腔室中的幼崽连接到视频脑电图监测系统。 注意:戴上耳机后，鼠标可以自由移动，并且不会表现出任何行为差异。一旦连接到电极丝上，必须在腔室系绳内调整导线，以提供适量的松弛度，以便幼犬可以在整个腔室中自由移动。</li>
	<li>使用草地放大器以 1K 增益对 1000 Hz 的 EEG 数据进行数字化处理。稍后使用软件（例如LabChart Pro）查看EEG信号（3-70 Hz之间的带通滤波器）。</li>
	<li>在断开动物进行颈动脉结扎手术之前，记录损伤前基线脑电图30分钟。</li>
</ol>4. 左颈动脉结扎
<ol><li>在向动的罩中用 4 - 5% 异氟醚麻醉幼犬（出生后第 10 天），并将它们放在水浴垫上特别布置的设置上。定位动物仰卧位并用纸胶带固定前肢。<ol>
<li>将异氟醚的流量降低至2-3%。捏住尾巴以获得疼痛反应，并在整个手术过程中监测呼吸。</li>
	</ol>
</li>
	<li>用甜菜碱和酒精（3个循环的交替碘和70%乙醇）对颈部左侧的切口区域（下颌骨和锁骨之间）进行消毒。</li>
	<li>使用微剪刀在颈部左侧做一个约1厘米长的切口。使用解剖显微镜，小心地缩回皮下组织和皮肤以暴露颈动脉。小心识别迷走神经（横向延伸到动脉），并小心地将其从动脉中分离并收回。</li>
	<li>使用微钳在动脉下缝合5厘米长的无菌丝线。在动脉周围系上双结缝合线以阻塞血流。</li>
	<li>切开多余的缝合线，并通过拉回皮下组织和皮肤来关闭暴露的动脉。使用兽医粘合来密封切口。</li>
	<li>将动物放回室中在室温下的连续脑电图监测中，该室放置在加热床垫上。对幼崽核心温度进行点红外温度检查，以避免打开腔室。让动物在缺氧前恢复1小时。</li>
</ol>5. 脑电图和缺氧
<ol><li>通过氧气监测仪连续监测腔室内的FiO2 （吸入氧气的分数）。</li>
	<li>用60升/分钟的100%N2 和0.415升/分钟冲洗腔室，100%O2。一旦腔室中的氧饱和度达到12%，则将N2流量降低至10 L / min，同时保持O2 流量不变。通过小幅调整，将FiO2 保持在8%45分钟。</li>
	<li>缺氧暴露45分钟后，将FiO2 返回至21%。</li>
	<li>让幼崽在腔室中恢复，并在缺氧后监测脑电图2小时。</li>
	<li>记录期结束后，断开小鼠与脑电图记录的连接并返回给母亲。</li>
</ol>6. 脑电图分析
<ol><li>在LabChart Pro中用视频分析EEG文件。让盲法研究人员在脑电图上标记癫痫发作和背景模式17。癫痫发作被定义为持续超过 10 秒的心电事件，具有高频有节奏的锐波放电（≥3 倍基线），且有明显的演变17。</li>
	<li>让第二个盲法研究人员随机审查标记的事件以达成一致。</li>
	<li>回顾每个标记的心电图事件的相关视频，并根据新生儿啮齿动物行为癫痫发作评分进行分析16。简而言之，该评分范围为0-6（不动到严重的强直阵挛行为）。为了进一步描述癫痫发作的符号学特征，分析侧向性行为（多灶性/双侧运动 vs. 局灶性/单侧性 vs. 混合性）。</li>
	<li>创建功率频谱图。将快速傅里叶变换与大小为 1024 个数据点的余弦-贝尔数据窗口结合使用。借助 87.5% 的窗口重叠，在频谱图中创建平滑的 x 轴。将功率表示为μV218。</li>
</ol>

