我们感谢张实验室的各位成员提供了激动人心的讨论, 以及站内实验室的成员提供技术援助。这项研究由中国国家自然科学基金、2012CB837700 (E.E.Z. 和霍山) 资助的 31500860 (到霍山) 的973个项目中的 m.o.s. T, 并由北京市政府提供资金支持。E.E.Z. 得到中国 "全球青年专家招募计划" 的支持。

本议定书的所有程序都是根据中国政府条例在北京国家生物科学研究所的机构动物保育和使用委员会 (IACUC) 的批准下进行的。
1. Cry1荧光记者的构建
注: 先前的昼夜节律研究使用生物发光系统2,12,13融合一个昼夜启动器与不稳定的荧光素酶 (dLuc), 以诱导有节奏的dLuc表达。为了开发荧光记者, 不稳定的荧光素酶取代了不稳定的萤光蛋白 (dVenus), 以诱导韵律dVenus表达。P (Cry1) d金星记者的世代被描述如下, 它的结构当前被递交到 Addgene 并且将很快是可利用的为学术用途。
<ol><li>放大功能小鼠Cry1启动子 (+ 328 到-1208 Cry1基因的 bp 区域, 转录起始点被指定为 "-1")。使用 pcr 酶 (点歌加新), 引物 F1 和 R1 (见表 2), 小鼠基因组作为模板和制造商的 PCR 协议, 以放大Cry1启动子 DNA 片段。</li>
	<li>放大Cry1基因的内含子 336, 这对于Cry114的昼夜节律功能是重要的。使用 pcr 酶, 引物 F2 和 R2, 小鼠基因组作为模板和制造商的 PCR 协议, 以放大内含物 336 DNA 片段。</li>
	<li>放大金星。以 pcr 酶、引物 F3 和 R3、pVENUS-N1 质粒为模板, 并利用制造商的 pcr 协议放大金星DNA 片段。</li>
	<li>放大 NLS-D2 DNA 片段。以 pcr 酶、引物 F4 和 R4、pcDNA3.3-d2eGFP 质粒为模板, 并利用制造商的 pcr 协议对 NLS-D2 DNA 片段进行放大。</li>
	<li>合成 DNA 片段1作为外显子1。</li>
	<li>将 dna 片段2合成为内含子336与金星dna 片段之间的链接器。</li>
	<li>用 mCherry 和 WPRE 酶切 pAAV-EF1a-double floxed-hChR2 (H134R)-HGHpA-MluI-EcoRI 载体质粒。使用凝胶萃取试剂盒净化较大的 DNA 片段。使用制造商的协议。</li>
	<li>使用吉布森装配组合及其标准协议, 组装所有的 DNA 片段一起生产 pAAV (Cry1)-intron336-Venus-NLS-D2-WPRE-HGHpA 记者。使用制造商的协议。</li>
</ol>2. 腺相关病毒的生产15
<ol><li>制备以下质粒 DNA 储存: 31.25 µg pRV1、pH21 31.25 µg、µg 125 pFdelta6 和µg-P (pAAV) Cry1 intron336-Venus-NLS 的 62.5 D2-WPRE-HGHpA。</li>
	<li>准备五15厘米293T 细胞的盘子, 等到细胞变成90% 汇合。</li>
	<li>染的质粒进入297T 细胞后转染试剂协议。对于每道菜, 使用6.25 µg 的 pRV1, 6.25 µg pH21, 25 µg pFdelta6, 12.5 µg 的 pAAV P (Cry1) intron336-Venus-NLS-D2-WPRE-HGHpA, 120 µL 的转染试剂。</li>
