This work was supported by a Grant-in-Aid for Scientific Research (B) and a Grant-in Aid for Scientific Research on Innovative Areas 'Systems Molecular Ethology' from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan. T.U. was the recipient of a Grant-Aid from the Japan Society for the Promotion of Science for Young Scientists.

1.单队列殖民地的制备<ol><li>准备三蜂群创建两个单队列菌落并获得足够数量的新出现的工人。 <ol><li>检查在梳皑皑的外周细胞的一些蛹有棕色的眼睛和开放使用镊子封顶梳子色素角质层。如果外围梳状细胞中存在这些蛹，在整个梳子最蛹会出现在大约1-3天。 </li><li>随后，收集所有含有成人贴壁蜂已经用刷子去除后这些蛹的梳子，混合来自三个殖民地梳子，以减少菌落中的变异。 </li></ol></li><li>放置收集梳子在一个空蜂巢框和在33℃潮湿条件下用C（&gt; 60％相对湿度）孵育。收集大约6000新出现过工人从收集的梳子3天（每殖民地3000名工人）。 </li><li>确定借机的量基于五个新兴工人的重量RS随机抽取未经处理的殖民地收集。 <ol><li>重5使用称重机未经处理的殖民地随机抽取新出现的工人。 注:五个新出现的工人的重量一般500-600毫克的。 </li><li>通过将所收集的工作人员的重量由平均重量五个新出现工人估计收集工人的数量。 </li></ol></li><li>涂漆标志使用水性漆的海报标志约1,800工人（每900菌落工人）的thoraces。 注意:可洗的标记被用于施加涂料中列出的材料的表 。 </li><li>介绍一个皇后和大约0-2天的工人3000到包含两个梳子，蜂蜜含有梳1和花粉如腌制食品和产蛋女王另一个空梳一个新的蜂巢框。收集含有蜂蜜和花粉FR梳OM所有贴蜜蜂陆续未经处理的殖民地被删除。 注:建议使用核蜂巢箱（小型蜂巢盒）的。 </li><li>收集护士蜜蜂觅食早熟用油漆标记6〜8天创造的单队列殖民地之后。 <ol><li>为了收集护士蜜蜂，拿起那把他们的头梳进入细胞采取育雏保健工作者。使用镊子从包含许多育雏和成年工人收集梳理护士蜜蜂。 注意:如果收集工人HPGS如步骤1.7中所述解剖时被开发，这些工人被定义为护士蜜蜂。拿住镊子thoraces建议从梳子拿起工人。 </li><li>为了收集征粮，使用防虫网，以捕获返回自己的蜂巢对他们的后腿花粉负载工人。 注意:如果在步骤1.7中所述解剖时拍摄的工人HPGS出现缩水，这些工人被定义为觅食。 </li></ol></li><li> CLASSIFY的HPG分成三组，"开发"，"中间"和"皱缩"通过在立体显微镜下解剖。 <ol><li>麻醉在昆虫笼10-15蜜蜂在冰箱10-15分钟，直到蜜蜂无法飞行或步行。然后，移动蜜蜂在冰上，并完成在冰上麻醉5分钟。 注意:大约需要总15-20分钟达到麻醉。如果麻醉时间需要缩短，蜜蜂在笼子的数目应减少到每笼1-3蜜蜂。 </li><li>用剪刀和镊子解剖从主体的头部。固定放置在培养皿销牙科蜡头。将两个引脚到天线的基地，以固定头部。浸泡固定磁头在昆虫盐水（130毫米氯化钠，5毫米氯化钾，1毫米氯化钙2）。 </li><li>双目显微镜下除去用细镊子头部和手术刀的角质层的前方面。 注:HPGS可以在头部很容易找到去除的C后uticle。该HPG的是，在头部左右脑存在葡萄状的器官。 </li><li>取出气管组织是表皮下的白色膜状组织。然后，用小镊子缓缓地提拉他们解剖从头部HPGS。 </li><li>分类HPGS大和循环腺泡为"发达"。分类HPGS小和歪曲腺泡为"皱缩"。相应的分类既不"发达"，也没有"皱缩"HPGS为"中级"。 注意:代表的照片，显示这三个类HPG状态在图2中表示。 </li><li>主题"发达"和"皱缩"HPGS以RNA提取为"护士蜂HPGS&#39;和&#39;觅食HPGS"，分别和护士蜜蜂觅食（第4节）之间的HPGS比较基因表达。 </li></ol></li></ol> 2. 20E注射<ol><li>从收集典型菌落为DESCR护士蜜蜂IBED在过程1.6.1。 注:HPGS不能在这些蜜蜂进行审查，作为蜜蜂会被饲养。 </li><li>麻醉在4℃蜜蜂在冰箱（未在冰上）。 注意:这需要大约30分钟，使麻醉。如果麻醉时间被缩短，在笼蜜蜂的数目应减少到每笼1-3蜜蜂。 </li><li>使用镊子，固定放置在培养皿上的牙模的蜜蜂。 </li><li>注入1微升20E溶液成使用来自用玻璃针拔出器校准的毛细管微量制成的注射尖端的头部的前方面。 <ol><li>溶解20E在乙醇-昆虫盐水（130毫摩尔NaCl，5mM的氯化钾，1mM的氯化钙2）的混合物（1:4）以2.5微克/微升。 </li><li>喷射末梢连接到橡胶管中，和一个黄色枪头连接到管的相反的一侧。 </li><li>使用微量上放置封口膜1微升20E溶液，保持黄色微量移液器尖端在口中，一个d拉伸溶液进入注射小费。 </li><li>插入喷射尖端进入天线的基础上，并通过经由黄色微量移液器尖端吹注入该溶液。 注意:在此步骤中显示的协议是代表注射方法，但建议在必要时20安全使用自动注射机。 </li></ol></li><li>后在33-36℃的昆虫笼注入蜜蜂（理想温度为35℃）进行1或3天阴暗潮湿条件下（&gt; 60％相对湿度）下。供应蜂蜜 - 水混合物（1:1），以蜜蜂。如果需要的话，算上注射后幸存的蜜蜂。 </li></ol> 3.烯虫酯中的应用<ol><li>收集包含从典型菌落蛹所有附着的成年蜜蜂用刷子去除后，梳子。孵育梳子在33-36℃（理想温度为35℃）在潮湿的条件下为1天（&gt; 60％相对湿度）。 </li><l我&gt;收集从收集的梳新兴蜜蜂和应用的标志漆用海报涂料作为在协议中用于制备单队列菌落描述其thoraces。返回标记的蜜蜂他们的殖民地。 </li><li> 6天后，收集来自克隆彩绘工人（6日龄）。通过举办他们的thoraces用镊子拿起从梳子彩绘工人。 </li><li>麻醉在4℃蜜蜂在冰箱（未在冰上），为在步骤2.2。使用镊子，固定放置在培养皿上的牙模蜜蜂和应用1-5微升溶于丙酮中使用微量头甲氧普林溶液（50-250微克/微升）。使用过滤嘴是向丙酮抗性。 注意:丙酮可能会对蜜蜂导致死亡的有害影响。如果观察蜜蜂的死亡率高，建议丙酮在最小的体积为1微升的降低。 </li><li>后在昆虫笼蜜蜂在33-36°C（理想Ťemperature是35℃）下阴暗潮湿的条件下7天（&gt; 60％相对湿度）。供应蜂蜜 - 水混合物（1:1），以蜜蜂。如果需要的话，算上局部应用后幸存的蜜蜂。 注:在本文中，幸存的蜜蜂申请（ 表3）后7天计数。 </li></ol> 4. RNA提取和定量RT-PCR <ol><li>麻醉蜜蜂和步骤1.7.1说明1.7.4解剖HPGS。 </li><li>收集在干冰上和存储微量​​离心管的解剖HPGS在-80℃直到使用。 </li><li>添加400微升试剂为蛋白质变性。 注:此试剂是市售的（见材料表 ）。 </li><li>使用均质与离心管内适合一个同质化的杵举行电动搅拌机一只手HPG组织。均质化在约300rpm下的旋转速度在冰上打破和裂解组织细胞1分钟。 材料表 ）。 </li><li>加入80微升氯仿拌匀。在室温下孵育5分钟。 </li><li>离心机离心管在14170 XG在4℃下15分钟。将水相转移至新管中。添加异丙醇和1μl糖原（5毫克/毫升）的等体积。孵育在-20℃至少20分钟。 </li><li>离心微量离心管在14170×g离心10分钟，在4℃下，去除上清。 </li><li>加入400微升75％乙醇的颗粒拌匀，在4℃，14170 XG离心离心管15分钟，然后取出上清。重复这些步骤一次。 </li><li>空气干燥沉淀5-10分钟，然后将其溶解于10μl无RNA酶的水。 注:沉淀完全干燥可能导致RNA沉淀的不溶化于水中。因此，这是微湿的沉淀是适当的溶解。 </li><li>使用分光光度计等的NanoDrop或类似测量RNA浓度。 </li><li>通过在37℃温育30分钟以降解任何污染的基因组DNA的治疗500ng的总RNA用DNase I的2.0ü。 </li><li>反向转录的200ng DNA酶I处理过的RNA，并使用市售的试剂盒进行实时PCR（见材料与试剂的表的列表）和基因特异性引物（MRJP2 5&#39;-AAATGGTCGCTCAAAATGACAGA-3&#39;和5 &#39;-ATTCATCCTTTACAGGTTTGTTGC-3&#39;，Hbg3 5&#39;-TACCTGGCTTCGTGTCAAC-3&#39;和5&#39;-ATCTTCGGTTTCCCTAGAGAATG-3&#39;）。执行的PCR如下:[95℃×30秒+（95℃×5秒+ 60℃×15秒+ 72℃×20秒）×45-55个循环]。 </li><li>规范化成绩单量与延伸因子1α-F2（EF1α˗F2）或核糖体蛋白49（RP49）9,16,21,22的。这些基因是可靠的控制功能g烯类在使用QRT-PCR的HPGS分析基因表达。 </li><li>执行双尾t检验来检测使用统计两个软件实验组（护士对蜜蜂觅食早熟，20E治疗蜜蜂与控制蜜蜂和烯虫酯处理与蜜蜂蜜蜂控制）之间的显著差异。 注意:如果F-检验假设方差的同质性，学生t检验可以使用。如果不是，可以使用韦尔奇氏t检验。 </li></ol>

