我们感谢N.萨维特尔（辛辛那提儿童医院医学中心，辛辛那提，俄亥俄州，美国），S. Efstathiou（英国剑桥的大学）和詹姆斯·希尔（路易斯安那州立大学健康科学中心，新奥尔良，美国）提供的样品从HSV-1感染的小鼠和兔子分别和试剂; H.增本（上总DNA研究所，千叶县，日本）和S. Khochbin（阿尔贝研究所Bonniot，格勒诺布尔，法国）的有益讨论。 这项工作是由该中心法国国家科学研究（CNRS）（ATIP程序，PL，http://www.cnrs.fr）资助，法国国家研究署（ANR）（ANR-05-MIIM- 008-01，CENTROLAT， <a href="http://www.agencenationale-recherche.fr">http://www.agencenationale-recherche.fr</a> ），该FINOVI基金会（ <a href="http://www.finovi.org/:fr:start">http://www.finovi.org/:fr:start</a> ），该LabEX DEVweCAN（ANR-10的LabX-61大学德里昂），在程序中“InvestissemenTS D&#39;艾文莉“（ANR-11-IDEX-0007）的ANR操作（ <a href="http://www.agence-nationale-recherche.fr">http://www.agence-nationale-recherche.fr</a> ），L&#39;协会倒拉RECHERCHE CONTRE乐巨蟹座（ARC-7979和ARC- 4910， <a href="http://www.arc-cancer.net">http://www.arc-cancer.net</a> ），LA法甲驳国立癌症勒（LNCC， <a href="http://www.ligue-cancer.net">http://www.ligue-cancer.net</a> ），和印加（EPIPRO程序， <a href="http://www.e-cancer.fr">http://www.e - cancer.fr</a> ），FC和PL是法国国家科学研究中心的研究人员。

作者宣称没有竞争的财务权益。

这里所描述的协议允许的小鼠神经组织切片的神经元内HSV-1潜伏基因组的检测。我们的调节病毒基因表达的途径的理解已经由缺乏方法来检测HSV-1基因组DNA 的原位神经组织内的限制。基因组拷贝数和被感染的神经元比例的信息主要来自PCR分析对分离的神经元11,12。在阐明作用的宿主细胞的核架构对HSV-1的延迟，我们设置确定潜伏的HSV-1基因组的国产化通过DNA-FISH，潜伏感染的小鼠神经?...

