这项研究是由美国国立卫生研究院资助（R15DA029863）的支持下，佛罗里达大西洋大学本科生科研补助金（M30014）和罗斯大学兽医研究资助。我们要感谢美国国家药物滥用研究所（马里兰州洛克维尔）为这项工作提供了（±）3,4-亚甲基（±MDMA）。

下面描述动物使用手续批准的机构动物护理和使用委员会（IACUC）在罗斯大学兽医学院和佛罗里达大西洋大学。虽然无菌操作技术和手套是必需的，而使用该协议的无RNA酶的环境是没有必要的。 1.准备<ol><li>组织编制和切片<ol><li>随机分配的大鼠分为三组:新鲜/生理盐水（SAL; 0.9％的NaCl），前缀/ SAL和前缀/ 3,4-亚甲基苯丙胺（MDMA， 见表 1详情）。 </li><li>施用三种剂量为1ml / kg的SAL和10mg / kg的腹膜内摇头丸，分别向SAL和MDMA基在2小时的时间间隔。让动物生存的治疗6天。 </li><li>在第7天，给药100mg / kg的氯胺酮以5mg组合/ kg的甲苯噻嗪腹膜内给大鼠为一深度麻醉（即，角膜眨眼和Ta的损失IL捏反射）。 </li><li>对于新鲜/ SAL组，迅速 ​​杀头大鼠并取出大脑新鲜的大脑测试（详情请参阅参考5）。 </li><li>为前缀/ SAL和前缀/摇头丸基，使沿胸骨约10 -12厘米的切口，然后暴露胸骨的末端。 </li><li>持用止血钳胸骨的端部和切断膜片上横向两侧锋利的剪刀，然后切向上穿过肋和平行于肺部。 </li><li>用一只手握住心脏的小镊子腹尖。用另一只手，刺穿左心室用18G的注射器针头（注意针的适配器侧带有Y形硅软管到冰冷的SAL和多聚甲醛的容器和蠕动泵相连;参见标号9）。 </li><li>打开SAL泵以流速（〜5毫升/分钟）的中等水平和穿刺右心房与镊子以允许返回环向的逸出ulation。保持SAL灌注30分钟。 </li><li>开启多聚甲醛泵在中等水平，并允许冰冷的4％多聚甲醛灌注到动物30分钟。 </li><li>除去灌注仪和杀头的动物。请通过头骨的中线切后前，然后扇动头骨暴露颅骨腔10。 </li><li>从头骨腔刮刀取出大脑，并放置在大脑中含有25毫升的4％多聚甲醛在50ml离心管中。保持含多聚甲醛管内的脑，在4ºC冰箱24小时。 </li><li>移动脑入含25毫升30％的蔗糖溶液，在4ºC3天50ml离心管中。最后，存放在塑料袋大脑在-80℃至使用。 </li><li>安装在卡盘的大脑嵌入媒体及在-26ºC温度冻结在低温恒温器含有脑嵌入媒体。切新鲜冻结ñ大脑到20微米的部分和多聚甲醛为前缀的脑成10微米的部分。通过触摸滑动部分转移部分到RT显微镜载玻片。 </li><li>风干在RT搅拌4小时。存储在紧张的密封袋的部分幻灯片在-80℃至使用。 </li></ol></li><li>脱水<ol><li>取从-80℃冷冻器的幻灯片和分配部分为三种测试:HT2A，PPIB（阳性对照）或dapB的 （阴性对照）;标记和标签部分用圆珠笔（不要用铅笔）。淹没在100％的乙醇的幻灯片在RT 5分钟。干燥载玻片5分钟在通风橱中。 </li></ol></li><li>预处理<ol><li>吸取20微升的预处理-1试剂（碱性磷酸酶灭活）对幻灯片的部分。将载玻片上的水分托盘的齿条导轨（注意，托盘覆盖有盖子，以保持高湿度，以防止蒸发从反应搜索解决方案N）。 </li><li>轻轻摇动在水平摇动器中的水分托盘以低速10分钟。 2分钟用双蒸水（DDH 2 O的）洗两次的幻灯片。