<ol>
	<li>Wilson, E. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The insect societies. , (1971).</li><li>Winston, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The biology of the honey bee. , (1987).</li><li>Lindauer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ein+Beitrag+zur+Frage+der+Arbeitsteilung+im+Bienenstaat.">Ein Beitrag zur Frage der Arbeitsteilung im Bienenstaat.</a> Zeitschrift f&#252;r vergleichende Physiologie. 34 (4), 299-345 (1952).</li><li>Sakagami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Untersuchungen+uber+die+Arbeitsteilung+in+einen+Zwergvolk+der+Honigbienen.+Beitrage+zur+Biologie+des+Bienenvolkes,+Apis+mellifera+L.">Untersuchungen uber die Arbeitsteilung in einen Zwergvolk der Honigbienen. Beitrage zur Biologie des Bienenvolkes, Apis mellifera L.</a> I. Jap J Zool. 11, 117-185 (1953).</li><li>Kubo, T., 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=Change+in+the+expression+of+hypopharyngeal-gland+proteins+of+the+worker+honeybees+(Apis+mellifera+L.)+with+age+and/or+role.">Change in the expression of hypopharyngeal-gland proteins of the worker honeybees (Apis mellifera L.) with age and/or role.</a> J Biochem. 119 (2), 291-295 (1996).</li><li>Ohashi, K., Natori, S., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Change+in+the+mode+of+gene+expression+of+the+hypopharyngeal+gland+cells+with+an+age-dependent+role+change+of+the+worker+honeybee+Apis+mellifera+L.">Change in the mode of gene expression of the hypopharyngeal gland cells with an age-dependent role change of the worker honeybee Apis mellifera L.</a> Eur J Biochem. 249 (3), 797-802 (1997).</li><li>Ohashi, K., Natori, S., Kubo, T. <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+amylase+and+glucose+oxidase+in+the+hypopharyngeal+gland+with+an+age-dependent+role+change+of+the+worker+honeybee+(Apis+mellifera+L).">Expression of amylase and glucose oxidase in the hypopharyngeal gland with an age-dependent role change of the worker honeybee (Apis mellifera L).</a> Eur J Biochem. 265 (1), 127-133 (1999).</li><li>Ohashi, K., Sawata, M., Takeuchi, H., Natori, S., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+cloning+of+cDNA+and+analysis+of+expression+of+the+gene+for+alpha-glucosidase+from+the+hypopharyngeal+gland+of+the+honeybee+Apis+mellifera+L.">Molecular cloning of cDNA and analysis of expression of the gene for alpha-glucosidase from the hypopharyngeal gland of the honeybee Apis mellifera L.</a> Biochem Biophys Res Commun. 221 (2), 380-385 (1996).</li><li>Ueno, T., Nakaoka, T., Takeuchi, H., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+gene+expression+in+the+hypopharyngeal+glands+of+worker+honeybees+(Apis+mellifera+L.)+associated+with+an+age-dependent+role+change.">Differential gene expression in the hypopharyngeal glands of worker honeybees (Apis mellifera L.) associated with an age-dependent role change.</a> Zoolog Sci. 26 (8), 557-563 (2009).</li><li>Robinson, G. E., Page, R. E., Strambi, C., Strambi, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hormonal+and+genetic+control+of+behavioral+integration+in+honey+bee+colonies.">Hormonal and genetic control of behavioral integration in honey bee colonies.</a> Science. 246 (4926), 109-112 (1989).</li><li>Sullivan, J. P., Fahrbach, S. E., Robinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juvenile+hormone+paces+behavioral+development+in+the+adult+worker+honey+bee.">Juvenile hormone paces behavioral development in the adult worker honey bee.</a> Horm Behav. 37 (1), 1-14 (2000).</li><li>Ben-Shahar, Y., Robichon, A., Sokolowski, M. B., Robinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+gene+action+across+different+time+scales+on+behavior.">Influence of gene action across different time scales on behavior.</a> Science. 296 (5568), 741-744 (2002).