<html>单纯疱疹病毒1型（HSV-1）是一种持久性人神经病毒，在周围神经系统，从它周期性地重新激活的复制和蔓延的三叉神经节（TG）的神经元建立长期潜伏感染。在HSV-1基因组是一个150 kb的双链DNA本地化主机神经元那里仍然是多拷贝chromatinized质粒，不整合到宿主细胞基因组1,2的核心。期间延时，HSV-1复制周期的遗传程序，强烈抑制，基因表达被限制在潜伏相关转录物（LAT）基因，从延迟建立到活化3的起始。土地增值税产生加工成一个主要的2 kb的稳定套索长8.5 kb的非编码RNA，和几个miRNA的4-7。因此，HSV-1的延迟为特征的病毒基因组DNA，RNA LAT，以及没有可检测的复制周期蛋白的存在。 ontent“&gt;动物模型，主要是鼠和兔，是实验模型扼要的人为延迟的几个特点，其中之一这些模型的主要利益是他们让学习HSV-1潜伏在免疫活性宿主的生理方面的问题。在过去的几十年许多实验性的工具，如遗传修饰的病毒和小鼠中，已经开发研究生理学，遗传学和HSV-1的延迟，从动物组织中的细胞生物学。到现在为止，病毒基因组DNA的检测和定量通过Southern印迹和从定量PCR分离甘油三酯，然而，目前还没有检测HSV-1基因组原位杂交组织切片8，因此，延迟是经常通过土地增值税的RNA的检测采用原位杂交的RNA，而不是评估的组织切片方法可用病毒基因组的检测，因为它已经不可能表征感染细胞中基于病毒的基因组的存在下，噻的技术限制是一个主要的缺点的宿主-病毒相互作用，例如病毒基因组和细胞和病毒基因的表达，或在宿主细胞介导的免疫应答9,10之间的关系的许多方面进行分析。 最重要的是，在潜伏感染的细胞-细胞间的异质性保持相对未开发的，并已被证明是延迟的一个重要特征在小鼠和在植入到SCID小鼠中11-17人体感觉神经节的神经元。通常情况下，它表明通过qPCR即每个细胞的HSV-1的基因组拷贝数变化从5到几百。尽管土地增值税表现为潜伏期和活化的关键调节因子，对离体神经元和原位 PCR定量PCR的数据显示，潜伏感染神经细胞的一个子集，低至30％，体现了土地增值税轨迹11,12,18-21。如何在宿主细胞内，并在病毒潜伏期成立组织影响的细胞环境和病毒基因表达仍不清楚。这里我们描述了原位杂交（FISH）方法用于有效地检测的动物的神经组织切片内的低拷贝的HSV-1基因组DNA的荧光健壮。这种方法已被设计和使用我们进入到高分辨率显微成像也就是要研究的病毒基因组的相互作用与宿主细胞内的核部件22。此外，我们描述了用于同时检测病毒DNA与RNA和蛋白质，这是一个独特的工具来描述，调节病毒基因表达的病毒 - 宿主相互作用的倍数染色方法。该方法也可以应用于范围广泛的要求的检测HSV-1的潜伏基因组中，如在大量切片的量化被感染的神经元的分析。一个关键的步骤是应用抗原修复治疗，使病毒DNA访问杂交。因此，该协议也可能是有效的，以检测其他双链DNA病毒，这是目前无法检测动物组织中传统的DNA-FISH方法。