	<li>72 h 在转染后, 用吸管枪将每个盘子中的细胞分离出来;将细胞分成两个50毫升的试管。</li>
	<li>将细胞以 800 x g 为10分钟, 然后丢弃上清。将20毫升磷酸盐缓冲的生理盐水 (PBS) 放入每管中冲洗细胞;将细胞以 800 x g 为10分钟, 并丢弃上清。</li>
	<li>并用重悬颗粒在150毫米氯化钠和20毫米三溶液, pH 8.0;使用25毫升的溶液/管。在每管中加入1.25 毫升新鲜制备的10% 脱氧钠 (dH2O)。添加 benzonase 核酸酶到最终浓度为每毫升50单位, 并彻底混合。孵育37°c 为1小时。</li>
	<li>用 3000 x g 离心15分钟, 去除细胞碎片。通过0.45 微米注射器过滤器, 进一步确保所有碎片被去除;这一步骤将有助于防止肝素柱堵塞。</li>
	<li>用蠕动泵建立肝素柱;将流量设置为1毫升/分钟, 确保不会将气泡引入列中。</li>
	<li>平衡柱与10毫升的150毫米氯化钠, 20 毫米三溶液, pH 值8.0。将包含病毒的解决方案添加到每一列。用20毫升的100毫米氯化钠, 20 毫米三溶液, pH 8.0 来洗涤柱。然后, 使用5毫升注射器, 用1毫升的200毫米氯化钠和20毫米三溶液 pH 值8.0 来冲洗柱, 接着是1毫升氯化钠和300毫米三溶液, pH 20。</li>
	<li>使用5毫升注射器洗脱的病毒与3毫升的400毫米氯化钠, 20 毫米三溶液, pH 8.0, 其次是4毫升的一个450毫米氯化钠, 20 毫米三溶液, pH 8.0, 最后由 3 ml 的500毫米氯化钠, 20 毫米三溶液, pH 值8.0。收集提取的材料。</li>
	<li>对于重组腺相关病毒 (rAAV) 浓度, 加载提取的材料, 每次4毫升, 进入离心过滤器单位的10万分子量截止。在室温下离心每4毫升样品 2000 x 克, 以5分钟的温度, 丢弃流经。</li>
	<li>当所有提取的材料都经过离心过滤单元, 加入4毫升的 PBS 和离心机的混合物约 250 ul。去除 rAAVs (病毒滴度应该是大约 2-8 x 1012病毒微粒每毫升) 从离心过滤器单位, 整除并且存放在-80 °c 直到使用。在这个阶段, 病毒可以储存几个月。</li>
</ol>3. rAAV 注射和光纤插入成年小鼠 SCN
<ol><li>rAAVs 的昼夜荧光报告携带者注射入 SCN<ol>
<li>负载5µL 的 rAAV (病毒滴度是大约 2-8 x 1012病毒粒子每毫升) 成一个微型注射器。</li>
		<li>麻醉一只成年老鼠 (2-5 月大, C57BL/6 背景) 与戊巴比妥 (80 毫克/千克) 通过腹腔注射。检查是否有足够的麻醉深度, 因为没有脚趾捏的反应。</li>
		<li>使用剪刀从鼠标头上取出皮毛, 然后将鼠标装入立体定向设备。通过循环热水毯或另一种加热装置, 为麻醉动物提供热源。 注: 或者, 使用脱毛奶油或电剪剪去去除毛皮。</li>
		<li>用适当的消毒剂清洁手术部位。例如, 这可能包括无菌水或酒精和碘, betadine, 或稀释洗必泰在交替磨砂。重复至少3次。把手术部位悬垂起来。</li>
		<li>用剪刀在头皮上做个切口, 露出头骨。</li>
		<li>调整立体定向装置, 使 bregma 和 lambda 在同一水平平面上;将两个对称点 bregma 在同一水平平面上。</li>