</li><li>Lehman, H. K., 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=Division+of+labor+in+the+honey+bee+(Apis+mellifera):+the+role+of+tyramine+beta-hydroxylase.">Division of labor in the honey bee (Apis mellifera): the role of tyramine beta-hydroxylase.</a> J Exp Biol. 209 (Pt 14), 2774-2784 (2006).</li><li>Mutti, N. S., Wang, Y., Kaftanoglu, O., Amdam, G. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Honey+bee+PTEN--description,+developmental+knockdown,+and+tissue-specific+expression+of+splice-variants+correlated+with+alternative+social+phenotypes).">Honey bee PTEN--description, developmental knockdown, and tissue-specific expression of splice-variants correlated with alternative social phenotypes).</a> PLoS One. 6 (7), e22195 (2011).</li><li>Whitfield, C. W., Cziko, A. M., Robinson, G. E. <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+profiles+in+the+brain+predict+behavior+in+individual+honey+bees.">Gene expression profiles in the brain predict behavior in individual honey bees.</a> Science. 302 (5643), 296-299 (2003).</li><li>Ueno, T., Takeuchi, H., Kawasaki, K., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+the+Gene+Expression+Profiles+of+the+Hypopharyngeal+Gland+of+Worker+Honeybees+in+Association+with+Worker+Behavior+and+Hormonal+Factors.">Changes in the Gene Expression Profiles of the Hypopharyngeal Gland of Worker Honeybees in Association with Worker Behavior and Hormonal Factors.</a> PLoS One. 10 (6), e0130206 (2015).</li><li>Paul, R. K., Takeuchi, H., Matsuo, Y., Kubo, T. <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+of+ecdysteroid-regulated+gene+E74+of+the+honeybee+in+ovary+and+brain.">Gene expression of ecdysteroid-regulated gene E74 of the honeybee in ovary and brain.</a> Insect Mol Biol. 14 (1), 9-15 (2005).</li><li>Takeuchi, 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=Identification+of+a+novel+gene,+Mblk-1,+that+encodes+a+putative+transcription+factor+expressed+preferentially+in+the+large-type+Kenyon+cells+of+the+honeybee+brain.">Identification of a novel gene, Mblk-1, that encodes a putative transcription factor expressed preferentially in the large-type Kenyon cells of the honeybee brain.</a> Insect Mol Biol. 10 (5), 487-494 (2001).</li><li>Takeuchi, H., Paul, R. K., Matsuzaka, E., Kubo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EcR-A+expression+in+the+brain+and+ovary+of+the+honeybee+(Apis+mellifera+L).">EcR-A expression in the brain and ovary of the honeybee (Apis mellifera L).</a> Zoolog Sci. 24 (6), 596-603 (2007).</li><li>Yamane, T., Miyatake, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reduced+female+mating+receptivity+and+activation+of+oviposition+in+two+Callosobruchus+species+due+to+injection+of+biogenic+amines.">Reduced female mating receptivity and activation of oviposition in two Callosobruchus species due to injection of biogenic amines.</a> Journal of Insect Physiology. 56 (3), 271-276 (2010).</li><li>Ben-Shahar, Y., Leung, H. T., Pak, W. L., Sokolowski, M. B., Robinson, G. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=cGMP-dependent+changes+in+phototaxis:+a+possible+role+for+the+foraging+gene+in+honey+bee+division+of+labor.">cGMP-dependent changes in phototaxis: a possible role for the foraging gene in honey bee division of labor.</a> J Exp Biol. 206 (Pt 14), 2507-2515 (2003).</li><li>Danforth, B. N., Ji, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elongation+factor-1+alpha+occurs+as+two+copies+in+bees:+implications+for+phylogenetic+analysis+of+EF-1+alpha+sequences+in+insects.">Elongation factor-1 alpha occurs as two copies in bees: implications for phylogenetic analysis of EF-1 alpha sequences in insects.</a> Mol Biol Evol. 15 (3), 225-235 (1998).</li><li>Pandey, A., Bloch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Juvenile+hormone+and+ecdysteroids+as+major+regulators+of+brain+and+behavior+in+bees.">Juvenile hormone and ecdysteroids as major regulators of brain and behavior in bees.</a> Current Opinion in Insect Science. 12, 26-37 (2015).</li></ol>