<table border="0" cellpadding="0" cellspacing="0" width="822">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:203px;">Balb/c mice</td>
			<td style="width:189px;">Janvier, France</td>
			<td style="width:93px;"> </td>
			<td style="width:337px;">6 week-old females</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;">HSV-1 strains</td>
			<td>SC16 strain (wild type)</td>
			<td></td>
			<td style="width:337px;">See Labetoule, M. et al. (2003) Invest Ophthalmol Vis Sci 44: 217&#8211;225, for details on HSV-1 strain and virus stock preparation.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ketamine hydrochloride</td>
			<td>Sigma</td>
			<td>K2753</td>
			<td rowspan="2" style="width:337px;">Intraperitoneal injection of a solution containing Ketamine (100mg/kg) and Xylazine (10mg/kg)</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Xylazine hydrochloride</td>
			<td>Sigma</td>
			<td>X1251</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Paraformaldehyde (PFA)</td>
			<td>Sigma</td>
			<td>158127</td>
			<td style="width:337px;">Suspend 4g of PFA in 90mL of water. Add 50&#181;L of 1N NaOH, and heat at 60&#176;C in a water bath with agitation. PFA dissolves in about 30min. Add 10mL of 10X PBS. This solution can be prepared in advance and stored at -20 &#176;C in 5mL tubes. Caution. Manipulate under a fume hood.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Physiological Saline</td>
			<td>Sigma</td>
			<td colspan="2">07982-100TAB-F</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">1X PBS, pH 7.4 (sterile)</td>
			<td>Life Technologies</td>
			<td>10010-015</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Sucrose</td>
			<td>Sigma</td>
			<td>84100</td>
			<td>Prepare a 20% sucrose solution in 1X PBS.</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Cryosectionning embedding medium - Tissue-Tek OCT Compound -</td>
			<td>SAKURA</td>
			<td>4583</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Large vector DNA purification kit</td>
			<td>Qiagen</td>
			<td>12462</td>
			<td style="width:337px;">To purify Cosmid or BAC vector containing HSV-1 genome and store at -20&#176;C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Nick translation kit</td>
			<td>Roche Applied Sciences</td>
			<td>10 976 776 001</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cy3-dCTP</td>
			<td>GE Healthcare</td>
			<td>PA53021</td>
			<td>Protect from light</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">0.5M EDTA</td>
			<td>Sigma</td>
			<td>E6758</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">G50 Mini spin column</td>
			<td>GE Healthcare</td>
			<td>27-5330-01 </td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Salmon sperm DNA 10mg/mL</td>
			<td>Invitrogen / Life Technologies</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ethanol molecular biology grade</td>
			<td>Sigma</td>
			<td>87047</td>
			<td>Prepare a 70% solution</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Salmon sperm DNA</td>
			<td>Invitrogen / Life Technologies</td>
			<td>15632-011</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Formamid Molecular biology grade</td>
			<td>Sigma</td>
			<td>F9037</td>
			<td>Caution. Manipulate under fume hood.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">HSV-1 biotinylated commercial probe </td>
			<td>Enzo Life Sciences</td>
			<td>ENZ-40838</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">ImmEdge hydrophobic pen</td>
			<td>Vector Laboratories</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">20X Saline Sodium Citrate (SSC)</td>
			<td>Sigma</td>
			<td>S6639</td>
			<td>Prepare a 2X SSC solution in ddH20.</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
			<td style="width:337px;">Prepare a 10% stock solution in water and store at +4&#176;C. Prepare the 0.5% solution in 1X PBS right before use.</td>
		</tr>
		<tr height="102">
			<td height="102" style="height:102px;">10mM sodium citrate pH 6.0</td>
			<td>Sigma</td>
			<td>S1804</td>
			<td style="width:337px;">Prepare a 100mM stock solution (10X). Weigh 10,5g of citric acid (MW 210.14. Caution, irritant and toxic, wear appropriate mask and gloves), and dissolve in 400mL water. Adjust pH at 6.0 with 1N NaOH (caution, irritant, wear gloves). Adjust to 500mL with distilled water. Dilute 10 times in distilled water before use.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Acetic Acid</td>
			<td>Sigma</td>
			<td>320099</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Methanol, molecular biology grade</td>
			<td>Sigma</td>
			<td>322415</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dextran sulfate - MW 500 000</td>
			<td>Euromedex</td>
			<td>EU0606-A</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Denhardt&#39;s solution (100X)</td>
			<td>Euromedex</td>
			<td>1020-A</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Rubber Cement &#34;FixoGum&#34;</td>
			<td>Marabut</td>
			<td>290110000</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;">DNA purification kit - Qiaquick PCR purification kit -</td>
			<td>Qiagen</td>
			<td>28104</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">T7 in vitro transcription kit</td>
			<td>Ambion / Life Technologies</td>
			<td>AM1314</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Biotin-16-UTP</td>
