NIH R01MH084894参与这项研究的经费。我们想感谢博士。优思明赫德和斯科特·鲁索在西奈山医学院学校小鼠的捐赠，这项工作的拍摄过程中使用其手术和行为的设施。

注:动物养殖和关心所有程序均按照西奈山伊坎医学院的机构动物护理和使用委员会（IACUC）的规定进行。一定要在整个过程中使用无菌手套。 1.药物和病毒制剂<ol><li> 药物研制 <ol><li>通过溶解1.35毫升100mg / ml的氯胺酮和0.75毫升20毫克/毫升甲苯噻嗪12.9毫升0.9％盐水溶液制备15.0毫升氯胺酮/赛拉嗪麻醉剂。彻底混合的解决方案。 </li></ol></li><li> 病毒制剂 <ol><li>克隆mGlu2和mGlu2ΔTM4N构造成双顺反子单纯疱疹病毒（HSV）载体以下先前描述6标准协议。如前所述6,13,14包装病毒颗粒。残留的替代丙氨酸- 677 4.40，丙氨酸- 681 4.44和4.48 Ala685在mGlu2为Ser686 4.40，pH值E690 4.44和甘氨酸-694在4.48 mGlu3（HA-mGlu2ΔTM4N）先前已经6中所述。 注意:这是以前表明，嵌合构建的HA-mGlu2ΔTM4N在含有完整G蛋白依赖的信号6质膜表达。 </li><li>在不使用时在商店病毒载体-80℃。在冰上解冻病毒载体，然后分装到10微升等分试样。对于手术过程中，保持在冰上。 </li></ol></li></ol> 2.手术<ol><li> 术前准备 <ol><li>权衡鼠标和氯胺酮/甲苯​​噻嗪鸡尾酒的适宜剂量注入小鼠（详见1.1.1）。 </li><li>检查小鼠，看是否正常麻醉，挤压脚和尾对疼痛的反应，如果无法引起反应，鼠标被适当麻醉。 </li><li>从头骨的基地使用快船鼻尖剃光头鼠标。应用眼用凝胶鼠标AFterward防止小鼠的失明。 </li><li>加载每个注射器上的立体框架。然后倾斜立体框架的每个臂的垂直部分，从而它们是从正常的10度距离。确保臂倾斜，使得针彼此面对。 </li><li>填充用70％乙醇将针清洁每个注射器。填充针至少三次，以确保该注射器是干净的。 </li><li>一旦针头已被清洗，通过用双蒸H 2 O填充针冲洗针一旦刷新，填写每针1.3微升双蒸H 2 O的拧在注射器的柱塞释放0.3微升双蒸H 2 O的如果水珠在针尖，小心翼翼地擦去水。如果没有散发出来的注射器，推动柱塞完全放下，然后重复注射器清洗。 </li><li>充水后，再拉起注射器填充注射器0.5微升空气。 </li><li>一旦空气和水的注射器，认真填写注射器用1.3微升病毒解决方案。在这点上保证在注射器中的总体积为2.8微升。再次扭动注射器的尖端释放0.3微升病毒。如果在针尖液珠，仔细拭去液体。如果没有散发出来的注射器，推动柱塞完全放下，然后重复注射器清洗。 </li></ol></li><li> 手术 <ol><li>装上鼠标的立体框架，确保使头骨是水平的平调整立体框架。适用povodine碘到暴露的头皮。使用手术刀，使沿着露出剃区域内的颅骨的中线矢状切口。然后装上夹子滴定管对皮肤的切口部位，以确保头骨保持暴露。 </li><li>使用H 2 O 2溶解掉骨膜暴露的缝线头骨。现在，前囟门和缝合线是可见的，一定要调整立体框架，以确保头骨是水平的。 </li><li>对准前囟的注射器的针的提示和记录前囟的坐标。计算的，其中针将要插入的坐标。 <ol><li>对于延髓-Cauldal（RC）面，加1.6毫米将录制的前囟门RC坐标（+1.6从囟门）。对于背腹（DV）飞机，减去记录的DV前囟坐标（-2.4来自前囟）2.4毫米。 </li><li>最后，对于内侧横向（ML）面，添加2.6毫米于所记录的ML囟坐标（+2.6从囟）。对于所有的坐标一定要记录左，右坐标，因为这是一个双侧注射。 </li></ol></li><li>使针到所需的坐标。标记出针将要被插入并用钻头，钻标记区域的地方。 </li><li>用棉签无线PE走任何多余的血液或骨头碎片。 </li><li>使针的头骨，其中针的尖端接触大脑表面。然后放下针到所需的坐标慢慢地降低他们。 </li><li>一旦针是在所希望的坐标，慢慢地在5分钟（总共0.5微升）的过程中扭曲针0.1微升每分钟的柱塞注入注射器的内容物。 </li><li>一旦注入已经取得了离开再过5分钟皮层注射器。 </li></ol></li><li> 收盘涨/护理 <ol><li>取下鼠标皮质针稳定，慢慢地。然后从立体框架删除鼠标。 </li><li>应用氰基丙烯酸酯（皮肤粘合剂），以从切口皮肤的基部翼片，然后用钳子抓住皮肤的护翼，并放置在一起。 </li><li>允许氰基丙烯酸酯干燥。放在笼子里的鼠标移到一个加热垫（电热垫是，如果外科可选iCal的套装保持RT下37°C - 否则没有必要）。请务必将鼠标放在纸巾，以确保床上用品不符合手术部位。 </li><li>取决于外科手术过程的长度，确保该小鼠是从30内麻醉 - 程序在60分钟后。手术后，将动物放置在其自己笼和监控，直到它变成返回到其室收回之前意识。没有止痛剂被用于手术后的，因为它们可通过修改一些大脑生化途径的改变我们的实验的结果。然而，动物被监控，直到它们从麻醉中恢复上手术当天，和感染和疼痛/窘迫评价体征每天后操作。 </li></ol></li></ol> 3.甩头响应试验<ol><li> 建立 <ol><li>进行所有行为测试上午10:00和下午2:00，2之间 - stereota后3天CTIC注射病毒颗粒。 </li><li>溶解（±）-1-（2,5-二甲氧基-4-碘苯基）-2-氨基丙烷盐酸盐（DOI）到0.9％生理盐水溶液，以2.0毫克/公斤。还制备0.9％盐溶液中。 </li><li>制备家笼（28×18×15厘米），没有任何寝具并使用三脚架，调整摄像机，使得摄像机的视图是直接上方的家笼中。 </li><li> Habituate小鼠的空间之前在实验的开始，至少4小时。 </li><li>设置一个摄像机来记录头抽搐。 </li></ol></li><li> 实验 <ol><li>所以，这是直接在一个笼子相机位置。校准相机，使整个家庭笼中的视场。 </li><li>称重小鼠并用任一0.9％盐水或DOI的适当剂量（0.01毫升/克）腹膜内注入小鼠。注:如果鼠标的重量为25克，给予剂量为0.25毫升总体积。 </li><li>将每个鼠标背部在他们的家笼FOR 10分钟。 10分钟后，鼠标的地方入空笼的中心和验证有在视摄像头领域无盲点。按摄像机记录。离开这个房间。 注意:鼠标移动和各种行为反应其中（。甩头，耳朵划伤等请参考补充数据表1 2007年冈萨雷斯Maeso 等为通过DOI诱导行为反应的完整列表）3，将被记录30分钟。因此，存在在视记录领域无盲点是重要的。 </li><li> 30分钟后，停在原来的家笼记录在摄像机和地点鼠标背部。重复此过程对于每个鼠标。 </li></ol></li><li> 评论 <ol><li>让每个裁判审查磁带盲目鼠标的实验条件（ 即 ，头部过程中使用的药物实验抽搐或颅内注射过程中使用的病毒））。手工记录每一个甩头通过量豪特的视频。 注:头肌的定义是由鼠标（补充视频）所进行的快速摇动头部运动。 </li><li>对于每一个鼠标，平均从盲目裁判的三个总数最终HTR响应。然后基和这些值由实验条件进行统计分析（ 即 ，t检验或方差分析）。 </li></ol></li></ol>