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我们已经提出了一种在缺氧缺血性癫痫发作期间对新生小鼠进行连续视频脑电图监测的模型。结合脑电图进行视频分析，可表征癫痫发作的符号学特征。脑电图分析允许提取功率谱图和背景振幅分析。
正确和小心地放置电极在此方案中至关重要，因为电极放置过程中的伤害或不正确的放置会显着影响结果。在损伤前评估正常的基线脑电图活动至关重要，因为在电极放置过程中可能会发生出血或损伤，虽然很少见。其次，为了确认正确的电极放置，可以对大脑进行切片并检查正确位置的电极轨迹。此外，未能成群结队（单独）将幼崽送回母亲身边可能导致电极耳机损坏或幼崽被母亲杀死或忽视。
这种方法的一个局限性是小型新生儿大脑中深度电极记录的空间定位的限制。这限制了在脑电图记录中定位特定癫痫发作病灶的能力。该缺氧缺血模型的另一个局限性是癫痫发作负担的变异性。这种缺氧缺血的啮齿动物模型中病变大小和行为缺陷的变异性之前已有很好的描述7，8，19。毫不奇怪，这种变异性存在于癫痫发作负担中（癫痫发作事件的长度和癫痫发作事件的次数）。然而，始终如一的是，该模型中100%的幼崽在缺氧期间表现出癫痫发作。最后，幼崽可以进行脑电图监测（远离母亲）的时间是有限的。因此，相对于损伤，我们无法在以后的时间点用连续脑电图来表征正在进行的癫痫发作。
虽然，我们在本手稿中使用了缺氧缺血癫痫发作模型，但这种用于新生儿小鼠幼崽连续视频脑电图监测的方法可以很容易地应用于其他疾病/癫痫发作模型。仅根据行为很难识别新生儿啮齿动物的癫痫发作，因此视频脑电图监测很重要。未来的研究可以使用这些技术来分析其他新生儿癫痫发作模型中的癫痫发作负担和符号学，或对治疗和神经保护措施的反应。

缺氧缺血性脑病 （HIE） 是一种每年影响 1000 名新生儿中 1.5 名新生儿的疾病，是新生儿癫痫发作的最常见原因1，2。存活下来的婴儿有患各种神经残疾的风险，如脑瘫、智力残疾和癫痫3，4，5。
动物模型在理解和研究缺氧缺血和新生儿癫痫发作的病理生理学方面起着关键作用6，7。改进的Vannucci模型用于在出生后第10天诱导缺氧缺血（HI）（p10）7，8。这个年龄段的小鼠幼崽在神经学上大致翻译为完整的足月人类新生儿9。
与该损伤模型结合使用的连续视频脑电图 （EEG） 监测可进一步了解和表征新生儿缺氧缺血性癫痫发作。以前的研究已经使用各种方法来分析啮齿动物的新生儿癫痫发作，包括视频记录，有限的脑电图记录和遥测脑电图记录10，11，12，13，14，15，16。在下面的手稿中，我们深入讨论了在缺氧缺血期间记录小鼠幼崽连续视频脑电图的过程。这种用于新生儿小鼠幼崽连续视频脑电图监测的技术可以应用于各种疾病和癫痫发作模型。