		<li>用不育的棉签浸泡在4% 小时2o2/小时2o 腐蚀脑骨膜。然后使用干净, 不育的棉签清除和干燥头骨表面。</li>
		<li>使用微钻, 使一个孔约1毫米直径0.46 毫米后, 0.25 毫米侧向 bregma。</li>
		<li>
fi使用干净的棉花消毒拭子与生理盐水溶液清除骨碎片从暴露的表面的大脑。</li>
		<li>将微型注射器插入大脑, 确保微注射器的尖端是0.46 毫米后部和0.25 毫米侧向 bregma, 5.7 毫米深的头骨表面。</li>
		<li>注入 500 rAAV (50 nl 分钟-1) 进入 SCN, 留下微注射器到位10分钟后注射, 以确保完全扩散的 rAAV。</li>
		<li>慢慢地取出微注射器。</li>
	</ol>
</li>
	<li>将光纤插入 SCN 区域。<ol>
<li>使用光纤刀, 在长度上切割17毫米光纤, 确保光纤的每一侧都是平滑的。</li>
		<li>将光纤插入陶瓷插针中, 并将光纤固定在带有甲基乙基酮过氧化物 (AB 胶) 的陶瓷箍上, 确保光纤和陶瓷插针的表面是冲洗的。</li>
		<li>用冷消毒剂溶液或环氧乙烷清洗纤维。</li>
		<li>rAAV 注射 (步骤 3.1.11) 后, 将陶瓷插箍光纤插入 SCN。</li>
		<li>用牙科树脂将陶瓷插针和光纤固定在头骨上, 使其干燥。</li>
		<li>用后指甲油涂抹牙科树脂的表面。重复3.1.2 中概述的步骤 3.2.5, 省略实际注入 rAAV 控制。</li>
	</ol>
</li>
	<li>术后护理<ol>
<li>为小鼠提供术后镇痛药, 如丁丙诺啡2毫克/千克体重, 一天两次。</li>
		<li>把老鼠放在康复笼里, 当他们从麻醉中出来的时候。</li>
		<li>小鼠单独在12小时的光/暗条件下 (光强度约100莱克丝在笼子底部) 与食物/水可用的广告随意。在进一步试验前, 应分配八天时间, 以便能有足够的恢复期, 并确保最佳荧光报告的表达。</li>
	</ol>
</li>
</ol>4.体内荧光信号监测和数据采集
<ol><li>手术后八天, 用荧光信号监测器将鼠标头上的纤维连接起来(例如, 基因观察者)。检查控制鼠的 SCN 中的荧光信号, 建立背景信号。利用荧光信号监测仪对该信号进行观测和测量。</li>
	<li>用控制鼠标的相同操作对实验小鼠的荧光信号进行评价。排除已接收病毒载体但只显示分析背景信号的老鼠, 因为这可能表明光纤的尖端已经错过了 SCN。在这些初步措施之后, 把老鼠送回自己的笼子3周, 让荧光信号稳定下来。</li>
	<li>3周后, 用荧光信号监测器将小鼠连接起来。每10分钟测量荧光量为十五年代, 频率为100赫兹。在这些测量过程中, 老鼠可以自由地在笼子里移动, 食物/水可用的广告随意。使用100莱克丝的笼子底部的光强度, 并在光纤尖端的激光功率在 15-20 µW 之间。</li>
	<li>所有录音完成后, 灌注小鼠与4% 多聚甲醛, 并删除和切片的大脑检查放置的光纤。排除小鼠的分析, 如果光纤尖端没有正确植入的 SCN。</li>
</ol>5. 数据分析和介绍
<ol><li>测量荧光信号每10分钟为十五年代, 频率为100赫兹, 产生1500个数据点。这1500点的平均值在任何给定的时间点转换为荧光信号。随着时间的推移, 绘制多个平均值会产生一个信号-时间曲线, 它对应于自由移动小鼠的Cry1下的控制光条件下的转录节律。</li>
</ol>