淹没在200毫升烧杯中含有150个毫升预处理-2试剂（断裂引起多聚甲醛固定的交联键的）的滑动。煮沸的幻灯片，总共5分钟，在100℃（RNA结构11的热引起的恢复）。 </li><li>与DDH 2 O的两倍洗净滑梯2分钟。放置在100％的乙醇的幻灯片5分钟。干燥载玻片5分钟，在通风橱。使用的疏水性笔来绘制所选择的区域周围的圆中的组织切片（例如，非皮质结构，包括丘脑和下丘脑，如图2所述）。 </li><li>移取10微升的预处理-3试剂（增加探头穿透）每个选定区域，并覆盖水分盘的盖子。放置于托盘中的杂交炉配备有水平摇动器并设定炉温度在80°C;轻轻摇动盘30分钟。与DDH 2 O的两倍洗净滑梯2分钟。 </li></ol></li></ol> 2.杂交和扩增<ol><li>移取10微升HTR2A，PPIB和dapB的探针试剂，分别对所选区域。覆盖水分托盘与盖以防止试剂的蒸发。轻轻摇动的水平摇动器设置为低速。孵育载玻片在80°C在烘箱2小时。 </li><li>用2分钟的清洗缓冲液洗两次的幻灯片。移取10微升的扩增-1试剂上的每个选定区域的，摇动孵育载玻片在80°C 30分钟。 </li><li>用2分钟的清洗缓冲液洗两次的幻灯片。移取10微升的扩增-2试剂的每个选定区域的，摇动孵育载玻片在80°C持续15分钟。 </li><li>两次W¯¯洗净滑梯第i 2分钟的清洗缓冲液。移取10微升的扩增-3试剂的每个选定区域的，摇动孵育载玻片在80°C 30分钟。 </li><li>用2分钟的清洗缓冲液洗两次的幻灯片。移取10微升的扩增-4试剂的每个选定区域的，摇动孵育载玻片在80°C持续15分钟。 </li><li>用2分钟的清洗缓冲液洗两次的幻灯片。移取10微升的扩增-5试剂上的每个选定区域的，摇动孵育载玻片在室温30分钟。 </li><li>用2分钟的清洗缓冲液洗两次的幻灯片。移取10微升的扩增-6试剂上的每个选定区域的，摇动孵育载玻片在室温15分钟。用2分钟的清洗缓冲液洗两次的幻灯片。 </li></ol> 3.信号检测<ol><li>移取10微升红试剂的每个选定区域的（注意，所述试剂是红B和红-A的混合物在一个1:60比率，应当用于我mmediately）。放置在滑动中，在水平摇动器上在室温水分托盘和轻轻摇动以低速10分钟。 </li><li> 10分钟，（DDH 2 O的）洗两次的幻灯片。干燥载玻片在60ºC在烤箱中15分钟。放置在二甲苯幻灯片通风柜5分钟下。吸取20微升二甲苯类的安装介质在每个选定区域，并盖上盖玻片。 </li></ol> 4.图像捕捉<ol><li>取所选择的区域 （例如，下丘脑，图1和3）与4×和20×物镜的倍率的数字显微照片。保存图像文件。 </li></ol> 5.图像颗粒分析<ol><li>拖放图像文件到主ImageJ的窗口。转到菜单栏并选择"图像"，然后选择从下拉菜单中选择"类型"，然后选择"8位"。 </li><li>选择"调整"，然后选择"苏eshold"，打开对话框。调整在对话框中的下栏值，以使不需要的背景被除去。转到菜单栏并选择"分析"，然后选择"分析粒子"打开第二个对话框。 </li><li>设置粒度为10，接下来选择"显示概述"，并选择"显示结果"和"汇总"框。最后，点击"确定"按钮，查看对话框粒子数据。 </li></ol> 6.电子表格计算<ol><li>将数据输入到Excel工作表，并作为基准的SAL组平均颗粒的数量。计算每个区段的百分率水平，并作出相应的曲线图（图4）。 </li></ol>