			<td>Roche Applied Sciences</td>
			<td>11388908910</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">RNA purification mini-column</td>
			<td>Qiagen</td>
			<td>73404</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Ribonucleoside Vanadyl Complex</td>
			<td>New England Biolabs</td>
			<td>S1402S</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">H2O2</td>
			<td>Sigma</td>
			<td>H3410</td>
			<td style="width:337px;">Prepare a 3% solution in distilled water. Store at +4 &#176;C and protect from light.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401011</td>
			<td style="width:337px;">prepare a 10mg/mL solution in RNAse free water</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Normal Goat Serum</td>
			<td>Invitrogen</td>
			<td>PCN5000</td>
			<td></td>
		</tr>
		<tr height="68">
			<td height="68" style="height:68px;">Primary antibodies</td>
			<td>Any supplier</td>
			<td></td>
			<td style="width:337px;">The following primary antibodies were used in the result section: anti-mouse CENP-A (rabbit mAb C51A7, Cell Signaling Technologies), and anti-ATRX H-300 (Santa Cruz Biotechnology)</td>
		</tr>
		<tr height="85">
			<td height="85" style="height:85px;">Secondary fluorescent antibodies</td>
			<td>Invitrogen / Life Technologies</td>
			<td></td>
			<td style="width:337px;">The fluorescent secondary antibodies routinely used in our protocol are AlexaFluor labeled goat antibodies (IgG H+L). The antibody used in the result section is an anti-rabbit goat antibody labaled with AlexaFluor 488 (reference A11001)</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Tyramide Signal Amplification (TSA) kit - Streptavidin + AlexaFluor 350 (blue fluorescence)</td>
			<td>Invitrogen / Life Technologies</td>
			<td>#T20937</td>
			<td rowspan="2" style="width:337px;">TSA kits are also available from Perkin Elmer</td>
		</tr>
		<tr height="51">
			<td height="51" style="height:51px;width:203px;">Tyramide Signal Amplification (TSA) kit - Streptavidin + AlexaFluor 488 (green fluorescence)</td>
			<td>Invitrogen / Life Technologies</td>
			<td>#T20932</td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;">Hoechst 33342</td>
			<td>Invitrogen / Life Technologies</td>
			<td>H3570</td>
			<td style="width:337px;">Prepare a 0.5&#181;g/mL solution in 1X PBS immediatly before use. Discard the remaining solution.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">22x50mm coverslip. n&#176;1.5 glass.</td>
			<td>Electron Microscopy Sciences</td>
			<td>72204-04</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:203px;"> Mounting medium with anti-fading agent - Vectashield -</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td style="width:337px;">Another conventional product is Fluoromount G from electron microscopy Science</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Superfrost glass slides</td>
			<td>FisherScientific</td>
			<td>12-550-15</td>
			<td style="width:337px;"> </td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;"> </td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EQUIPMENT</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="18">
			<td height="18" style="height:18px;">Equipment / material</td>
			<td>Company</td>
			<td>Reference</td>
			<td>Note</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Needle for infection</td>
			<td></td>
			<td></td>
			<td>Glass micropipette hot drawn. Home made.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dissection equipement</td>
			<td>Moria, France</td>
			<td></td>
			<td>Microsurgical scissors and forceps</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Peristaltic pump</td>
			<td style="width:189px;">Cole Palmer Instruments</td>
			<td></td>
			<td>Easyload Masterflex</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Micro-syringe pump device (Nano Pump)</td>
			<td>kdScientific</td>
			<td>KDS310</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Cryostat</td>
			<td>Leica France</td>
			<td>CM 1510-1</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">-80 &#176;C freezer</td>
			<td>Sanyo</td>
			<td></td>
			<td>Ultra Low -80&#176;C</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Domestic microwave oven</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Dry block heater</td>
			<td>Eppendorf</td>
			<td>022670204</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Incubator Slide moat</td>
			<td>Boekel Scientific</td>
			<td>240000</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Coplin Jar</td>
			<td>Dominique Dutscher</td>
			<td>68512</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">Staining glass container</td>
			<td>Dominique Dutscher</td>
			<td>68506</td>
			<td></td>
		</tr>
		<tr height="136">
			<td height="136" style="height:136px;">Fluorescent microscope</td>
			<td>Zeiss</td>
			<td></td>
			<td style="width:337px;">The images presented in the result section were collected with a Zeiss AxioObserver with objective x40 LD NeoFluor N.A 0.6, and x100 PlanApochromat N.A 1.3. Filter set #38, #43 and #43. HXP 120 fluorescence light source. Photometrics CoolSNAP HQ2 CCD camera. Signal will be more easily observed on a recent high efficiency microscope such as Zeiss AxioImager/AxioObserver series, Nikon Ti-E/Ni-E series or Leica DM/DMI6000 series</td>
		</tr>
	</tbody>
</table>