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作者没有披露的利益冲突。

与前体方法相比, 如切片培养4,5, rt-pcr16,原位杂交17, 这要求动物被杀死,体内记录方法允许研究者们研究活着的动物的昼夜节律基因表达。因此, 这种技术提供了评估不同物理扰动 (如睡眠剥夺、压力、食物摄入等) 对神经昼夜节律的影响的能力。与体内生物发光7、8、10方法相比, 只能在恒定的暗条件下工作, 荧光在体内记录方法可以在光下应用。情况-技术的一个关键的好处作为光信号再夹带 SCN 昼夜节律时钟18。虽然这种方法可以检测到相对恒定、健壮的昼夜节律, 但应该提到, 记者技术并没有提供定量信息, 因此不能用于测量基因转录水平和翻译。
该协议有许多关键步骤。浓缩 rAAV 滴度应高达 2-8x 1012病毒粒子每毫升;较低的病毒滴度会发出暗淡甚至无法察觉的荧光信号。当将鼠标头固定到立体定向装置中时, 确保头骨水平, 以保证病毒注射和光纤植入的准确定位。注射的病毒应该慢慢做 (≤50-1), 以确保病毒仍然在 SCN。陶瓷插针和光纤应牢固固定在颅骨上, 牙科树脂不应与皮肤接触。如果陶瓷插针和光纤没有足够的固定, 它们会逐渐从鼠标头上分离出来。
这种技术可以推广到包括其他基因记者 (例如, Per2, Bmal1, c-fos, Pomc等) 通过改变启动子和相应的内含19。该技术通过反转两个反转液氧序列的 flag-inton336-Venus-NLS-D2 盒, 并将该报告器与一只小鼠线相结合, 可用于记录特定神经元的活动。例如, 将这种方法与 Vip 的小鼠线结合在一起, 可以记录Cry1转录节律在血管活性肠肽表达神经元的 SCN。此方法还可以使用 GCaMP6 作为 ca2 +指示器记录 ca2 +昼夜节律的大脑。

在哺乳动物中, 视交叉上核 (SCN) 支配着整个身体的昼夜节律, 以协调个体对外部环境变化的反应 (如光照、温度、压力等)1。核心时钟组件由Per1-3、 Cry1-2、时钟和Bmal1组成, 在调节每个细胞的昼夜节律时起着中枢作用。每个细胞在 SCN 包含转录激活剂, CLOCK/BMAL1, 作为一个 heterodimer, 以诱导表达的每和哭。每/哭复合, 然后抑制 CLOCK/BMAL1 的功能, 形成一个转录转换反馈回路, 约24小时完成2,3。
以往对 scn 的研究主要采用了前体4、5、6的体外营养切片培养方法, 虽然这种方法提供了有价值的信息, 但它的局限性抑制了我们获取有关其他脑核对 SCN 的影响的数据, 以及外源性刺激 (如光照) 对居住在这个关键区域的细胞的影响。在 2001年, 仁冈村的组是第一个使用生物发光系统在体内监测昼夜节律基因表达的 SCN 在自由移动小鼠7。肯-本间的小组在过去几年里进一步开发了生物荧光在体内记录系统在 SCN8,9,10。这些研究共同为研究人员提供了在恒定的黑暗或光脉冲后监测生物钟的能力。然而, 由于生物发光太暗, 无法在光/暗循环期间进行连续监测, 加上光是11昼夜节律性时钟所需的主要信号, 因此有不断增加的需求, 开发的实验方法, 克服了与生物发光记录相关的限制。本报告描述了一种基于荧光的系统, 用于监测自由移动小鼠体内的 SCN 生物钟。这种易于使用的方法允许在光/暗循环期间进行连续监测, 并允许在实时和高时间分辨率下长期观察 SCN 中生物钟基因的转录。