<table>
	<tbody>
		<tr>
			<td>SURGERY</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ball Point Applicator</td>
			<td>Metrex Research</td>
			<td>8300-F</td>
			<td>i-bond applicator</td>
		</tr>
		<tr>
			<td>Cranioplast (Powder/Resin)</td>
			<td>Coltene</td>
			<td>H00383</td>
			<td>Perm Reline/Power</td>
		</tr>
		<tr>
			<td>I-Bond</td>
			<td>Kulzer GmbH, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LOOK Silk Suture</td>
			<td>Surgical Specialities Corporation</td>
			<td>SP115</td>
			<td>LOOK SP115 Black Braided Silk Non absorbable surgical suture</td>
		</tr>
		<tr>
			<td>RS-5168 Botvin Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5168</td>
			<td>Forcep for surgery/ligation</td>
		</tr>
		<tr>
			<td>RS-5138 Graefe Forceps</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5138</td>
			<td>Forcep for surgery/ligation</td>
		</tr>
		<tr>
			<td>UV light for I-Bond</td>
			<td>Blast Lite By First Media</td>
			<td>BL778</td>
			<td>UV ligth for I-bond</td>
		</tr>
		<tr>
			<td>Vannas Microdissecting Scissor</td>
			<td>Roboz Surgical Instrument</td>
			<td>RS5618</td>
			<td>Scissor for ligation</td>
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			<td>Vet Bond</td>
			<td>3M Vetbond</td>
			<td>1469SB</td>
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			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HYPOXIA</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hypoxidial</td>
			<td>Starr Life Science</td>
			<td></td>
			<td></td>
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			<td>Oxygen sensor</td>
			<td>Medical Products</td>
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			<td>MiniOxI- oxygen analyzer/sensor for hypoxia rig</td>
		</tr>
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			<td></td>
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			<td>EEG RECORDING</td>
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			<td>Female receptacle connector 0.079&#34;</td>
			<td>Mill-Max Manufacturing Corp</td>
			<td>832-10-024-10-001000</td>
			<td>Ordered from Digikey</td>
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			<td>Grass Amplifier</td>
			<td>Natus Neurology Incorporated</td>
			<td></td>
			<td>Grass Product</td>
		</tr>
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			<td>LabChart Pro</td>
			<td>ADI Instruments</td>
			<td></td>
			<td>Software to run the system</td>
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			<td>Male Socket Connector 0.079&#34;</td>
			<td>Mill-Max Manufacturing Corp</td>
			<td>833-43-024-20-001000</td>
			<td>Ordered from Digikey</td>
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		<tr>
			<td>Operational Amplifier</td>
			<td>Texas Instruments, Dallas, TX, USA</td>
			<td>TLC2274CD</td>
			<td>TLC2274 Quad Low&#8208;Noise Rail&#8208;to Rail Operational Amplifier</td>
		</tr>
		<tr>
			<td>Operational Amplifier</td>
			<td>Texas Instruments, Dallas, TX, USA</td>
			<td>TLC2272ACDR</td>
			<td>TLC2274 Quad Low&#8208;Noise Rail&#8208;to Rail Operational Amplifier</td>
		</tr>
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			<td>Stainless Steel wire</td>
			<td>A-M Systems</td>
			<td>791400</td>
			<td>0.005&#34; Bare/0.008&#34; Coated 100 ft</td>
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			<td>Ultra-Flexible Wire</td>
			<td>McMaster-Carr</td>
			<td>9564T1</td>
			<td>36 Gauze wire of various color</td>
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	</tbody>
</table>

continuous video electroencephalogram during hypoxia-ischemia in neonatal mice

缺氧缺血是新生儿癫痫发作的最常见原因。动物模型对于了解新生儿癫痫发作和缺氧缺血的机制和生理学至关重要。本文介绍了一种在新生小鼠中连续进行视频脑电图（EEG）监测的方法，以检测癫痫发作并分析缺氧缺血期间的脑电图背景。同时使用视频和脑电图可以描述癫痫发作的符号学和确认癫痫发作。该方法还允许分析实验时间段内的功率谱图和EEG背景模式趋势。在这种缺氧缺血模型中，该方法允许在损伤前进行脑电图记录，以获得规范基线，并在损伤和恢复期间进行。总监测时间受到限制，因为无法将幼崽与母亲分开超过四个小时。虽然，我们在本文中使用了缺氧缺血性癫痫发作的模型，但这种用于新生儿视频脑电图监测的方法可以应用于啮齿动物的各种疾病和癫痫发作模型。