<ol>
	<li>Muzyk, A. J., Jakel, R. J., Preud'homme, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+syndrome+after+a+massive+overdose+of+controlled-release+paroxetine.">Serotonin syndrome after a massive overdose of controlled-release paroxetine.</a> Psychosomatics. 51, 437-442 (2010).</li><li>Davies, O., Batajoo-Shrestha, B., Sosa-Popoteur, J., Olibrice, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Full+recovery+after+severe+serotonin+syndrome,+severe+rhabdomyolysis,+multi-organ+failure+and+disseminated+intravascular+coagulopathy+from+MDMA.">Full recovery after severe serotonin syndrome, severe rhabdomyolysis, multi-organ failure and disseminated intravascular coagulopathy from MDMA.</a> Heart Lung. 43, 117-119 (2014).</li><li>Bosch, O. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Verbal+memory+deficits+are+correlated+with+prefrontal+hypometabolism+in+18FDG+PET+of+recreational+MDMA+users.">Verbal memory deficits are correlated with prefrontal hypometabolism in 18FDG PET of recreational MDMA users.</a> PLoS On. 8, e61234 (2013).</li><li>Smithies, V., Broadbear, J., Verdejo-Garcia, A., Conduit, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dysfunctional+overnight+memory+consolidation+in+ecstasy+users.">Dysfunctional overnight memory consolidation in ecstasy users.</a> J Psychopharmaco. 28, 751-762 (2014).</li><li>Winzer-Serhan, U. H., Broide, R. S., Chen, Y., Leslie, F. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+sensitive+radioactive+in+situ+hybridization+using+full+length+hydrolyzed+riboprobes+to+detect+&#945;2+adrenoceptor+subtype+mRNAs+in+adult+and+developing+rat+brain.">Highly sensitive radioactive in situ hybridization using full length hydrolyzed riboprobes to detect &#945;2 adrenoceptor subtype mRNAs in adult and developing rat brain.</a> Brain Res Brain Res Proto. 3, 229-241 (1999).</li><li>Zoeller, R. T., Fletcher, D. L., Butnariu, O., Lowry, C. A., Moore, F. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-ethylmaleimide+(NEM)+can+significantly+improve+in+situ+hybridization+results+using+35S-labeled+oligodeoxynucleotide+or+complementary+RNA+probes.">N-ethylmaleimide (NEM) can significantly improve in situ hybridization results using 35S-labeled oligodeoxynucleotide or complementary RNA probes.</a> J Histochem Cytoche. 45, 1035-1041 (1997).</li><li>Wang, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope:+a+novel+in+situ+RNA+analysis+platform+for+formalin-fixed,+paraffin-embedded+tissues.">RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues.</a> J Mol Diag. 14, 22-29 (2012).</li><li>Wang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNAscope+for+in+situ+detection+of+transcriptionally+active+human+papillomavirus+in+head+and+neck+squamous+cell+carcinoma.">RNAscope for in situ detection of transcriptionally active human papillomavirus in head and neck squamous cell carcinoma.</a> J Vis E. , (2014).</li><li>Gage, G. J., Kipke, D. R., Shain, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+animal+perfusion+fixation+for+rodents.">Whole animal perfusion fixation for rodents.</a> J Vis E. , (2012).</li><li>Mishra, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+pericytes+and+capillary+diameter+in+brain+slices+and+isolated+retinae.">Imaging pericytes and capillary diameter in brain slices and isolated retinae.</a> Nat Proto. 9, 323-336 (2014).</li><li>Shi, S. R., Cote, R. J., Taylor, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen+retrieval+techniques:+current+perspectives.">Antigen retrieval techniques: current perspectives.</a> J Histochem Cytoche. 49, 931-937 (2001).</li><li>Qin, Y., Heine, V. M., Karst, H., Lucassen, P. J., Joels, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+patterns+in+rat+dentate+granule+cells:+comparison+between+fresh+and+fixed+tissue.">Gene expression patterns in rat dentate granule cells: comparison between fresh and fixed tissue.</a> J Neurosci Method. 131, 205-211 (2003).</li><li>Carter, B. S., Fletcher, J. S., Thompson, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+messenger+RNA+expression+by+in+situ+hybridization+using+RNA+probes+synthesized+via+in+vitro+transcription.">Analysis of messenger RNA expression by in situ hybridization using RNA probes synthesized via in vitro transcription.</a> Method. 52, 322-331 (2010).</li><li>Mijnster, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+and+cellular+distribution+of+serotonin+5-hydroxytryptamine2a+receptor+mRNA+in+the+nucleus+accumbens,+olfactory+tubercle,+and+caudate+putamen+of+the+rat.">Regional and cellular distribution of serotonin 5-hydroxytryptamine2a receptor mRNA in the nucleus accumbens, olfactory tubercle, and caudate putamen of the rat.</a> J Comp Neuro. 389, 1-11 (1997).</li><li>Li, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brain+region-specific+alterations+of+5-HT2A+and+5-HT2C+receptors+in+serotonin+transporter+knockout+mice.">Brain region-specific alterations of 5-HT2A and 5-HT2C receptors in serotonin transporter knockout mice.</a> J Neuroche. 84, 1256-1265 (2003).</li><li>Horner, K. A., Gilbert, Y. E., Noble, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+5-HT2A+receptor+mRNA+expression+following+withdrawal+from+a+chronic+escalating+dose+regimen+of+D-amphetamine.">Differential regulation of 5-HT2A receptor mRNA expression following withdrawal from a chronic escalating dose regimen of D-amphetamine.</a> Brain Re. 1390, 10-20 (2011).</li><li>Mocci, G., Jimenez-Sanchez, L., Adell, A., Cortes, R., Artigas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+5-HT2A+receptors+in+prefrontal+cortex+pyramidal+neurons+projecting+to+nucleus+accumbens.+Potential+relevance+for+atypical+antipsychotic+action.">Expression of 5-HT2A receptors in prefrontal cortex pyramidal neurons projecting to nucleus accumbens. Potential relevance for atypical antipsychotic action.</a> Neuropharmacolog. 79, 49-58 (2014).</li><li>Reneman, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+acute+and+chronic+effects+of+MDMA+('Ecstasy')+on+cortical+5-HT2A+receptors+in+rat+and+human.">The acute and chronic effects of MDMA ('Ecstasy') on cortical 5-HT2A receptors in rat and human.</a> 26, 387-396 (2002).</li></ol>