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			<td height="19" style="height:19px;width:627px;">KOD Plus Neo</td>
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			<td style="width:245px;">Reagent</td>
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			<td>addgene</td>
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			<td height="19" style="height:19px;">pcDNA3.3_d2eGFP</td>
			<td>addgene</td>
			<td>#26821</td>
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			<td height="20" style="height:21px;">pAAV-EF1a-double floxed-hChR2(H134R)-mCherry-WPRE-HGHpA</td>
			<td>addgene</td>
			<td>#20297</td>
			<td>Plasmid</td>
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			<td height="19" style="height:19px;">EcoRI</td>
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			<td>FD0274</td>
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			<td height="19" style="height:19px;">Gibson Assembly Mix</td>
			<td>NEB</td>
			<td>E2611s</td>
			<td>Reagent</td>
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			<td height="19" style="height:19px;">Lipofectamine 2000</td>
			<td>Thermo Scientific</td>
			<td>12566014</td>
			<td>Reagent</td>
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			<td height="19" style="height:19px;">Syringe Filter</td>
			<td>EMD Millipore</td>
			<td>SLHV033RS</td>
			<td>0.45&#160;&#181;m&#160;</td>
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			<td height="19" style="height:19px;">HiTrap heparin columns</td>
			<td>gelifesciences</td>
			<td>17-0406-01</td>
			<td>1 mL&#160;</td>
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			<td>EMD Millipore</td>
			<td>&#160;UFC810024</td>
			<td>100,000 MWCO</td>
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			<td height="19" style="height:19px;">Benzonase nuclease</td>
			<td>Sigma-Aldrich</td>
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			<td height="19" style="height:19px;">Sodium deoxycholate</td>
			<td>Sigma-Aldrich</td>
			<td>D5670</td>
			<td>Make fresh solution for each batch</td>
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			<td height="19" style="height:19px;">mouse stereotaxic apparatus</td>
			<td>B&#38;E TEKSYSTEMS LTD</td>
			<td>#SR-5M/6M</td>
			<td>Equipment</td>
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			<td>SigmaAldrich</td>
			<td>#1507002</td>
			<td>Reagent</td>
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			<td height="19" style="height:19px;">mouse stereotaxic apparatus</td>
			<td>B&#38;E TEKSYSTEMS LTD</td>
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			<td height="19" style="height:19px;">Hydrogen peroxide solution</td>
			<td>SigmaAldrich</td>
			<td>#216763</td>
			<td>Reagent</td>
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			<td height="19" style="height:19px;">Optical Fiber</td>
			<td>Thorlabs</td>
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			<td>0.39 NA, &#216;200 &#181;m</td>
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			<td height="19" style="height:19px;">microsyringe pump</td>
			<td>Nanoliter 2000 Injector, WPI</td>
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			<td>Equipment</td>
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			<td>Shanghai Fiblaser</td>
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			<td>230&#8201;&#956;m I.D., 2.5 mm O.D.</td>
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			<td>BiolinkOptics</td>
			<td></td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

in vivo monitoring of circadian clock gene expression in the mouse suprachiasmatic nucleus using fluorescence reporters

该技术将光纤介导荧光记录与重组腺相关病毒基因记者的精确交付结合在一起。这种新的和易于使用的体内荧光监测系统, 以记录的转录节律的时钟基因, Cry1, 在视交叉上核 (SCN) 的自由移动小鼠。为此, Cry1转录荧光报告器被设计和包装成腺相关病毒。纯化, 浓缩病毒注射到小鼠的 SCN, 然后插入一根光纤, 然后固定在大脑的表面上。这些动物被送回自己的笼子, 并允许1月的手术后恢复期, 以确保足够的记者表达。荧光然后记录在自由移动的小鼠通过体内监测系统, 在我们的机构建设。在体内记录系统中, 488 nm 激光与 1 x 4 光束分配器耦合, 将光分成四个同等功率的激光励磁输出。这个设置使我们能够同时记录四动物。每个发射的荧光信号都是通过光电倍增管和数据采集卡收集的。与先前的活体生物钟记录技术的生物发光相比, 这种荧光体内记录系统允许在光周期中记录生物钟基因的表达。

