作者感谢 Arnim Jenett, Aljoscha 海滨, 和鲁宾实验室的其他成员的建议和共享的未发表的试剂和 Janelia 飞光项目组产生共焦图像。作者还感谢高卢实验室的成员对手稿的评论。

1. 为多色 FlpOut (MCFO) 实验准备苍蝇 注意: MCFO 技术指的是所谓的 Flp 介导的停止盒式切除 (FlpOut) 的修改版本。转基因 MCFO 蝇携带热休克启动子 (hsp)-Flp 重组和不同的记者在无人控制下。每个记者都由一个 myristoylated (myr) 超级文件夹绿色荧光蛋白 (sfGFP) 的共同骨干组成, 其中10个表位标签 ( e. g ., HA, 旗或 V5) 的副本已经插入。所产生的荧光蛋白的命名和 #34; 意大利面条怪兽协调和 #34; (smGFPs) 14 , 可以使用针对不同表位标记的特定抗体检测到. 
<ol> <li> 与 MCFO 磁带和 hsp Flp (hsp-Flp; 10 xuas-首次登记税-停止-首次登记税-myr-smGFP-房委会, 10 xuas-首次登记税-停止-首次登记税-myr-smGFP-旗帜, 10 xuas-首次登记-停止-首次登记-myr-smGFP-V5) 到适当的神经胶质 GAL4 驱动程序线.
	<ol> <li> 由于 hsp 在25和 #176 已处于活动状态; c 和少数细胞可能会进行重组, 导致不标记, 设置十字在18和 #176; c, 以避免系统泄漏。在18和 #176 等待大约20天; C 要获得 F1 生成。在热冲击以后 (参见步骤 2), 维护飞行在25和 #176; C ( 图 3 ). </li>
	</ol> </li> </ol> 2。热冲击 注意: 根据插入点的不同, Flp 的效率可能会有所不同, 因此, 最佳的热冲击时间必须优化。对于这个协议, 在 X 染色体上插入热休克蛋白-Flp 的 MCFO 飞行储存 (参见 材料表 ). 
<ol> <li> 在步骤 1.1 (F1 代蝇、3-4 天) 中将十字的子代转移到包含食品和热休克的新瓶中37和 #176; C 在水浴. 
	注: 幼蝇 (1-2 天) 对热休克非常敏感。等待1或2天以上的热休克, 以降低死亡率的治疗造成的。使用一些 F1 代苍蝇作为一个控制, 没有热休克. </li>
	<li> 在热休克期间, 在正常的小瓶中维持苍蝇的食物, 以降低压力导致的死亡率。把雌性和雄性放在不同的瓶子里. </li>
	<li> 请务必将整个瓶子浸入水中, 以确保均匀的热休克。使用5-8 分钟休克作为一个适当的出发点, 稀疏标签的胶质细胞与许多 GAL4 驱动线;冲击持续时间必须优化每一个驱动程序和实验 (更短或更长的热休克时间可能是必要的). </li>
	<li> 热休克后, 将小瓶水平放置在长凳上几分钟, 以便让苍蝇从热休克中恢复. 
	注: 不需要更换药瓶. </li>
	<li> 保持25和 #176 的苍蝇; C 和解剖 (见步骤3和 4) 他们2或更多天后, 热休克的最佳记者表达. </li>
</ol> 3。解剖准备和解决方案 <ol> <li> 解剖的当天, 在200和 #181 中准备新的固定液; 通过混合180和 #181 的 S2 细胞培养基, 以20和 #181; l. 20% 甲醛 (粉煤灰), 以获得2% 的粉煤灰溶液。在解剖之前和期间保持固定液在冰上. 
	注意: 粉煤灰是有毒的, 必须小心处理. 
	注: 混合160和 #181; l S2 细胞培养基与40和 #181; l 20% 甲醛 (粉煤灰) 在200和 #181; l PCR 管制备4% 的煤灰溶液而不是2% 的溶液. </li>
	<li> 使用一个 PCR 管每个基因型与最多8-10 大脑每管最佳染色结果. </li>
	<li> 准备成人大脑洗涤解决方案: 0.5% 牛血清白蛋白, 0.5% X-100 在 PBS. 