The authors have nothing to disclose.

单队列殖民地的制备在这里，我们描述了我们先前的研究16用于为与工蜂的角色转变相关的生理HPG分析准备单队列殖民地的协议。在单队列菌落（ 表1）中观察到护士蜜蜂和早熟觅食出满足在程序1.6-1.7和图2中所描述的标准。在HPG分类为基准规定的图2中的照片是有用的。基于这两个工人行为和HPG发展的标准是有效的，用于定义典型...

欧洲蜜蜂， 蜜蜂 ，是一种完全社会性昆虫具有高度组织性的社会1。工蜂（劳动等级）从事各种工作，以保持集落的活性，并且这些任务根据羽化后工蜂的年龄，这被称为劳动2-4的年龄相关的分割发生变化。青年工人（&lt;13日龄）照顾在蜂巢的育雏通过分泌蜂王浆（蜂护士），而年纪较大的工人（&gt; 15天）采集花蜜和花粉蜂巢（征粮）2-4之外。工人的生理与这个角色转变的关联变化。例如，下咽腺（HPGS）的功能，配对位于头外分泌腺，改变与护理的作用转变为觅食2,5关联。护士蜂HPGS主要合成王浆主蛋白，是蜂王浆主要成分。另一方面，觅食HPGS主要合成碳水化合物代谢酶，如α葡糖苷酶III，通过将蔗糖转化为葡萄糖和果糖处理花蜜到蜂蜜。我们以前的研究显示，MRJP2的表达，其编码的一个主要蜂王浆蛋白质和Hbg3，它的作用移6-9中编码α葡糖苷酶III，变更。 要确定是否在工人组织中的生理变化，包括HPGS，与角色的转变或与工人的年龄，这将是操纵蜜蜂殖民地的人口组成，如准备单队列有用菌落在几乎相同年龄的工人执行不同的任务10,11。 Robinson 等人。（1989）描述了用于建立单一的队列菌落10的方法。单队列殖民地最初包括女王和0-2日龄工人。建立殖民地几天后，ALM工人OST同龄承担不同的任务。有些工人进行护理任务，如在典型菌落，而其他工作人员执行任务觅食比往年提前，并因此被称为早熟觅食。护士蜜蜂觅食早熟之间的基因表达的比较将提供有关工作的组织12-16的相关角色的生理有用的信息。这里，我们描述了制备单队列菌落HPGS 16的角色和/或与年龄相关的生理分析的详细协议。我们还简要介绍了如何通过定量逆转录-聚合酶链反应（RT-PCR）检查MRJP2和Hbg3的基因的表达来评价HPG生理学。 除了工人生理学单队列菌落的分析，内分泌系统的检查是用于分析相关联的角色的工人生理学的调节机制重要。幼激素（JH），其硝酸钾WN在昆虫幼虫的&#39;现状&#39;的激素，加速从护理的作用，在工蜂11觅食的转变。此外，蜕皮激素，这是变态过程称为蜕皮激素，可能参与的作用移作为编码蘑菇体被表达，工人脑17-19的较高中心蜕皮激素信号传导分子的基因。因此，我们还描述了在我们以前的研究16用于与20E治疗工人的详细协议，它是蜕皮激素的活性形式，和烯虫酯，一JH类似物，为的内分泌系统的上HPG生理效应分析（表达MRJP2和Hbg3）。