图 1A显示了Cry1的荧光记者设计。使用上述方法, 500 rAAV (Cry1) intron336-Venus-NLS-D2 成功地注入成年鼠的 SCN, 并表现出强健的金星表达 (图 1B, 1C)。在12小时/12 小时的光/暗 (LD) 和暗/暗 (DD) 条件下记录的荧光信号 (图 2) 在两种情况下产生了强健的昼夜节律。最后, 将荧光信号记录在长周期和短光照条件下 (图 3)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56765/56765fig1.jpg"> 图1。荧光报告器的设计及其在 SCN 中的表达 (A) Cry1的荧光记者设计。(B) 显微照片鼠脑显示8天后病毒注射后的表现。(C) 在 (B) 所示的显微照片上以红色包装的区域的放大倍数。<a href="/files/ftp_upload/56765/56765fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56765/56765fig2.jpg"> 图2。LD 和 DD 条件下的 SCN体内荧光记录 (A) 在 LD 和 DD 条件下, 在 SCN 的背景荧光显示没有可检测的节律。白色和灰色区域分别表示光和光的周期。(B) P (Cry1)-intron336-Venus-NLS-D2 的表达在7天的过程中, 在 12-12 LD 条件和7天的 DD 条件。(C) 分析 LD 情况下的昼夜节律 (24.4 小时);红线表示正弦曲线中的会聚拟合 (匹配的结果显示在左面板中)。(D) 分析 DD 条件下的昼夜节律 (~ 23.25 小时);红线表示正弦曲线中的会聚拟合 (匹配的结果显示在左面板中)。<a href="/files/ftp_upload/56765/56765fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56765/56765fig3.jpg"> 图3。在体内荧光记录在20-4 长和4-20 短光周期条件下的 SCN 荧光记录 P (Cry1)-intron336-Venus-NLS-D2 表达在7天在 12-12 LD 条件下, 其次是7天在20-4 长光周期条件下, 3 天在 DD 条件下, 3 天在 12-12 LD 情况下, 和然后在4-20 短光周期条件下7天。白色和灰色区域分别表示光照和光的周期。<a href="/files/ftp_upload/56765/56765fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
表1。协议所需的特定试剂(见材料表)
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>底漆名称</td>
			<td colspan="3">序列</td>
		</tr>
<tr>
<td>底漆 F1</td>
			<td colspan="3">cactaggggttcctgcggccgcACGCGTGTAAAGATGCACATGTG</td>
		</tr>
<tr>
<td>底漆 R1</td>
			<td colspan="3">TTGTAACCTTGATACTTACCTACTTAGATCGCAGATCTCGTCCGG</td>
		</tr>
<tr>
<td>底漆 F2</td>
			<td colspan="3">GTTATGACACAGTGTAGAAACTATGGCATAGGACAGATGACTGTG</td>
		</tr>
<tr>
<td>底漆 R2</td>
			<td colspan="3">CATGGTCTTTGTAGTCCATGGTGGGTACCtCTTGACAGCTCTACC</td>
		</tr>
<tr>
<td>底漆 F3</td>
			<td colspan="3">ctgtattttcagggcCCTGCAGGtGTGAGCAAGGGCGAGGAGCTG</td>
		</tr>
<tr>
<td>底漆 R3</td>
			<td colspan="3">TACCTTTCTCTTCTTTTTTGGAGGCTTGTACAGCTCGTCCATGCC</td>
		</tr>
<tr>
<td>底漆 F4</td>
			<td colspan="3">GCATGGACGAGCTGTACAAGCCTCCAAAAAAGAAGAGAAAGGTAG</td>
		</tr>
<tr>
<td>底漆 R4</td>
			<td colspan="3">tgatatcgaattcGGATCCCTACACATTGATCCTAGCAGAAGCAC</td>
		</tr>
</tbody></table>表2。协议中使用的底漆
表3。在协议中使用的 DNA 序列<a href="/files/ftp_upload/56765/Table3.xlsx">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters at JoVE.com

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

这种新开发的荧光技术能够长期监测自由运动小鼠视交叉上核 (SCN) 中昼夜节律基因的转录。

体内荧光记者对小鼠视交叉上细胞核生物钟基因表达的监测

This technique combines optical fiber mediated fluorescence recordings with the precise delivery of recombinant adeno-associated virus based gene ...

in-vivo-monitoring-circadian-clock-gene-expression-mouse

Nanyang Technological University

Research

JoVE Journal

Neuroscience

In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

8.2K 次观看.  Peking University. 这种新开发的荧光技术能够长期监测自由运动小鼠视交叉上核 (SCN) 中昼夜节律基因的转录。

视频: In Vivo Monitoring of Circadian Clock Gene Expression in the Mouse Suprachiasmatic Nucleus Using Fluorescence Reporters