癫痫发作符号学
新生儿缺氧缺血暴露导致小鼠全身性和局灶性癫痫发作（图1A-C）。视频脑电图记录允许将电图结果与视频行为相关联。这些行为使用先前发表的新生儿啮齿动物行为癫痫发作评分（BSS）进行评分16。除BSS外，我们还根据行为是局灶性/单侧，双侧还是混合性对事件进行分类（图1B）。
在该模型中，小鼠通常表现出3种癫痫发作符号学模式:1）重复绕到结扎一侧并延伸对侧四肢，2）身体屈曲和尾巴卷曲到结扎一侧的姿势丧失，或3）单侧或双侧四肢划桨的姿势丧失（不同严重程度和长度）。大多数观察到的事件涉及局灶性/单侧或混合行为（图1B）。此外，在缺氧期间，一部分小鼠表现出非惊厥性癫痫发作活动，其中幼崽在脑电图上持续癫痫发作活动不动（图1C）。
电气记录
在颈动脉结扎前30分钟开始进行脑电图记录，以获得损伤前基线。基线活性（图1A 和 图2A）与先前描述的p10小鼠幼崽17的背景相似。结扎后，幼崽立即被放回视频脑电图。在结扎和缺氧开始之间的时间段内，一部分小鼠表现出惊厥性癫痫发作（图1A-C）。
缺氧诱导后，脑电图的背景振幅减小（图3B），间歇性地表现出尖峰波放电的爆发，随后是抑制（图2A）。小鼠表现出电图癫痫发作，其从抑制的背景中出现，作为节律性尖峰波放电，并进展为变得更加复杂和频繁，伴有多尖峰波（图2B）。在缺氧期间，功率谱图分析显着于缺血和对侧半球之间的不对称性（图3A，B）。缺血半球表现出爆发抑制模式，对侧半球表现出抑制背景（图1A 和 图3A，B）。平均而言，癫痫发作在诱导缺氧后5.5±8.1分钟开始，每次事件持续56±57秒。缺氧期间死亡率为13%（n=4/30），所有死亡均在惊厥性（BSS=5-6）癫痫发作后发生。
在复氧和恢复期间，一部分小鼠在记录期的剩余时间内（缺氧后2小时）继续癫痫发作。与缺氧后的基线相比，脑电图背景受到抑制（图1A 和 图3），在缺氧后记录期间逐渐恢复。在整个记录期间，小鼠平均表现出9±5次癫痫发作事件，每次持续54±57.7秒。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61346/61346fig1v2.jpg"> 图1:暴露于新生儿缺氧缺血的p10小鼠的癫痫发作特征。 （A）代表性功率谱图从缺血性顶叶皮层电极通过实验时间轴。（振幅彩色热图比例 x10–6）。箭头表示频谱图下方的原始脑电图示踪所表示的时间。（B）整个实验的癫痫发作行为，即二次充血后/缺氧前期，缺氧期间和Hypoxia后。（C）所有癫痫发作事件的行为癫痫发作评分（BSS）和时间（n=30只小鼠，每只小鼠都有一个独特的符号，每个点是离散的癫痫发作事件）。100%的小鼠在缺氧期间被抓住（蓝框;时间=−60分钟是颈动脉结扎的完成，时间=0是缺氧的开始）。13% 的人死于惊厥性癫痫发作后的缺氧（5-6 级）。这个数字是从Burnsed等人13修改而来的。 <a href="https://www.jove.com/files/ftp_upload/61346/61346fig1v2large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61346/61346fig2v2.jpg"> 图2:缺氧缺血期间的特征性脑电图（EEG）模式。 （A）脑电图背景从左到右:损伤前基线，缺氧期间爆发抑制，缺氧后抑制。从同侧顶叶皮层深度电极记录。（B）缺氧期间癫痫发作的演变。从同侧海马深度电极记录。阴影框（I-V）对应于（B）右侧扩展的脑电图摘录。这个数字是从Burnsed等人13修改而来的。 <a href="https://www.jove.com/files/ftp_upload/61346/61346fig2v2large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>
<img alt="Figure 3" class="bigfig" src="/files/ftp_upload/61346/61346fig03.jpg"> 图3:缺血和对侧半球之间脑电图背景的不对称性。 （A）HI小鼠缺氧（45分钟）缺血皮层（左）和对侧皮层（右;振幅刻度x10-6）期间的非对称功率谱图。突发抑制模式和缺血半球癫痫发作，CL半球抑制。（B）IL和CL半球缺氧和再氧合期间的背景抑制。将实验时间段（基线，结扎后30分钟，缺氧期间- 开始后15分钟和30分钟，再氧合后15分钟和60分钟 - 开始后15分钟和60分钟）从脑电图随机摘录中获取的所有平均电压测量值与基线进行比较。每只动物的基线作为自己的对照，数据报告为基线的百分比（n = 5只小鼠）。测量是从皮质电极进行的。这个数字是从Burnsed等人13修改而来的。<a href="https://www.jove.com/files/ftp_upload/61346/61346fig03large.jpg" target="_blank">请点击此处查看此图的放大版本。</a>