detection of the genome and transcripts of a persistent dna virus in neuronal tissues by fluorescent in situ hybridization combined with immunostaining

的基因的单细胞codetection，其RNA产物和细胞调节蛋白是关键的，研究基因表达的调控。这是在病毒学领域的挑战，特别对于核复制的持久性DNA病毒，涉及动物模型的研究。单纯疱疹病毒1型（HSV-1）建立在神经元末梢终身潜伏感染。潜隐病毒作为水库，从它重新激活并诱导新的疱疹发作。的HSV-1的等待时间的细胞生物学仍知之甚少，部分原因是由于缺乏方法来检测HSV-1基因组原位的动物模型。我们描述了DNA的荧光原位杂交（FISH）方法有效地检测来自被感染的动物模型神经组织切片，在低拷贝病毒的基因组。该方法依赖于基于热的抗原修复，和直接标记的自制的DNA探针，或可商购的探针。我们开发了一个三重的领带宁方法，结合DNA-FISH与RNA-FISH和免疫荧光，使用基于过氧化物酶信号放大，以适应每个染色要求。一个主要的改进是获得，在10μm的组织切片，低背景信号，可以在高分辨率的共聚焦显微镜和宽视场常规落射荧光成像的能力。此外，三重染色加工具有广泛的针对细胞和病毒蛋白质的抗体。完整的协议需要2.5天，适应组织内抗体和探针的渗透。

经过几个月的广泛测试，我们发现，热基化学揭露原位杂交技术制成潜伏的HSV-1基因组可用于荧光。在这个过程中，我们尝试了各种去掩蔽过程，并仅基于热的处理（ 即加热部分到子沸点温度在微波炉中）出现的效率。然后，我们测试所使用的常规免疫组织化学（IHC）和电子显微镜来检索表位31,32，包括0.01M的pH值为6.0柠檬酸缓冲液（我们在所有的研究中使用），1×PBS中，1mM?...

Watch this Scientific Journal Video about Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining at JoV

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

我们原位杂交协议建立一个荧光供于动物模型的组织切片中的持久DNA病毒基因组的检测。这个协议允许研究感染过程由codetection病毒基因组，它的RNA产物，以及单细胞内的病毒或细胞蛋白。

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

Single cell codetection of a gene, its RNA product and cellular regulatory proteins is critical to study gene expression regulation. This is a challenge ...

detection-genome-transcripts-persistent-dna-virus-neuronal-tissues

Research

JoVE Journal

Neuroscience

Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

18.2K 次观看.  CNRS UMR 5534. 我们原位杂交协议建立一个荧光供于动物模型的组织切片中的持久DNA病毒基因组的检测。这个协议允许研究感染过程由codetection病毒基因组，它的RNA产物，以及单细胞内的病毒或细胞蛋白。 

视频: Detection of the Genome and Transcripts of a Persistent DNA Virus in Neuronal Tissues by Fluorescent In situ Hybridization Combined with Immunostaining

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

Fast Micro-iontophoresis of Glutamate and GABA: A Useful Tool to Investigate Synaptic Integration

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of the dendritic tree, which eventually leads to neuronal output of action potentials at the axon. The influence of diverse spatial and temporal parameters of specific synaptic input on neuronal output is currently under investigation, e.g. the distance-dependent attenuation of dendritic inputs, the location-dependent interaction of spatially segregated inputs, the influence of GABAergig inhibition on excitatory integration, linear and non-linear integration modes, and many more.
With fast micro-iontophoresis of glutamate and GABA it is possible to precisely investigate the spatial and temporal integration of glutamatergic excitation and GABAergic inhibition. Critical technical requirements are either a triggered fluorescent lamp, light-emitting diode (LED), or a two-photon scanning microscope to visualize dendritic branches without introducing significant photo-damage of the tissue. Furthermore, it is very important to have a micro-iontophoresis amplifier that allows for fast capacitance compensation of high resistance pipettes. Another crucial point is that no transmitter is involuntarily released by the pipette during the experiment.
Once established, this technique will give reliable and reproducible signals with a high neurotransmitter and location specificity. Compared to glutamate and GABA uncaging, fast iontophoresis allows using both transmitters at the same time but at very distant locations without limitation to the field of view. There are also advantages compared to focal electrical stimulation of axons: with micro-iontophoresis the location of the input site is definitely known and it is sure that only the neurotransmitter of interest is released. However it has to be considered that with micro-iontophoresis only the postsynapse is activated and presynaptic aspects of neurotransmitter release are not resolved. In this article we demonstrate how to set up micro-iontophoresis in brain slice experiments.