Slice Preparation, Organotypic Tissue Culturing and Luciferase Recording of Clock Gene Activity in the Suprachiasmatic Nucleus

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the anterior hypothalamus. The clock is directly synchronized by light via the retina and optic nerve. Circadian oscillations are generated by interacting negative feedback loops of a number of so called "clock genes" and their protein products, including the Period (Per) genes. The core clock is also dependent on membrane depolarization, calcium and cAMP 1. The SCN shows daily oscillations in clock gene expression, metabolic activity and spontaneous electrical activity. Remarkably, this endogenous cyclic activity persists in adult tissue slices of the SCN 2-4. In this way, the biological clock can easily be studied in vitro, allowing molecular, electrophysiological and metabolic investigations of the pacemaker function.
The SCN is a small, well-defined bilateral structure located right above the optic chiasm 5. In the rat it contains ~8.000 neurons in each nucleus and has dimensions of approximately 947 &mu;m (length, rostrocaudal axis) x 424 &mu;m (width) x 390 &mu;m (height) 6. To dissect out the SCN it is necessary to cut a brain slice at the specific level of the brain where the SCN can be identified. Here, we describe the dissecting and slicing procedure of the SCN, which is similar for mouse and rat brains. Further, we show how to culture the dissected tissue organotypically on a membrane 7, a technique developed for SCN tissue culture by Yamazaki et al. 8. Finally, we demonstrate how transgenic tissue can be used for measuring expression of clock genes/proteins using dynamic luciferase reporter technology, a method that originally was used for circadian measurements by Geusz et al. 9. We here use SCN tissues from the transgenic knock-in PERIOD2::LUCIFERASE mice produced by Yoo et al. 10. The mice contain a fusion protein of PERIOD (PER) 2 and the firefly enzyme LUCIFERASE. When PER2 is translated in the presence of the substrate for luciferase, i.e. luciferin, the PER2 expression can be monitored as bioluminescence when luciferase catalyzes the oxidation of luciferin. The number of emitted photons positively correlates to the amount of produced PER2 protein, and the bioluminescence rhythms match the PER2 protein rhythm in vivo 10. In this way the cyclic variation in PER2 expression can be continuously monitored real time during many days. The protocol we follow for tissue culturing and real-time bioluminescence recording has been thoroughly described by Yamazaki and Takahashi 11.

The procedure of preparing slices containing the adult mouse hypothalamic suprachiasmatic nucleus (SCN), and a rapid way to culture the SCN tissue in organotypic culture condition, are reported. Further, the measurement of oscillatory clock gene protein expression using dynamic luciferase reporter technology is described.

切片的制备，组织器官型培养，并记录在视交叉上核时钟基因活性的荧光素酶

A central circadian (~24 hr) clock coordinating daily rhythms in physiology and behavior resides in the suprachiasmatic nucleus (SCN) located in the ...

科研

神经科学

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

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

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

脊髓电二:外吸电极的制作

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

High-resolution Live Imaging of Cell Behavior in the Developing Neuroepithelium

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central nervous system (CNS). Although much is known about the molecular mechanisms that pattern the spinal cord and elicit neuronal differentiation1, 2, we lack a deep understanding of these early events at the level of cell behavior. It is thus critical to study the behavior of neural progenitors in real time as they undergo neurogenesis.
In the past, real-time imaging of early embryonic tissue has been limited by cell/tissue viability in culture as well as the phototoxic effects of fluorescent imaging. Here we present a novel assay for imaging such tissue for long periods of time, utilizing a novel ex vivo slice culture protocol and wide-field fluorescence microscopy (Fig. 1). This approach achieves long-term time-lapse monitoring of chick embryonic spinal cord progenitor cells with high spatial and temporal resolution.
This assay may be modified to image a range of embryonic tissues3, 4 In addition to the observation of cellular and sub-cellular behaviors, the development of novel and highly sensitive reporters for gene activity (for example, Notch signaling5) makes this assay a powerful tool with which to understand how signaling regulates cell behavior during embryonic development.