<ol>
	<li>Fribourg, M., 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=Decoding+the+Signaling+of+a+GPCR+Heteromeric+Complex+Reveals+a+Unifying+Mechanism+of+Action+of+Antipsychotic+Drugs.">Decoding the Signaling of a GPCR Heteromeric Complex Reveals a Unifying Mechanism of Action of Antipsychotic Drugs.</a> Cell. 147 (5), 1011-1023 (2011).</li><li>Gonzalez-Maeso, 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=Identification+of+a+serotonin/glutamate+receptor+complex+implicated+in+psychosis.">Identification of a serotonin/glutamate receptor complex implicated in psychosis.</a> Nature. 452 (7183), 93-97 (2008).</li><li>Gonzalez-Maeso, 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=Hallucinogens+Recruit+Specific+Cortical+5-HT(2A)+Receptor-Mediated+Signaling+Pathways+to+Affect+Behavior.">Hallucinogens Recruit Specific Cortical 5-HT(2A) Receptor-Mediated Signaling Pathways to Affect Behavior.</a> Neuron. 53 (3), 439-452 (2007).</li><li>Gonzalez-Maeso, 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=Transcriptome+fingerprints+distinguish+hallucinogenic+and+nonhallucinogenic+5-hydroxytryptamine+2A+receptor+agonist+effects+in+mouse+somatosensory+cortex.">Transcriptome fingerprints distinguish hallucinogenic and nonhallucinogenic 5-hydroxytryptamine 2A receptor agonist effects in mouse somatosensory cortex.</a> J Neurosci. 23 (26), 8836-8843 (2003).</li><li>Moreno, J. L., Holloway, T., Albizu, L., Sealfon, S. C., Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metabotropic+glutamate+mGlu2+receptor+is+necessary+for+the+pharmacological+and+behavioral+effects+induced+by+hallucinogenic+5-HT2A+receptor+agonists.">Metabotropic glutamate mGlu2 receptor is necessary for the pharmacological and behavioral effects induced by hallucinogenic 5-HT2A receptor agonists.</a> Neurosci Lett. 493 (3), 76-79 (2011).</li><li>Moreno, J. 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=Identification+of+Three+Residues+Essential+for+5-HT2A-mGlu2+Receptor+Heteromerization+and+its+Psychoactive+Behavioral+Function.">Identification of Three Residues Essential for 5-HT2A-mGlu2 Receptor Heteromerization and its Psychoactive Behavioral Function.</a> J Biol Chem. 287, 44301-44319 (2012).</li><li>Geyer, M. A., Vollenweider, F. 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+research:+contributions+to+understanding+psychoses.">Serotonin research: contributions to understanding psychoses.</a> Trends Pharmacol Sci. 29 (9), 445-453 (2008).</li><li>Nichols, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hallucinogens.">Hallucinogens.</a> Pharmacol Ther. 101 (2), 131-181 (2004).</li><li>Hanks, J. B., Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+serotonergic+psychedelics.">Animal models of serotonergic psychedelics.</a> ACS Chem Neurosci. 4 (1), 33-42 (2013).</li><li>Fiorella, D., Rabin, R. A., Winter, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+5-HT2A+and+5-HT2C+receptors+in+the+stimulus+effects+of+hallucinogenic+drugs.+II:+Reassessment+of+LSD+false+positives.">Role of 5-HT2A and 5-HT2C receptors in the stimulus effects of hallucinogenic drugs. II: Reassessment of LSD false positives.</a> Psychopharmacology (Berl). 121 (3), 357-363 (1995).</li><li>Vollenweider, F. X., Vollenweider-Scherpenhuyzen, M. F., Babler, A., Vogel, H., Hell, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Psilocybin+induces+schizophrenia-like+psychosis+in+humans+via+a+serotonin-2+agonist+action.">Psilocybin induces schizophrenia-like psychosis in humans via a serotonin-2 agonist action.</a> Neuroreport. 9 (17), 3897-3902 (1998).</li><li>Moreno, J. L., Sealfon, S. C., Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Group+II+metabotropic+glutamate+receptors+and+schizophrenia.">Group II metabotropic glutamate receptors and schizophrenia.</a> Cell Mol Life Sci. 66 (23), 3777-3785 (2009).</li><li>Kurita, M., 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=HDAC2+regulates+atypical+antipsychotic+responses+through+the+modulation+of+mGlu2+promoter+activity.">HDAC2 regulates atypical antipsychotic responses through the modulation of mGlu2 promoter activity.</a> Nat Neurosci. 15 (9), 1245-1254 (2012).</li><li>Kurita, M., 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=Repressive+Epigenetic+Changes+at+the+mGlu2+Promoter+in+Frontal+Cortex+of+5-HT2A+Knockout+Mice.">Repressive Epigenetic Changes at the mGlu2 Promoter in Frontal Cortex of 5-HT2A Knockout Mice.</a> Mol Pharmacol. 83 (6), 1166-1175 (2013).</li><li>Rives, M. 