<table border="0" cellpadding="0" cellspacing="0" width="769">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:231px;">UNIPOSCA</td>
			<td style="width:120px;">Mitsubishi pencil</td>
			<td style="width:91px;">PC-5M</td>
			<td style="width:327px;">Marker pen for the application of marks to bees&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">20-hydroxyecdysone</td>
			<td>Sigma Aldrich</td>
			<td>H5142</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methoprene</td>
			<td>Sigma Aldrich</td>
			<td>33375</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Breeding case insect</td>
			<td>IRIS OHYAMA</td>
			<td>CP-SS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electromotion mixier&#160;</td>
			<td>ISO</td>
			<td>23M-R25</td>
			<td>homogenization of tissue</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIZol Reagent</td>
			<td>Invitrogen</td>
			<td>15596-026</td>
			<td>the reagent for total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DNase I&#160;</td>
			<td>Takara</td>
			<td>2270A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PrimeScript RT reagent kit</td>
			<td>Takara</td>
			<td>RR037A</td>
			<td>the reagent for reverse transcription</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SYBR Premix ExTaq II</td>
			<td>Takara</td>
			<td>RR820A</td>
			<td>the reagent for real-time PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycle 1.2 Instrument</td>
			<td>Roche</td>
			<td>12011468001</td>
			<td>the instrument for real-time PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">LightCycle Capillaries (20&#956;l)</td>
			<td>Roche</td>
			<td>4929292001</td>
			<td>the material for real-time PCR</td>
		</tr>
	</tbody>
</table>

preparation of single-cohort colonies and hormone treatment of worker honeybees to analyze physiology associated with role and/or endocrine system

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related division of labor). Young workers are engaged in nursing the brood (nurse bees), while older workers are engaged in foraging for nectar and pollen (foragers). The physiology of the workers changes in association with this role shift. For example, the main function of the hypopharyngeal glands (HPGs) changes from the secretion of major royal jelly proteins (MRJPs) to the secretion of carbohydrate-metabolizing enzymes. Because worker tasks change as the workers age in typical colonies, it is difficult to discriminate the physiological changes that occur with aging from those that occur with the role shift. To study the physiological changes in worker tissues, including the HPGs, in association with the role shift, it would be useful to manipulate the honeybee colony population by preparing single-cohort colonies in which workers of almost the same age perform different tasks. Here we describe a detailed protocol for preparing single-cohort colonies for this analysis. Six to eight days after single-cohort colony preparation, precocious foragers that perform foraging tasks earlier than usual appear in the colony. Representative results indicated role-associated changes in HPG gene expression, suggesting role-associated HPG function. In addition to manipulating the colony population, analysis of the endocrine system is important for investigating role-associated physiology. Here, we also describe a detailed protocol for treating workers with 20-hydroxyecdysone (20E), an active form of ecdysone, and methoprene, a juvenile hormone analogue. The survival rate of treated bees was sufficient to examine gene expression in the HPGs. Gene expression changes were observed in response to 20E- and/or methoprene-treatment, suggesting that hormone treatments induce physiological changes of the HPGs. The protocol for hormone treatment described here is appropriate for examining hormonal effects on worker physiology.

该协议用 ​​于制备单队列菌落的概述图1A中示出。从制备单队列菌落样品收集实验时间过程示于图1B。该纳护理行为或取食行为的行为准则工人从单队列菌落收集，并且HPG发展这些工人估计表1示出的HPG发展的分类为三组。 "开发"，"中间"和"皱缩"根据大小。示出这三个类HPGS的代表性照片在图2中所示，结果表明，这两个?...

Watch this Scientific Journal Video about Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System at JoVE.com

工蜂单群菌落的制备和激素处理以分析与角色和/或内分泌系统相关的生理学|神经科学 |视频

Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

在这里，我们描述详细的协议单队列蜂群的准备 - 分析相关角色工人生理的有用工具。我们还描述了用于治疗工人幼激素和蜕皮激素评估这些激素在工作者行为和/或生理调控的参与详细协议。

单队列殖民地和工蜂的激素治疗的准备来分析生理学与关联作用和/或内分泌系统

Honeybee workers are engaged in various tasks related to maintaining colony activity. The tasks of the workers change according to their age (age-related ...

preparation-single-cohort-colonies-hormone-treatment-worker-honeybees

Research

Education

JoVE Journal

JoVE Core

Biology

Neuroscience

Human Biology

Endocrine System

SCIENTISTS IN ACTION

15.7K 次观看.  The University of Tokyo. 在这里，我们描述详细的协议单队列蜂群的准备 - 分析相关角色工人生理的有用工具。我们还描述了用于治疗工人幼激素和蜕皮激素评估这些激素在工作者行为和/或生理调控的参与详细协议。 