作者宣称，他们有没有竞争的财务权益。

One of the major concerns of in situ hybridization test is to choose appropriate techniques used for RNA preservation because of RNase enzymes. It is well known that these enzymes are widely present in the cells and environment which can affect the results. However, enzyme activity can be quickly distinguished by placing the tissue in the dry ice/alcohol solution5,12. Although quick preservation is critical for hybridization using a riboprobe13, little is known about experimental conditions...

Serotonin (5-hydroxytryptamine; 5-HT) syndrome is an acute neurologic disorder caused by 5-HT-promoting drugs such as antidepressants1, while also occurring in situations of MDMA use for recreational purposes2. Molecular mechanisms responsible for mood swings, learning and memory deficits that occur in association with the acute syndrome are not well understood3,4. In situ hybridization is a powerful research tool allowing the detection and quantification of specific mRNAs expressed potentially at a single-cell level. The conventional way to perform in situ hybridization is to utilize a radioactive-labeled riboprobe that specifically hybridizes the gene of interest. However, a major drawback is that the method requires complicated and time-consuming probe preparation and hybridization steps, as well as access to fresh frozen tissues maintained in an RNase-free environment5,6. 
Oligonucleotide probes have been recently developed to hybridize shorter RNA fragments than those required for riboprobes7. Furthermore, the probes produce a low background signal without sacrificing specificity8. This newly-developed probe technology can be used in situ hybridization on paraformaldehyde-prefixed brain tissues commonly available in immunocytochemical laboratories.
Here, we describe a protocol for in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed rat brain and compare findings with those noted in a fresh frozen brain5,6. This protocol is used to test the hypothesis that MDMA substance abuse causes changes in 5-HT2A receptor gene htr2a mRNA in the brain. We began the procedure with MDMA treatment followed by paraformaldehyde tissue perfusion of the animal, in situ hybridization of the thr2a probe, and data analysis. Note that dapB (the bacterial gene coding for dihydrodipicolinate reductase) is used as a negative control, and ppiB (housekeeping gene coding for peptidylprolyl isomerase B) as a positive control.

<table border="0" cellpadding="0" cellspacing="0" width="873">
	<tbody>
		<tr>
			<td>RNAscope Negative Control Probe-DapB</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310098</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Pretreat 4</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>320046</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope 2.0 HD Reagent Kit - Red</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>310036</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAscope Probe - Rn-Ppib</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>313921</td>
			<td></td>
		</tr>
		<tr>
			<td>Rat Htr2a</td>
			<td>Advanced cell diagnostic, INC</td>
			<td>300031</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM 1850</td>
			<td></td>
		</tr>
		<tr>
			<td>Horizontal shaker</td>
			<td>VWR</td>
			<td>88032-088</td>
			<td></td>
		</tr>
		<tr>
			<td>Hybridization oven</td>
			<td>Thermo Fisher Scientific</td>
			<td>222000</td>
			<td></td>
		</tr>
		<tr>
			<td>Superfrost Plus microscope slide</td>
			<td>Fisher Scientific</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrophobic pen</td>
			<td>Vector</td>
			<td>H-4000</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Olympus</td>
			<td>Provis AX70</td>
			<td></td>
		</tr>
	</tbody>
</table>

rapid in situ hybridization using oligonucleotide probes on paraformaldehyde-prefixed brain of rats with serotonin syndrome