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=Crosstalk+between+GABAB+and+mGlu1a+receptors+reveals+new+insight+into+GPCR+signal+integration.">Crosstalk between GABAB and mGlu1a receptors reveals new insight into GPCR signal integration.</a> Embo J. 28 (15), 2195-2208 (2009).</li><li>Milligan, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Prevalence,+Maintenance+and+Relevance+of+GPCR+Oligomerization.">The Prevalence, Maintenance and Relevance of GPCR Oligomerization.</a> Mol Pharmacol. (84), 158-169 (2013).</li><li>Ferre, S., 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=G+protein-coupled+receptor+oligomerization+revisited:+functional+and+pharmacological+perspectives.">G protein-coupled receptor oligomerization revisited: functional and pharmacological perspectives.</a> Pharmacol Rev. 66 (2), 413-434 (2014).</li><li>Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GPCR+oligomers+in+pharmacology+and+signaling.">GPCR oligomers in pharmacology and signaling.</a> Mol Brain. 4 (1), 20 (2011).</li><li>Gonzalez-Maeso, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Family+a+GPCR+heteromers+in+animal+models.">Family a GPCR heteromers in animal models.</a> Front Pharmacol. 5, 226 (2014).</li><li>Dragulescu-Andrasi, A., Chan, C. T., De, A., Massoud, T. F., Gambhir, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioluminescence+resonance+energy+transfer+(BRET)+imaging+of+protein-protein+interactions+within+deep+tissues+of+living+subjects.">Bioluminescence resonance energy transfer (BRET) imaging of protein-protein interactions within deep tissues of living subjects.</a> Proceedings of the National Academy of Sciences of the United States of America. 108 (29), 12060-12065 (2011).</li><li>Calebiro, D., 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=Single-molecule+analysis+of+fluorescently+labeled+G-protein-coupled+receptors+reveals+complexes+with+distinct+dynamics+and+organization.">Single-molecule analysis of fluorescently labeled G-protein-coupled receptors reveals complexes with distinct dynamics and organization.</a> Proc Natl Acad Sci U S A. 110 (2), 743-748 (2013).</li><li>Fonseca, J. M., Lambert, N. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Instability+of+a+class+a+G+protein-coupled+receptor+oligomer+interface.">Instability of a class a G protein-coupled receptor oligomer interface.</a> Mol Pharmacol. 75 (6), 1296-1299 (2009).</li><li>Hern, J. 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=Formation+and+dissociation+of+M1+muscarinic+receptor+dimers+seen+by+total+internal+reflection+fluorescence+imaging+of+single+molecules.">Formation and dissociation of M1 muscarinic receptor dimers seen by total internal reflection fluorescence imaging of single molecules.</a> Proc Natl Acad Sci U S A. 107 (6), 2693-2698 (2010).</li><li>Hlavackova, V., 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=Sequential+inter-+and+intrasubunit+rearrangements+during+activation+of+dimeric+metabotropic+glutamate+receptor+1.">Sequential inter- and intrasubunit rearrangements during activation of dimeric metabotropic glutamate receptor 1.</a> Sci Signal. 5 (237), 59 (2012).</li><li>Irannejad, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conformational+biosensors+reveal+GPCR+signalling+from+endosomes.">Conformational biosensors reveal GPCR signalling from endosomes.</a> Nature. 495 (7442), 534-538 (2013).</li><li>Calebiro, D., Nikolaev, V. O., Persani, L., Lohse, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Signaling+by+internalized+G-protein-coupled+receptors.">Signaling by internalized G-protein-coupled receptors.</a> Trends Pharmacol Sci. 31 (5), 221-228 (2010).</li><li>Celada, P., Puig, M. V., Diaz-Mataix, L., 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=The+hallucinogen+DOI+reduces+low-frequency+oscillations+in+rat+prefrontal+cortex:+reversal+by+antipsychotic+drugs.">The hallucinogen DOI reduces low-frequency oscillations in rat prefrontal cortex: reversal by antipsychotic drugs.</a> Biol Psychiatry. 64 (5), 392-400 (2008).</li><li>B&#233;&#239;que, J. -. C., Imad, M., Mladenovic, L., Gingrich, J. A., Andrade, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+the+5-hydroxytryptamine+2A+receptor-mediated+facilitation+of+synaptic+activity+in+prefrontal+cortex.">Mechanism of the 5-hydroxytryptamine 2A receptor-mediated facilitation of synaptic activity in prefrontal cortex.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (23), 9870-9875 (2007).</li></ol>