视频: Preparation of Single-cohort Colonies and Hormone Treatment of Worker Honeybees to Analyze Physiology Associated with Role and/or Endocrine System

Preparation and Culture of Chicken Auditory Brainstem Slices

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at discreet periods of development 1-4 as well as mechanisms for temporal coding in the central nervous system 5-7.
Here we present a method to prepare chicken auditory brainstem slices that can be used for acute experimental procedures or to culture organotypic slices for long-term experimental manipulations.
The chicken auditory brainstem is composed of nucleus angularis, magnocellularis, laminaris and superior olive. These nuclei are responsible for binaural sound processing and single coronal slice preparations preserve the entire circuitry. Ultimately, organotypic slice cultures can provide the opportunity to manipulate several developmental parameters such as synaptic activity, expression of pre and postsynaptic components, expression of aspects controlling excitability and differential gene expression
This approach can be used to broaden general knowledge about neural circuit development, refinement and maturation.

The chicken auditory brainstem is comprised of nuclei responsible for binaural sound processing. A single coronal slice preparation maintains the entire circuitry while the cultured approach provides a unique preparation to study the development of neuronal structure and auditory function at the molecular, cellular and network levels.

鸡脑干切片的制备及文化

The chicken auditory brainstem is a well-established model system that has been widely used to study the anatomy and physiology of auditory processing at ...

科研

神经科学

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

钙成像的急性下区片的制备

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent and divergent anatomical connections including feed forward and feedback wiring. Furthermore, information of the same origin is partially sent via parallel pathways to different and sometimes into the same brain areas. To understand the evolutionary benefits as well as the computational advantages of these wiring strategies and especially their temporal dependencies on each other, it is necessary to have simultaneous access to single neurons of different tracts or neuropiles in the same preparation at high temporal resolution. Here we concentrate on honeybees by demonstrating a unique extracellular long term access to record multi unit activity at two subsequent neuropiles1, the antennal lobe (AL), the first olfactory processing stage and the mushroom body (MB), a higher order integration center involved in learning and memory formation, or two parallel neuronal tracts2 connecting the AL with the MB.&#160;The latter was chosen as an example and will be described in full. In the supporting video the construction and permanent insertion of flexible multi&#160;channel wire electrodes is demonstrated. Pairwise differential amplification of the micro&#160;wire electrode channels drastically reduces the noise and verifies that the source of the signal is closely related to the position of the electrode tip. The mechanical flexibility of the used wire electrodes allows stable invasive long&#160;term recordings over many hours up to days, which is a clear advantage compared to conventional extra&#160;and intracellular in vivo recording techniques.

Simultaneous extracellular long term recordings from two different brain neuropiles or two different anatomical tracts were established in honeybees. These recordings allow the investigation of temporal aspects of neuronal processing across different brain areas at the single neuron as well as at the ensemble level in a behaving animal.

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

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 ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Video Imaging and Spatiotemporal Maps to Analyze Gastrointestinal Motility in Mice

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

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

视频成像和时空地图来分析胃肠动力小鼠

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

Methods and Tips for Intravenous Administration of Adeno-associated Virus to Rats and Evaluation of Central Nervous System Transduction

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), optogenetics, cre-lox targeting, etc. The purpose of this manuscript is to aid the investigator attempting expansive central nervous system (CNS) gene transfer in the rat via tail vein injection of AAV. Wide-scale expression is relevant for conditions with widespread pathology, and a rat model is significant due to its greater size and physiologic similarities to humans compared to mice. In this example application, a wide-scale neuronal transduction is used to mimic a neurodegenerative disease that affects the entire spinal cord, amyotrophic lateral sclerosis (ALS). The efficient wide-scale CNS transduction can also be used to deliver therapeutic protein factors in pre-clinical studies. After a post-injection expression interval of several weeks, the effects of the transduction are evaluated. For a green fluorescent protein (GFP) control vector, the amount of GFP in the cerebellum is estimated quickly and reliably by a basic imaging program. For motor disease phenotypes that are induced by the ALS related protein transactive response DNA-binding protein of 43 kDa (TDP-43), the deficits are scored by escape reflex and rotarod. Beyond disease modeling and gene therapy, there are diverse potential applications for the wide-scale gene targeting described here. The expanded use of this method will aid in expediting hypothesis testing in the neurosciences and neurogenetics.