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin (5-hydroxytryptamine; 5-HT) syndrome. This hypothesis can be tested using in situ hybridization to detect the serotonin 5-HT2A receptor gene htr2a. In the past, such procedures, utilizing radioactive riboprobe, were difficult because of the complicated workflow that needs several days to perform and the added difficulty that the technique required the use of fresh frozen tissues maintained in an RNase-free environment. Recently, the development of short oligonucleotide probes has simplified in situ hybridization procedures and allowed the use of paraformaldehyde-prefixed brain sections, which are more widely available in laboratories. Here, we describe a detailed protocol using non-radioactive oligonucleotide probes on the prefixed brain tissues. Hybridization probes used for this study include dapB (a bacterial gene coding for dihydrodipicolinate reductase), ppiB (a housekeeping gene coding for peptidylprolyl isomerase B), and htr2a (a serotonin gene coding for 5-HT2A receptors). This method is relatively simply, cheap, reproducible and requires less than two days to complete.

使用寡核苷酸RNA探针（&lt;25个核苷酸），杂交可以被检测为红点从多聚甲醛前缀和新鲜冷冻大脑​​下丘脑制备细胞。所述HTR2A mRNA分子存在于一些细胞（用实线箭头表示），而不是其他（空心箭头）。我们观察到，有在多聚甲醛加前缀和新鲜冷冻组织（图1）之间没有差别。一个成功的杂交可先用肉眼（图2）进行评价。我们发现，所选择的区域（标有圆圈）杂交的...

Watch this Scientific Journal Video about Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome at JoVE.com

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

快速<em&gt;原位</em使用寡核苷酸探针对大鼠多聚甲醛为前缀的大脑5-羟色胺综合征&gt;杂交

3,4-Methylenedioxymethamphetamine (MDMA; ecstasy) toxicity may cause region-specific changes in serotonergic mRNA expression due to acute serotonin ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

8.8K 次观看.  Ross University School of Veterinary Medicine. This protocol describes a rapid and simplified in situ hybridization method ideal forparaformaldehyde-prefixed brain, thus reducing the need for prolonged complex steps while using fresh frozen tissues. The method is validated using the identification of the serotonin 5-HT2A receptor gene htr2a in rats.

视频: Rapid In Situ Hybridization using Oligonucleotide Probes on Paraformaldehyde-prefixed Brain of Rats with Serotonin Syndrome

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

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS:使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

结合亲脂性染料，原位杂交，免疫组织化学和组织学

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Rapid Determination of the Thermal Nociceptive Threshold in Diabetic Rats

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity to normally innocuous stimuli1. Hyperalgesia and allodynia have been studied in many different rodent models of diabetes mellitus2. However, as stated by B&ouml;lcskei et al, determination of "pain" in animal models is challenging due to its subjective nature3. Moreover, the traditional methods used to determine behavioral responses to noxious thermal stimuli usually lack reproducibility and pharmacological sensitivity3. For instance, by using the hot-plate method of Ankier4, flinch, withdrawal and/or licking of either hind- and/or fore-paws is quantified as reflex latencies at constant high thermal stimuli (52-55 &deg;C). However, animals that are hyperalgesic to thermal stimulus do not reproducibly show differences in reflex latencies using those supra-threshold temperatures3,5. As the recently described method of B&ouml;lcskei et al.6, the procedures described here allows for the rapid, sensitive and reproducible determination of thermal nociceptive thresholds (TNTs) in mice and rats. The method uses slowly increasing thermal stimulus applied mostly to the skin of mouse/rat plantar surface. The method is particularly sensitive to study anti-nociception during hyperalgesic states such as PDN. The procedures described bellow are based on the ones published in detail by Alm&aacute;si et al 5 and B&ouml;lcskei et al 3. The procedures described here have been approved the Laboratory Animal Care and Use Committee (LACUC), Wright State University.

Here, we describe a rapid reliable and simple procedure to determine the lowest temperature at which rats or mice show nocifensive behavior, i.e. the thermal nociceptive threshold (TNT). This method applies a slowly increasing thermal stimulus allowing precise and reproducible estimation of TNTs with minimum, if any, stress to the animals.

糖尿病大鼠的热痛觉阈值的快速测定

Painful diabetic neuropathy (PDN) is characterized by hyperalgesia i.e., increased sensitivity to noxious stimulus, and allodynia i.e., hypersensitivity ...