The authors have nothing to disclose.

连同mGlu2-KO小鼠5以前的研究结果，与mGlu2和mGlu2 / mGlu3嵌合构建的结果不形成在培养的细胞对5-HT 2A -mGlu2受体复合表明对5-HT 2A -mGlu2在异聚受体复合需要鼠标额叶皮层由LSD样致幻5-HT 2A受体激动剂诱导头部抽动行为。这种方法的一个限制是，它不会在天然组织亚细胞水平测量接近分子的接近。此外，也有应注意各种临界点。因为将小鼠用HSV病毒载体注射，即要执行的实验的时间范围是2 - 注射后4天。病毒载体的位置和表达应与初次注射后不超过4天切片脑的免疫荧光染色进行验证。小鼠中的坐标不匹配或不表达病毒载体应该可以从实验数据中排除，因为它们不表达mGlu2或mGlu2ΔTM4N。关心立体定向手术后也至关重要，因为磁头伤口不当闭合可导致感染可引起在体内实验和免疫荧光染色这两个问题。注射病毒颗粒后4 -最后，立体定向注射任何行为范式（开放场交替t检验， 等 ）后，可以，只要它是2内使用。此外，协调和表达应该通过免疫荧光确认。 该GPCR的功能均聚物和/或在活细胞中异聚现已很好地建立16-18的概念。然而，尽管取得了一些进展，还需要更多的研究来确定GPCR异聚复合物的整体动物模型19的确切作用（S）。方法，如BRET和/或FRET成像研究小动物的深部组织中的蛋白质 - 蛋白质物理接近模型可以提供手段，加强活体20 GPCR异聚的功能作用的认识。使用GPCR的HSV介导的表达任一天然的或改良的跨膜蛋白，提供在体内模型来评估的GPCR的功能和相互作用。 还需要在啮齿动物模型中进一步研究，以检查它们的部件24，以及受体内化25,26后潜在的G蛋白偶联之间21-23，结构重排的GPCR异聚的稳定性和生命时间（形成和离解）。鉴于细胞信号传导和功能的GPCR的基础性作用，它很可能是这方面的可能导致有趣的基础和翻译研究。 虽然进一步调查，需要与先前的STU定量表征在人类和小鼠的CNS两种受体的超微结构共定位，一起模具是令人信服地证明了电，蜂窝，和在小鼠模型中诱导的致幻行为反应是固有的5-HT 2A受体表达皮层锥体神经元3,27,28，这里描述利用HSV介导的表达方法获得的研究结果表明是需要由致幻5-HT 2A受体激动剂DOI诱导头部抽搐精神病样行为在小鼠额叶皮层5-HT 2A和mGlu2受体之间的异形成。 HSV介导的mGlu2的表达，但不mGlu2ΔTM4N，在mGlu2-KO小鼠的额叶皮层神经元救援由致幻5-HT 2A受体激动剂DOI感应头肌行为。这个翻译工具可能是有利的临床前研究，以评估GPCR异聚的行为表型。