In this article we introduce fast micro-iontophoresis of neurotransmitters as a technique to investigate integration of postsynaptic signals with high spatial and temporal precision.

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

One of the fundamental interests in neuroscience is to understand the integration of excitatory and inhibitory inputs along the very complex structure of ...

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be stimulated by neuronally-released transmitter. However, the ability of astrocytic receptors to exhibit plasticity in response to changes in neuronal activity has received little attention. Here we describe a model system that can be used to globally scale up or down astrocytic group I metabotropic glutamate receptors (mGluRs) in acute brain slices. Included are methods on how to prepare parasagittal hippocampal slices, construct chambers suitable for long-term slice incubation, bidirectionally manipulate neuronal action potential frequency, load astrocytes and astrocyte processes with fluorescent Ca2+ indicator, and measure changes in astrocytic Gq GPCR activity by recording spontaneous and evoked astrocyte Ca2+ events using confocal microscopy. In essence, a &#8220;calcium roadmap&#8221; is provided for how to measure plasticity of astrocytic Gq GPCRs. Applications of the technique for study of astrocytes are discussed. Having an understanding of how astrocytic receptor signaling is affected by changes in neuronal activity has important implications for both normal synaptic function as well as processes underlying neurological disorders and neurodegenerative disease.

Here we describe an adaptation of protocols used to induce homeostatic plasticity in neurons for the study of plasticity of astrocytic G protein-coupled receptors. Recently used to examine changes in astrocytic group I mGluRs in juvenile mice, the method can be applied to measure scaling of various astrocytic GPCRs, in tissue from adult mice in situ and in vivo, and to gain a better appreciation of the sensitivity of astrocytic receptors to changes in neuronal activity.

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

Close to two decades of research has established that astrocytes in situ and in vivo express numerous G protein-coupled receptors (GPCRs) that can be ...

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 ...

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for maintenance, repair or neurodegeneration in postdevelopmental periods. High throughput analyses have recently revealed that axons have a more dynamic and complex transcriptome than previously expected. These analysis, however have been mostly done in cultured neurons where axons can be isolated from the somato-dendritic compartments. It is virtually impossible to achieve such isolation in whole tissues in vivo. Thus, in order to verify the recruitment of mRNAs and their functional relevance in a whole animal, transcriptome analyses should ideally be combined with techniques that allow the visualization of mRNAs in situ. Recently, novel ISH technologies that detect RNAs at a single-molecule level have been developed. This is especially important when analyzing the subcellular localization of mRNA, since localized RNAs are typically found at low levels. Here we describe two protocols for the detection of axonally-localized mRNAs using a novel ultrasensitive RNA ISH technology. We have combined RNAscope ISH with axonal counterstain using fluorescence immunohistochemistry or histological dyes to verify the recruitment of Atf4 mRNA to axons in vivo in the mature mouse and human brains.

Detection of Axonally Localized mRNAs in Brain Sections Using High-Resolution In Situ Hybridization

RNA in situ hybridization (ISH) enables the visualization of RNAs in cells and tissues. Here we show how combination of RNAscope ISH with immunohistochemistry or histological dyes can be successfully used to detect mRNAs localized to axons in sections of mouse and human brains.

在脑切片检测轴突本地化mRNAs的高空间分辨率<em&gt;原位</em&gt;杂交

mRNAs are frequently localized to vertebrate axons and their local translation is required for axon pathfinding or branching during development and for ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Extracellular Recording of Neuronal Activity Combined with Microiontophoretic Application of Neuroactive Substances in Awake Mice