Methods for a wide-scale central nervous system gene delivery in the rat are covered. In this example, the purpose is to mimic a disease that affects the entire spinal cord. The widespread transduction can be used to deliver a therapeutic protein to the CNS from a one-time, peripheral administration.

腺相关病毒对大鼠的静脉管理方法和技巧及中枢神经系统转导的评价

Adeno-associated virus (AAV) vectors are a key reagent in the neurosciences for clustered regularly interspaced short palindromic repeats (CRISPR), ...

Online Transcranial Magnetic Stimulation Protocol for Measuring Cortical Physiology Associated with Response Inhibition

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) characterization of primary motor cortex (M1) excitability and inhibition. Motor response inhibition prevents unwanted actions and is abnormal in several neuropsychiatric conditions. TMS is a non-invasive technology that can quantify M1 excitability and inhibition using single- and paired-pulse protocols and can be precisely timed to study cortical physiology with high temporal resolution. We modified the original Slater-Hammel (S-H) stop signal task to create a &#34;racecar&#34; version with TMS pulses time-locked to intra-trial events. This task is self-paced, with each trial initiating after a button push to move the racecar towards the 800 ms target. GO trials require a finger-lift to stop the racecar just before this target. Interspersed randomly are STOP trials (25%) during which the dynamically adjusted stop signal prompts subjects to prevent finger-lift. For GO trials, TMS pulses were delivered at 650 ms after trial onset; whereas, for STOP trials, the TMS pulses occurred 150 ms after the stop signal. The timings of the TMS pulses were decided based on electroencephalography (EEG) studies showing event-related changes in these time ranges during stop signal tasks. This task was studied in 3 blocks at two study sites (n=38) and we recorded behavioral performance and event-related motor-evoked potentials (MEP). Regression modelling was used to analyze MEP amplitudes using age as a covariate with multiple independent variables (sex, study site, block, TMS pulse condition [single- vs. paired-pulse], trial condition [GO, successful STOP, failed STOP]). The analysis showed that TMS pulse condition (p&#60;0.0001) and its interaction with trial condition (p=0.009) were significant. Future applications for this online S-H/TMS paradigm include the addition of simultaneous EEG acquisition to measure TMS-evoked EEG potentials. A potential limitation is that in children, the TMS pulse sound could&#160;affect behavioral task performance.

We describe an experimental procedure to quantify excitability and inhibition of primary motor cortex during a motor response inhibition task by using Transcranial Magnetic Stimulation throughout the course of a Stop Signal Task.

在线经颅磁刺激测量与反应抑制相关的皮质生理

We describe the development of a reproducible, child-friendly motor response inhibition task suitable for online Transcranial Magnetic Stimulation (TMS) ...

Convection Enhanced Delivery of Optogenetic Adeno-associated Viral Vector to the Cortex of Rhesus Macaque Under Guidance of Online MRI Images

In non-human primate (NHP) optogenetics, infecting large cortical areas with viral vectors is often a difficult and time-consuming task. Here, we demonstrate the use of magnetic resonance (MR)-guided convection enhanced delivery (CED) of optogenetic viral vectors into primary somatosensory (S1) and motor (M1) cortices of macaques to obtain efficient, widespread cortical expression of light-sensitive ion channels. Adeno-associated viral (AAV) vectors encoding the red-shifted opsin C1V1 fused to yellow fluorescent protein (EYFP) were injected into the cortex of rhesus macaques under MR-guided CED. Three months post-infusion, epifluorescent imaging confirmed large regions of optogenetic expression (&#62;130 mm2) in M1 and S1 in two macaques. Furthermore, we were able to record reliable light-evoked electrophysiology responses from the expressing areas using micro-electrocorticographic arrays. Later histological analysis and immunostaining against the reporter revealed widespread and dense optogenetic expression in M1 and S1 corresponding to the distribution indicated by epifluorescent imaging. This technique enables us to obtain expression across large areas of the cortex within a shorter period of time with minimal damage compared to the traditional techniques and can be an optimal approach for optogenetic viral delivery in large animals such as NHPs. This approach demonstrates great potential for network-level manipulation of neural circuits with cell-type specificity in animal models evolutionarily close to humans.&#160;

Here, we demonstrate magnetic resonance (MR)-guided convection enhanced delivery (CED) of viral vectors into the cortex as an efficient and simplified approach for achieving optogenetic expression across large cortical areas in the macaque brain.