致幻剂，如LSD，裸盖菇碱和麦司卡林引起人的意识，认知和情感7-9显著的变化。无论是由遗传或药物的方法羟色胺5-HT 2A受体信号的失活会导致明显减弱，以致幻剂的行为反应在两种啮齿类动物模型3,10和人类11。虽然致幻剂结合其他受体亚型8，对5-HT 2A受体被认为是必要的这些化学物质的独特的行为活动。 II组代谢型谷氨酸受体（ 即 ，mGlu2和mGlu3）一直是相当大的注意关于致幻剂的分子机制和其底层的精神病12整体作用的目标。以前，已经显示，与mGlu2蛋白（mGlu2-KO小鼠）中不表达小鼠是不敏感的HAL的细胞和行为效应lucinogens 5。还已经提出，对5-HT 2A和mGlu2受体形成一个特定的异聚复合物，通过该血清素和谷氨酸的配体调节在活细胞中的1,2- G蛋白偶联的图案。 在结构上，跨膜结构域4和mGlu2的5异的形成中发挥基础性作用与5-HT 2A受体5。此外，进一步的研究表明，三个残基位于mGlu2的TM4的细胞内端是必要的，以形成在活细胞6对5-HT 2A -mGlu2受体heterocomplex。 基于这些发现在异源表达系统中观察到，在这里，我们描述了使用野生型mGlu2和mGlu2-KO小鼠的额皮质mGlu2 / mGlu3嵌合构建的HSV介导的表达来测试是否5-HT 2A之间异形成和mGlu2是必要的通过致幻5-HT 2A受体激动剂诱导头肌的行为。

<table border="0" cellpadding="0" cellspacing="0" width="2329">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:423px;">mGlu2 bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td style="width:1716px;">mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mGlu2&#916;TM4N bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GFP bicitronic herpes simplex virus (HSV) vector&#160;</td>
			<td style="width:71px;">MIT Core</td>
			<td style="width:119px;"></td>
			<td>mGlu2 and mGlu2DTM4N were subcloned into the bicistronic HSV-GFP virus vector p1005+ HSV expressing GFP under the control of the CMV promoter. Viral particles were produced by the Viral Core Facility at the McGovern Institute (MIT). For more information, please contact the director, Dr. Rachael Neve (rneve@mit.edu)</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:423px;">xylazine&#160;</td>
			<td style="width:71px;">Lloyd</td>
			<td style="width:119px;">List no. 4811-20ml, NADA #139-236, NDC Code(s): 61311-481-10</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">ketamine&#160;</td>
			<td style="width:71px;">Vedco</td>
			<td style="width:119px;">KetaVed-10ml, NADA #200-029, NDC Code(s): 50989-161-06</td>
			<td>1.35 mL of 100mg/ml of ketamine+.75 mL of 20mg/ml of xylazine are diluted in 12.0 mL of .9% saline solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">ophthalmic gel</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC0550805</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">burret clips</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9268369</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Feather surgical blade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9032736</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:423px;">Hydrogen Peroxide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>19-898-919&#160;</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton syringe</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14815203</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Hamilton&#8482; Small Hub Removable Needles (33 Ga)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>14816206</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Cordless Micro Drill</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>NC9089241</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dermabond Dermal Adhesive</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC0690470</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">(&#177;)-1-(2,5-Dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride (DOI)</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>42203-78-1</td>
			<td>Dissolved in .9% saline solution to the concentration of 2.0 mg/kg</td>
		</tr>
	</tbody>
</table>

hsv-mediated transgene expression of chimeric constructs to study behavioral function of gpcr heteromers in mice