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several weeks, and this is accompanied by short distance regeneration (a few mm) of propriospinal axons and spinal-projecting axons from the brainstem. Among the 36 large identifiable spinal-projecting neurons, some are good regenerators and others are bad regenerators. These neurons can most easily be identified in wholemount CNS preparations. In order to understand the neuron-intrinsic mechanisms that favor or inhibit axon regeneration after injury in the vertebrates CNS, we determine differences in gene expression between the good and bad regenerators, and how expression is influenced by spinal cord transection. This paper illustrates the techniques for housing larval and recently transformed adult sea lampreys in fresh water tanks, producing complete spinal cord transections under microscopic vision, and preparing brain and spinal cord wholemounts for in situ hybridization. Briefly, animals are kept at 16&#160;&#176;C and anesthetized in 1% Benzocaine in lamprey Ringer. The spinal cord is transected with iridectomy scissors via a dorsal approach and the animal is allowed to recover in fresh water tanks at 23 &#176;C. For in situ hybridization, animals are reanesthetized and the brain and cord removed via a dorsal approach.

Complete Spinal Cord Injury and Brain Dissection Protocol for Subsequent Wholemount In Situ Hybridization in Larval Sea Lamprey

Lampreys recover locomotion after a complete spinal cord injury. However, some spinal-projecting neurons are good regenerators and others are not. This paper illustrates the techniques for housing sea lamprey larvae (and recently transformed adults), producing complete spinal cord transections and preparing wholemount brains and spinal cords for in situ hybridization.

完全性脊髓损伤和脑解剖协议后续Wholemount<em&gt;原位</em&gt;杂交苗种海七鳃鳗

After a complete spinal cord injury, sea lampreys at first are paralyzed below the level of transection. However, they recover locomotion after several ...

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 ...

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 ...

Recording EEG in Freely Moving Neonatal Rats Using a Novel Method

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such as epilepsy and neurodegenerative disorders. However, it is technically difficult to obtain EEG recordings in neonates as it requires specialized handling and great care. Here, we present a novel method to record EEG in neonatal rat pups (P8-P15). We designed a simple and reliable electrode using computer pin loci; it can be easily implanted into the skull of a rat pup to record high-quality EEG signals in the normal and epileptic brain. Pups were given an intraperitoneal (i.p.) injection of the neurotoxin kainic acid (KA) to induce epileptic seizures. The surgical implantation performed in this procedure is less expensive than other EEG procedures for neonates. This method allows one to record high-quality and stable EEG signals for more than 1 week. Furthermore, this procedure can also be applied to adult rats and mice to study epilepsy or other neurological disorders.

Here, we introduce a novel technique designed to record electroencephalography (EEG) in freely moving neonatal epileptic pups and describe its procedures, features, and applications. This method allows one to record EEG for more than 1 week.

使用新颖的方法在自由移动的新生大鼠中记录脑电图

EEG is a useful method to detect electrical activity in the brain. Moreover, it is a widely used diagnostic tool for various neurological conditions, such ...

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 ...

Induction of Diffuse Axonal Brain Injury in Rats Based on Rotational Acceleration

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large percentage of TBI patients requiring hospitalization. DAI involves widespread axonal damage from shaking, rotation or blast injury, leading to rapid axonal stretch injury and secondary axonal changes that are associated with a long-lasting impact on functional recovery. Historically, experimental models of DAI without focal injury have been difficult to design. Here we validate a simple, reproducible and reliable rodent model of DAI that causes widespread white matter damage without skull fractures or contusions.

This protocol validates a reliable, easy-to-perform and reproducible rodent model of brain diffuse axonal injury (DAI) that induces widespread white matter damage without skull fractures or contusions.

基于旋转加速大鼠漫射性无羟基脑损伤的诱导

Traumatic brain injury (TBI) is a major cause of death and disability. Diffuse axonal injury (DAI) is the predominant mechanism of injury in a large ...

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双荧光原位杂交食脑切片

TMS:使用的Theta突发协议，以探索个人与脆性X综合征与孤独症的可塑性Mechasnism

结合亲脂性染料，原位杂交，免疫组织化学和组织学

糖尿病大鼠的热痛觉阈值的快速测定

完全性脊髓损伤和脑解剖协议后续Wholemount

在脑切片检测轴突本地化mRNAs的高空间分辨率

结合双荧光

使用新颖的方法在自由移动的新生大鼠中记录脑电图

通过RNA定位蝗虫天线中的气味受体基因

基于旋转加速大鼠漫射性无羟基脑损伤的诱导