Watch this Scientific Journal Video about Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice at JoVE.com

Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

本手稿描述了一种在经历缺氧缺血的新生小鼠中使用多个深度电极进行连续视频脑电图记录的方法。

新生小鼠缺氧缺血期间的连续视频脑电图

Hypoxia ischemia is the most common cause of neonatal seizures. Animal models are crucial for understanding the mechanisms and physiology underlying ...

continuous-video-electroencephalogram-during-hypoxia-ischemia

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

3.3K 次观看.  University of Virginia. 本手稿描述了一种在经历缺氧缺血的新生小鼠中使用多个深度电极进行连续视频脑电图记录的方法。

视频: Continuous Video Electroencephalogram during Hypoxia-Ischemia in Neonatal Mice

Video-oculography in Mice

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the mouse, have a limited capacity to lock their eyes onto a target. In contrast to these target directed eye movements, compensatory ocular eye movements are easily elicited in afoveate animals1,2,3,4. Compensatory ocular movements are generated by processing vestibular and optokinetic information into a command signal that will drive the eye muscles. The processing of the vestibular and optokinetic information can be investigated separately and together, allowing the specification of a deficit in the oculomotor system. The oculomotor system can be tested by evoking an optokinetic reflex (OKR), vestibulo-ocular reflex (VOR) or a visually-enhanced vestibulo-ocular reflex (VVOR). The OKR is a reflex movement that compensates for "full-field" image movements on the retina, whereas the VOR is a reflex eye movement that compensates head movements. The VVOR is a reflex eye movement that uses both vestibular as well as optokinetic information to make the appropriate compensation. The cerebellum monitors and is able to adjust these compensatory eye movements. Therefore, oculography is a very powerful tool to investigate brain-behavior relationship under normal as well as under pathological conditions (f.e. of vestibular, ocular and/or cerebellar origin).
Testing the oculomotor system, as a behavioral paradigm, is interesting for several reasons. First, the oculomotor system is a well understood neural system5. Second, the oculomotor system is relative simple6; the amount of possible eye movement is limited by its ball-in-socket architecture ("single joint") and the three pairs of extra-ocular muscles7. Third, the behavioral output and sensory input can easily be measured, which makes this a highly accessible system for quantitative analysis8. Many behavioral tests lack this high level of quantitative power. And finally, both performance as well as plasticity of the oculomotor system can be tested, allowing research on learning and memory processes9.
Genetically modified mice are nowadays widely available and they form an important source for the exploration of brain functions at various levels10. In addition, they can be used as models to mimic human diseases. Applying oculography on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors under normal and pathological conditions. Here, we describe how to measure video-oculography in mice8.

Video-oculography is a very quantitative method to investigate ocular motor performance as well as motor learning. Here, we describe how to measure video-oculography in mice. Applying this technique on normal, pharmacologically-treated or genetically modified mice is a powerful research tool to explore the underlying physiology of motor behaviors.