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2. Consequently, investigation of structural details of the interaction between 5-HT2A and mGlu2 affecting schizophrenia-related behaviors represents a powerful translational tool. As previously shown, the head-twitch response (HTR) in mice is elicited by hallucinogenic drugs and this behavioral response is absent in 5-HT2A knockout (KO) mice3,4. Additionally, by conditionally expressing the 5-HT2A receptor only in cortex, it was demonstrated that 5-HT2A receptor-dependent signaling pathways on cortical pyramidal neurons are sufficient to elicit head-twitch behavior in response to hallucinogenic drugs3. Finally, it has been shown that the head-twitch behavioral response induced by the hallucinogens DOI and lysergic acid diethylamide (LSD) is significantly decreased in mGlu2-KO mice5. These findings suggest that mGlu2 is at least in part necessary for the 5-HT2A receptor-dependent psychosis-like behavioral effects induced by LSD-like drugs. However, this does not provide evidence as to whether the 5-HT2A-mGlu2 receptor complex is necessary for this behavioral phenotype. To address this question, herpes simplex virus (HSV) constructs to express either mGlu2 or mGlu2&#916;TM4N (mGlu2/mGlu3 chimeric construct that does not form the 5-HT2A-mGlu2 receptor complex) in the frontal cortex of mGlu2-KO mice were used to examine whether this GPCR heteromeric complex is needed for the behavioral effects induced by LSD-like drugs6.

以前的研究结果表明，该头部抽搐鼠行为反应被可靠和鲁棒由致幻剂引起，并且它在5-HT 2A -KO小鼠3是不存在。此外，已经表明由致幻5-HT 2A激动剂DOI和LSD引起头部抽搐反应在mGlu2-KO小鼠5被显著降低。然而，虽然以前的研究结果令人信服地证明，5-HT 2A和mGlu2被组装成在转染细胞1,2,15 体外异聚复合物，此结构布置是否表现为这样在活小鼠中仍未解决。要充分认识在mGlu2-KO小鼠额叶皮质致幻5-HT 2A受体激动剂，无论是mGlu2或mGlu2ΔTM4N表达引起的精神样作用的5-HT 2A受体-mGlu2的heterocomplex的作用，研究这是否MANIPulation调节行为。 小鼠接受双顺反子的HSV帧内额叶皮质注射表达单独绿色荧光蛋白（GFP），要么mGlu2或mGlu2ΔTM4N，或绿色荧光蛋白。首先，它证实了病毒过表达在小鼠额叶皮质mGlu2或mGlu2ΔTM4N（ 图1A和1B）。正如以前证明5，通过DOI诱导头部抽搐的行为与空载体HSV-GFP注射mGlu2-KO小鼠中不存在。值得注意的是，由致幻5-HT 2A激动剂DOI诱导头部抽搐反应在mGlu2-KO小鼠过表达mGlu2获救，但不mGlu2ΔTM4N，额叶皮层相比，在表达GFP（ 图1C）的动物看到。总之，这些研究结果表明，在额皮质中的5-HT 2A -mGlu2受体复合物是用于调节精神病样状态的关键。 图1. mGlu2表达的 mGlu2 的受体Heterocomplex。表达与5-HT 2A受体是必要的致幻通过诱导药物精神病样的行为。 （ 一 ）额叶皮层HSV介导的转基因表达的形象代表。 HSV-mGlu2，这也表达绿色荧光蛋白，注入额叶皮层，和GFP表达，免疫细胞化学，比例尺，200微米透露。 （B）中的HSV介导的转基因表达在mGlu2-KO小鼠的小鼠额叶皮层和抗mGlu2反应性通过Western印迹测定。对mGlu2受体第一抗体的特异性先前已证实了在敲除小鼠6。代谢型谷氨酸受体是形成共价连接的同二聚体的GPCR。我们测量mGlu2的免疫反应性作为单体（100 kDa）的6。 （C </stronmGlu2克&gt;）病毒介导的表达，但不mGlu2ΔTM4N，在mGlu2- KO小鼠的额皮质显著抢救由致幻5-HT 2A激动剂DOI（每组n = 4）诱导的头部抽搐反应。 *** P &lt;0.001; NS，不显著;邦费罗尼的的单向ANOVA事后检验。误差棒代表SEM图由莫雷诺等人 （2012）修改6。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53717/53717fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53717/HeadTwitchResponse.wmv" target="_blank">补充视频1.甩头响应。 （点击下载）。</a> CD-1野生型小鼠用2.0毫克/千克DOI注射，并置于笼（壁涂黑两个笼之间）以引发头颤搐反应（后行为引起*）。

Watch this Scientific Journal Video about HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice at JoVE.com

HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

本文介绍如何注入病毒载体插入鼠标额叶皮质测试需要GPCR异形成行为检测。

研究小鼠GPCR异聚的行为功能嵌合构造的HSV介导的转基因表达

The heteromeric receptor complex between 5-HT2A and mGlu2 has been implicated in some of the behavioral phenotypes in mouse models of psychosis1,2.  ...

hsv-mediated-transgene-expression-chimeric-constructs-to-study

Nanyang Technological University

Research

JoVE Journal

Neuroscience

7.2K 次观看.  Icahn School of Medicine at Mount Sinai. 本文介绍如何注入病毒载体插入鼠标额叶皮质测试需要GPCR异形成行为检测。 