Imaging embryonic tissue in real-time is challenging over long periods of time. Here we present an assay for monitoring cellular and sub-cellular changes in chick spinal cord for long periods with high spatial and temporal resolution. This technique can be adapted for other regions of the nervous system and developing embryo.

高分辨率实时成像发展中的神经上皮细胞的行为

The embryonic spinal cord consists of cycling neural progenitor cells that give rise to a large percentage of the neuronal and glial cells of the central ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

使用超声波血脑屏障破坏和锰增强MRI的脑功能成像

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

Manipulation of gene expression in tissues is required to perform functional studies. In this paper, we demonstrate the cerebroventricular microinjection (CVMI) technique as a means to modulate gene expression in the adult zebrafish brain. By using CVMI, substances can be administered into the cerebroventricular fluid and be thoroughly distributed along the rostrocaudal axis of the brain. We particularly focus on the use of antisense morpholino oligonucleotides, which are potent tools for knocking down gene expression in vivo. In our method, when applied, morpholino molecules are taken up by the cells lining the ventricular surface. These cells include the radial glial cells, which act as neurogenic progenitors. Therefore, knocking down gene expression in the radial glial cells is of utmost importance to analyze the widespread neurogenesis response in zebrafish, and also would provide insight into how vertebrates could sustain adult neurogenesis response. Such an understanding would also help the efforts for clinical applications in human neurodegenerative disorders and central nervous system regeneration. Thus, we present the cerebroventricular microinjection method as a quick and efficient way to alter gene expression and neurogenesis response in the adult zebrafish forebrain. We also provide troubleshooting tips and other useful information on how to carry out the CVMI procedure.

In this article, we demonstrate a method for manipulation of gene expression in the ventricular cells of the adult zebrafish telencephalon using antisense morpholino oligonucleotides. We present this method as an efficient and quick protocol that can be used for functional studies in the adult vertebrate brain.

显微吗啉代寡核苷酸脑室微量注射成年斑马鱼大脑中的基因表达

Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Analysis of Gene Expression Changes in the Rat Hippocampus After Deep Brain Stimulation of the Anterior Thalamic Nucleus

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective treatment for several movement disorders that have failed to respond to medication. Recent progress in the field of DBS surgery has begun to extend the application of this surgical technique to other conditions as diverse as morbid obesity, depression and obsessive compulsive disorder. Despite these expanding indications, little is known about the underlying physiological mechanisms that facilitate the beneficial effects of DBS surgery. One approach to this question is to perform gene expression analysis in neurons that receive the electrical stimulation. Previous studies have shown that neurogenesis in the rat dentate gyrus is elicited in DBS targeting of the anterior nucleus of the thalamus1. DBS surgery targeting the ATN is used widely for treatment refractory epilepsy. It is thus of much interest for us to explore the transcriptional changes induced by electrically stimulating the ATN. In this manuscript, we describe our methodologies for stereotactically-guided DBS surgery targeting the ATN in adult male Wistar rats. We also discuss the subsequent steps for tissue dissection, RNA isolation, cDNA preparation and quantitative RT-PCR for measuring gene expression changes. This method could be applied and modified for stimulating the basal ganglia and other regions of the brain commonly clinically targeted. The gene expression study described here assumes a candidate target gene approach for discovering molecular players that could be directing the mechanism for DBS.

The mechanism underlying the therapeutic effects of Deep Brain Stimulation (DBS) surgery needs investigation. The methods presented in this manuscript describe an experimental approach to examine the cellular events triggered by DBS by analyzing the gene expression profile of candidate genes that can facilitate neurogenesis post DBS surgery.

基因表达的变化大鼠海马后，前丘脑核脑深部刺激分析

Deep brain stimulation (DBS) surgery, targeting various regions of the brain such as the basal ganglia, thalamus, and subthalamic regions, is an effective ...