视频oculography小鼠

Eye movements are very important in order to track an object or to stabilize an image on the retina during movement. Animals without a fovea, such as the ...

科研

神经科学

Neonatal Subventricular Zone Electroporation

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal cells1. Understanding the molecular pathways responsible for NSC self-renewal, commitment, and differentiation is critical for harnessing their unique potential to repair the brain and better understand central nervous system disorders. Previous methods for the manipulation of mammalian systems required the time consuming and expensive endeavor of genetic engineering at the whole animal level2. Thus, the vast majority of studies have explored the functions of NSC molecules in vitro or in invertebrates.
Here, we demonstrate the simple and rapid technique to manipulate neonatal NPCs that is referred to as neonatal subventricular zone (SVZ) electroporation. Similar techniques were developed a decade ago to study embryonic NSCs and have aided studies on cortical development3,4 . More recently this was applied to study the postnatal rodent forebrain5-7. This technique results in robust labeling of SVZ NSCs and their progeny. Thus, postnatal SVZ electroporation provides a cost and time effective alternative for mammalian NSC genetic engineering.

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem cells

新生儿脑室下区电穿孔

Neural stem cells (NSCs) line the postnatal lateral ventricles and give rise to multiple cell types which include neurons, astrocytes, and ependymal ...

Neonatal Pial Surface Electroporation

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and gliogenesis, including after injury. However, methods for genetically interrogating these progenitor populations and tracking their lineages had been limited owing to a lack of specificity or time consuming production of viruses. Thus, progress in this region has been relatively slow with only a handful of investigations of this location. Electroporation has been used for over a decade to study neural stem cell properties in the embryo, and more recently in the postnatal brain. Here we describe an efficient, rapid, and simple technique for the genetic manipulation of pial surface progenitors based on an adapted electroporation approach. Pial surface electroporation allows for facile genetic labeling and manipulation of these progenitors, thus representing a time-saving and economical approach for studying these cells.

The pial surface is a unique progenitor zone in the CNS that is receiving increasing attention. Herein, we detail a method for rapid genetic manipulation of this progenitor zone using a modified electroporation method. This procedure can be used for cellular and molecular investigations of cell lineages and signaling pathways involved in cell differentiation and to elucidate the fate and properties of daughter cells.

新生儿软脑膜表面电穿孔

Over the past several years the pial surface has been identified as a germinal niche of importance during embryonic, perinatal and adult neuro- and ...

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 ...

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 ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central nervous system. Changes in ENS function are a major cause of GI symptoms and disease and may contribute to GI symptoms reported in neuropsychiatric disorders including autism. It is well established that isolated colon segments generate spontaneous, rhythmic contractions known as Colonic Migrating Motor Complexes (CMMCs). A procedure to analyze the enteric neural regulation of CMMCs in ex vivo preparations of mouse colon is described. The colon is dissected from the animal and flushed to remove fecal content prior to being cannulated in an organ bath. Data is acquired via a video camera positioned above the organ bath and converted to high-resolution spatiotemporal maps via an in-house software package. Using this technique, baseline contractile patterns and pharmacological effects on ENS function in colon segments can be compared over 3-4 hr. In addition, propagation length and speed of CMMCs can be recorded as well as changes in gut diameter and contraction frequency. This technique is useful for characterizing gastrointestinal motility patterns in transgenic mouse models (and in other species including rat and guinea pig). In this way, pharmacologically induced changes in CMMCs are recorded in wild type mice and in the Neuroligin-3R451C mouse model of autism. Furthermore, this technique can be applied to other regions of the GI tract including the duodenum, jejunum and ileum and at different developmental ages in mice.

This article describes a video imaging technique and high-resolution spatiotemporal mapping to identify changes in the neural regulation of colonic motility in adult mice. Subtle effects on gastrointestinal (GI) function can be detected using this approach in isolated tissue preparations to advance our understanding of GI disease.