视频: HSV-Mediated Transgene Expression of Chimeric Constructs to Study Behavioral Function of GPCR Heteromers in Mice

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

科研

神经科学

Local Application of Drugs to Study Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent neuropsychiatric disorder that stems from the biophysical and cellular actions of nicotine on nicotinic acetylcholine receptors (nAChRs) throughout the central nervous system. Understanding the various nAChR subtypes that exist in brain areas relevant to nicotine addiction is a major priority.
Experiments that employ electrophysiology techniques such as whole-cell patch clamp or two-electrode voltage clamp recordings are useful for pharmacological characterization of nAChRs of interest. Cells expressing nAChRs, such as mammalian tissue culture cells or Xenopus laevis oocytes, are physically isolated and are therefore easily studied using the tools of modern pharmacology. Much progress has been made using these techniques, particularly when the target receptor was already known and ectopic expression was easily achieved. Often, however, it is necessary to study nAChRs in their native environment: in neurons within brain slices acutely harvested from laboratory mice or rats. For example, mice expressing "hypersensitive" nAChR subunits such as &alpha;4 L9&prime;A mice 1 and &alpha;6 L9&prime;S mice 2, allow for unambiguous identification of neurons based on their functional expression of a specific nAChR subunit. Although whole-cell patch clamp recordings from neurons in brain slices is routinely done by the skilled electrophysiologist, it is challenging to locally apply drugs such as acetylcholine or nicotine to the recorded cell within a brain slice. Dilution of drugs into the superfusate (bath application) is not rapidly reversible, and U-tube systems are not easily adapted to work with brain slices.
In this paper, we describe a method for rapidly applying nAChR-activating drugs to neurons recorded in adult mouse brain slices. Standard whole-cell recordings are made from neurons in slices, and a second micropipette filled with a drug of interest is maneuvered into position near the recorded cell. An injection of pressurized air or inert nitrogen into the drug-filled pipette causes a small amount of drug solution to be ejected from the pipette onto the recorded cell. Using this method, nAChR-mediated currents are able to be resolved with millisecond accuracy. Drug application times can easily be varied, and the drug-filled pipette can be retracted and replaced with a new pipette, allowing for concentration-response curves to be created for a single neuron. Although described in the context of nAChR neurobiology, this technique should be useful for studying many types of ligand-gated ion channels or receptors in neurons from brain slices.

In this paper, we describe a useful method to study ligand-gated ion channel function in neurons of acutely isolated brain slices. This method involves the use of a drug-filled micropipette for local application of drugs to neurons recorded using standard patch clamp techniques.

局部应用药物研究烟碱型乙酰胆碱受体功能的小鼠脑片

Tobacco use leads to numerous health problems, including cancer, heart disease, emphysema, and stroke. Addiction to cigarette smoking is a prevalent ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

树突分支中的海马区对小鼠的齿状回评

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

Quantification of Filamentous Actin (F-actin) Puncta in Rat Cortical Neurons

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich structures suggest alterations in synaptic integrity and connectivity. Here we provide a detailed protocol for culturing primary rat cortical neurons, Phalloidin staining for F-actin puncta, and subsequent quantification techniques. First, the frontal cortex of E18 rat embryos are dissociated into low-density cell culture, then the neurons grown in vitro for at least 12-14 days. Following experimental treatment, the cortical neurons are stained with AlexaFluor 488 Phalloidin (to label the dendritic F-actin puncta) and microtubule-associated protein 2 (MAP2; to validate the neuronal cells and dendritic integrity). Finally, specialized software is used to analyze and quantify randomly selected neuronal dendrites. F-actin rich structures are identified on second order dendritic branches (length range 25-75 &#181;m) with continuous MAP2 immunofluorescence. The protocol presented here will be a useful method for investigating changes in dendritic synapse structures subsequent to experimental treatments.

Quantification of Filamentous Actin F-actin Puncta in Rat Cortical Neurons

Filamentous actin (F-actin) plays an important role in spinogenesis, synaptic plasticity, and synaptic stability. Quantification of F-actin puncta is therefore a useful tool to study the integrity of synaptic structures. This protocol describes the procedures of quantifying F-actin puncta labeled with Phalloidin in low-density primary cortical neuronal cultures.

大鼠皮层神经元丝状肌动蛋白（F-肌动蛋白）泪点的量化

Filamentous actin protein (F-actin) plays a major role in spinogenesis, synaptic plasticity, and synaptic stability. Changes in dendritic F-actin rich ...