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and awake animals. Therefore, methods allowing the manipulation of synaptic systems in awake animals are required in order to determine the contribution of synaptic inputs to neuronal processing unaffected by anesthetics. Here, we present methodology for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. By combining these procedures, we performed microiontophoretic injections of gabazine to selectively block GABAA receptors in neurons of the inferior colliculus of head-restrained mice. Gabazine successfully modified neural response properties such as the frequency response area and stimulus-specific adaptation. Thus, we demonstrate that our methods are suitable for recording single-unit activity and for dissecting the role of specific neurotransmitter receptors in auditory processing.
The main limitation of the described procedure is the relatively short recording time (~3 hr), which is determined by the level of habituation of the animal to the recording sessions. On the other hand, multiple recording sessions can be performed in the same animal. The advantage of this technique over other experimental procedures used to manipulate the level of neurotransmission or neuromodulation (such as systemic injections or the use of optogenetic models), is that the drug effect is confined to the local synaptic inputs to the target neuron. In addition, the custom-manufacture of electrodes allows adjustment of specific parameters according to the neural structure and type of neuron of interest (such as the tip resistance for improving the signal-to-noise ratio of the recordings).

We present methods for the construction of electrodes to simultaneously record extracellular neural activity and release multiple neuroactive substances at the vicinity of the recording sites in awake mice. This technique allows the detailed analysis of putative local synaptic inputs to the neuron of interest.

神经元电活动外记录与神经活性物质在清醒小鼠微电泳应用相结合

Differences in the activity of neurotransmitters and neuromodulators, and consequently different neural responses, can be found between anesthetized and ...

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can be hampered by the lack of specific primary or secondary antibodies for immunostaining. Here we describe a protocol to detect the expression of three different genes in the same brain section using double fluorescence in situ hybridization with two gene-specific probes followed by immunostaining with an antibody of high specificity directed against the protein encoded by a third gene. The Aspartoacyclase (ASPA) gene, mutations of which can lead to a rare human white matter disease - Canavan disease - is thought to be expressed in oligodendrocytes and microglia but not in astrocytes and neurons. However, the precise expression pattern of ASPA in the brain has yet to be established. This protocol has allowed us to determine that ASPA is expressed in a subset of mature oligodendrocytes and it can be generally applied to a wide range of gene expression pattern studies.

Combining Double Fluorescence In Situ Hybridization with Immunolabelling for Detection of the Expression of Three Genes in Mouse Brain Sections

Localizing gene expression to specific cell types can be challenging due to the lack of specific antibodies. Here we describe a protocol for simultaneous triple detection of gene expression by combining double fluorescence RNA in situ hybridization with immunostaining.

结合双荧光<em&gt;原位</em&gt;杂交免疫标记为三个基因在小鼠大脑切片中表达的检测

Detection of gene expression in different types of brain cells e.g., neurons, astrocytes, oligodendrocytes, oligodendrocyte precursors and microglia, can ...

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

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

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

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

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

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

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

In the central nervous system, the D1-alpha subtype receptor (Drd1&#945;) is the most abundant dopamine (DA) receptor, which plays a vital role in regulating neuronal growth and development. However, the mechanisms underlying Drd1&#945; receptor abnormalities mediating behavioral responses and modulating working memory function are still unclear. Using a novel RNA in situ hybridization assay, the current study identified dopamine Drd1&#945; receptor and tyrosine hydroxylase (TH) RNA expression from DA-related circuitry in the nucleus accumbens (NAc) area and substantia nigra region (SNR), respectively. Drd1&#945; expression in the NAc shows a &#34;discrete dot&#34; staining pattern. Clear sex differences in Drd1&#945; expression were observed. In contrast, TH shows a &#34;clustered&#34; staining pattern. Regarding TH expression, female rats displayed a higher signal expression per cell relative to male animals. The methods presented here provide a novel in situ hybridization technique for investigating changes in dopamine system dysfunction during the progression of central nervous system diseases.

Identification of Dopamine D1-Alpha Receptor Within Rodent Nucleus Accumbens by an Innovative RNA In Situ Detection Technology

Identification of dopamine D1-alpha receptor in the nucleus accumbens is critical for clarifying D1 receptor dysfunction during a central nervous system disease. We performed a novel RNA in situ hybridization assay to visualize single RNA molecules in a specific brain area.

检测持久性病毒的DNA在神经组织​​中的荧光的基因组与成绩单原位杂交结合免疫组化染色

检测持久性病毒的DNA在神经组织中的荧光的基因组与成绩单原位杂交结合免疫组化染色