视频成像和时空地图来分析胃肠动力小鼠

The enteric nervous system (ENS) plays an important role in regulating gastrointestinal (GI) motility and can function independently of the central ...

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 ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Optogenetic Manipulation of Neural Circuits During Monitoring Sleep/wakefulness States in Mice

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 (ChR2), is expressed by a virus vector in a particular type of neuronal cells in various Cre-driver mice. Activation of these opsins is triggered by application of light pulses which are delivered by laser or LED through optic cables, and the effect of activation is observed with very high time resolution. Experimenters are able to acutely stimulate neurons while monitoring behavior or another physiological outcome in mice. Optogenetics can enable useful strategies to evaluate function of neuronal circuits in the regulation of sleep/wakefulness states in mice. Here we describe a technique for examining the effect of optogenetic manipulation of neurons with a specific chemical identity during electroencephalogram (EEG) and electromyogram (EMG) monitoring to evaluate the sleep stage of mice. As an example, we describe manipulation of GABAergic neurons in the bed nucleus of the stria terminalis (BNST). Acute optogenetic excitation of these neurons triggers a rapid transition to wakefulness when applied during NREM sleep. Optogenetic manipulation along with EEG/EMG recording can be applied to decipher the neuronal circuits that regulate sleep/wakefulness states.

Here, we describe methods of optogenetic manipulation of particular types of neurons during monitoring of sleep/wakefulness states in mice, presenting our recent work on the bed nucleus of the stria terminalis as an example.

监测小鼠睡眠/觉醒状态期间神经回路的光遗传学操作

In recent years, optogenetics has been widely used in many fields of neuroscientific research. In many cases, an opsin, such as channel rhodopsin 2 ...

Disruption of Frontal Lobe Neural Synchrony During Cognitive Control by Alcohol Intoxication

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance imaging (fMRI) studies indicates that the anterior cingulate (ACC) and the lateral prefrontal cortices (latPFC) are essential nodes subserving cognitive control. However, because of its limited temporal resolution, fMRI cannot accurately reflect the timing and nature of their presumed interplay. The present study combines distributed source modeling of the temporally precise magnetoencephalography (MEG) signal with structural MRI in the form of &#34;brain movies&#34; to: (1) estimate the cortical areas involved in cognitive control (&#34;where&#34;), (2) characterize their temporal sequence (&#34;when&#34;), and (3) quantify the oscillatory dynamics of their neural interactions in real time. Stroop interference was associated with greater event-related theta (4 - 7 Hz) power in the ACC during conflict detection followed by sustained sensitivity to cognitive demands in the ACC and latPFC during integration and response preparation. A phase-locking analysis revealed co-oscillatory interactions between these areas indicating their increased neural synchrony in theta band during conflict-inducing incongruous trials. These results confirm that theta oscillations are fundamental to long-range synchronization needed for integrating top-down influences during cognitive control. MEG reflects neural activity directly, which makes it suitable for pharmacological manipulations in contrast to fMRI that is sensitive to vasoactive confounds. In the present study, healthy social drinkers were given a moderate alcohol dose and placebo in a within-subject design. Acute intoxication attenuated theta power to Stroop conflict and dysregulated co-oscillations between the ACC and latPFC, confirming that alcohol is detrimental to neural synchrony subserving cognitive control. It interferes with goal-directed behavior that may result in deficient self-control, contributing to compulsive drinking. In sum, this method can provide insight into real-time interactions during cognitive processing and can characterize the selective sensitivity to pharmacological challenge across relevant neural networks.

This experiment uses an anatomically-constrained magnetoencephalography (aMEG) method to examine brain oscillatory dynamics and long-range functional synchrony during engagement of cognitive control as a function of acute alcohol intoxication.

酒精中毒在认知控制过程中对前小管神经同步的破坏

Decision making relies on dynamic interactions of distributed, primarily frontal brain regions. Extensive evidence from functional magnetic resonance ...