A Free-breathing fMRI Method to Study Human Olfactory Function

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, evaluation of the functions of the human central olfactory system with functional magnetic resonance imaging (fMRI) is still a challenge because of several technical difficulties. There are some significant variables to take into account when considering an effective method for mapping the function of the central olfactory system using fMRI, including proper odorant selection, the interaction between odor presentation and respiration, and potential anticipation of or habituation to odorants. An event-related, respiration-triggered olfactory fMRI technique can accurately administer odorants to stimulate the olfactory system while minimizing potential interference. It can effectively capture the precise onsets of fMRI signals in the primary olfactory cortex using our data post-processing method. The technique presented here provides an efficient and practical means for generating reliable olfactory fMRI results. Such a technique can ultimately be applied in the clinical realm as a diagnostic tool for diseases associated with olfactory degeneration, including Alzheimer&#39;s and Parkinson&#39;s disease, as we begin to further understand the complexities of the human olfactory system.

We present the technical challenges and solutions for obtaining reliable functional magnetic resonance imaging (fMRI) data from the human central olfactory system. This includes special considerations in olfactory fMRI paradigm design, descriptions of fMRI data acquisition with an MRI-compatible olfactometer, odorant selection, and a special software tool for data post-processing.

一种免费呼吸功能磁共振成像方法研究人类嗅觉功能

The study of human olfaction is a highly complex and valuable field with applications ranging from biomedical research to clinical evaluation. Currently, ...

Adeno-associated Virus-mediated Transgene Expression in Genetically Defined Neurons of the Spinal Cord

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or triple transgenic mice are generated in order to achieve selective expression of a reporter or effector gene (e.g., from the Rosa26 locus) in the desired spinal population. 2) Intraspinal injection of Cre-dependent recombinant adeno-associated virus (rAAV); here Cre-dependent AAV vectors coding for the reporter or effector gene of choice are injected into the spinal cord of mice expressing Cre recombinase in the desired neuronal subpopulation. This protocol describes how to generate Cre-dependent rAAV vectors and how to transduce neurons in the dorsal horn of the lumbar spinal cord segments L3-L5 with rAAVs. As the lumbar spinal segments L3-L5 are innervated by those peripheral sensory neurons that transmit sensory information from the hindlimbs, spontaneous behavior and responses to sensory tests applied to the hindlimb ipsilateral to the injection side can be analyzed in order to interrogate the function of the manipulated neurons in sensory processing. We provide examples of how this technique can be used to analyze genetically defined subsets of spinal neurons. The main advantages of virus-mediated transgene expression in Cre transgenic mice compared to classical reporter mouse-induced transgene expression are the following: 1) Different Cre-dependent rAAVs encoding various reporter or effector proteins can be injected into a single Cre transgenic line, thus overcoming the need to create several multiple transgenic mouse lines. 2) Intraspinal injection limits manipulation of Cre-expressing cells to the injection site and to the time after injection. The main disadvantages are: 1) Reporter gene expression from rAAVs is more variable. 2) Surgery is required to transduce the spinal neurons of interest. Which of the two methods is more appropriate depends on the neuron population and research question to be addressed.

Intraspinal injection of recombinase dependent recombinant adeno-associated virus (rAAV) can be used to manipulate any genetically labelled cell type in the spinal cord. Here we describe how to transduce neurons in the dorsal horn of the lumbar spinal cord. This technique enables functional interrogation of the manipulated neuron subtype.

腺相关病毒介导的脊髓基因神经元的转基因表达

Selective manipulation of spinal neuronal subpopulations has mainly been achieved by two different methods: 1) Intersectional genetics, whereby double or ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

间刺激间隔对啮齿类动物时间处理评估的力量

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

坐骨神经粉碎损伤大鼠模型中功能评估的3D运动学分析

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Ballistic Labeling of Pyramidal Neurons in Brain Slices and in Primary Cell Culture

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of pyramidal neurons and dendritic spines, a ballistic labeling technique can be utilized. In the present protocol, pyramidal neurons are labeled with DilC18(3) dye and analyzed using neuronal reconstruction software to assess neuronal morphology and dendritic spines. To investigate neuronal structure, dendritic branching analysis and Sholl analysis are performed, allowing researchers to draw inferences about dendritic branching complexity and neuronal arbor complexity, respectively. The evaluation of dendritic spines is conducted using an automatic assisted classification algorithm integral to the reconstruction software, which classifies spines into four categories (i.e., thin, mushroom, stubby, filopodia). Furthermore, an additional three parameters (i.e., length, head diameter, and volume) are also chosen to assess alterations in dendritic spine morphology. To validate the potential of wide application of the ballistic labeling technique, pyramidal neurons from in vitro cell culture were successfully labeled. Overall, the ballistic labeling method is unique and useful for visualizing neurons in different brain regions in rats, which in combination with sophisticated reconstruction software, allows researchers to elucidate the possible mechanisms underlying neurocognitive dysfunction.

We present a protocol to label and analyze pyramidal neurons, which is critical for evaluating potential morphological alterations in neurons and dendritic spines that may underlie neurochemical and behavioral abnormalities.

脑切片和原细胞培养中金字塔神经元的弹道标记

It has been reported that the size and shape of dendritic spines is related to their structural plasticity. To identify the morphological structure of ...