	注: 根据染色, 可能需要一个包含 1% X-100 的溶液。更高浓度的海卫一 X-100 增加了抗体的渗透到组织和它是必要的染色区域深处的组织 ( 例如, 突触区域在成人的 果蝇 脑). </li>
	<li> 准备堵塞解决方案: 3% 正常山羊血清, 3% 正常驴血清, 0.5% X-100 在 PBS. </li>
	<li> 成人洗脑液和阻断溶液可存储在4和 #176; C 几个星期. </li>
	<li> 将深凹陷井玻璃板放在冰上, 并用以下溶液填充, 其中有70% 乙醇, 一个带有 PBS, 一个带有 S2 细胞培养基. </li>
</ol> 4。成人大脑解剖 <ol> <li> 在显微镜的舞台上放置一个3厘米的解剖盘, 并用冷 S2 细胞培养基填充。在这个培养基中进行所有的解剖, 以确保组织在解剖过程中保持健康. 
	注: 使用解剖盘内衬几毫米黑色硅胶提供了良好的背景对比 (在图像), 并保存在解剖钳. </li>
	<li> 麻醉所需基因型的苍蝇与 CO 2 . </li>
	<li> 用镊子, 抓住苍蝇的翅膀和洗涤它在冷70% 乙醇三十年代, 然后与冷 PBS 额外三十年代. </li>
	<li> 将苍蝇移入深凹陷, 充满 S2 细胞培养基. </li>
	<li> 对相同基因型的所有果蝇重复相同的过程, 并在冷 S2 细胞培养基中保持它们直到解剖, 这将保存组织. 
	注: 麻醉在 S2 细胞培养基中, 只有在30分钟长潜伏期的时间内可以解剖的苍蝇数量可能会损伤组织. </li>
	<li> 用镊子抓苍蝇, 在显微镜下, 将苍蝇浸入 S2 细胞培养基中. </li>
	<li> 将头部与身体其他部分分离。丢弃身体, 将头部浸入 S2 细胞培养基中。在整个解剖过程中, 用钳子钳住头部是极其重要的。如果头部开始漂浮, 就很难在不损坏组织的情况下进行检索. </li>
	<li> 通过拉出一只眼睛开始解剖, 同时始终保持头部至少有一个镊子。确保镊子的尖端就在视网膜下面, 以避免破坏下面的组织。拉出另一只眼睛, 开始去除角质层, 直到大脑出现. </li>
	<li> 将所有气管组织和角质层移到大脑周围, 直到组织出现清洁。确保将所有的气管组织移到大脑周围, 以获得最佳的染色效果;现在, 这些组织已经准备好被修复和染色. </li>
	<li> 在冷 S2 细胞培养基中保持所有解剖的脑部, 然后将其转移到粉煤灰溶液中, 以便时间固定. </li>
</ol> 5。成人脑部染色 ol> 在室温 (RT) 中, 用 P10 吸管将分离的大脑转移到粉煤灰溶液中。为了避免组织损伤, 永远不要用镊子在解剖后转移大脑。在200和 #181 中执行所有步骤, 在 nutator 上进行 PCR. </li>
	<li> 对于所有步骤, 在用 P200 吸管丢弃旧的解决方案之前, 允许大脑在管的底部进行沉淀。试着除去上清液而不吸进组织。保护组织免受光照射. </li>
	<li> 在200和 #181 中修复大脑; 2% 粉煤灰在 S2 细胞培养基中为1小时或4% 粉煤灰30分钟。在样品间保持固定时间相同, 以提高重现性. </li>
	<li> 3 (或更多) 洗涤15分钟, 每200和 #181; 成人脑冲洗液, 用200和 #181 阻断组织; l 阻塞溶液30分钟。更长的潜伏期在阻拦解答是可能的. </li>
	<li> 在4和 #176 中一夜之间孵化大脑; C. 在成人脑洗涤液中稀释的初级抗体, 总容积为200和 #181; L. 
	注意: 潜伏期可能会因所使用的抗体类型而异。可能需要更长的孵化。
	<ol> <li> 用于标记三 MCFO 标记, 用反 HA (1:500)、反旗 (DYKDDDDK 表位) (1:100) 和 anti-V5 主抗体孵育大脑. </li>
		<li> 主直接共轭抗体可用于替代正常的主 + 次组合 ( e. g ., anti-V5:DyLight 549 (1:200))。在这种情况下, 将直接共轭抗体视为二次抗体. 
		注意: 对于每个基因型, 保持一些大脑作为对照, 以验证染色的特异性。跳过主抗体培养, 并直接进入下一步. </li>
	</ol> </li> <li> 在200和 #181 中为1小时清洗组织3次; 成人洗脑液 (步骤 3.3), 并在成人脑洗涤液中用二次荧光-共轭/原直接共轭抗体进行孵育, 在晚上4#176; C 或在 RT 上为4小时, 总容量为200和 #181; L 注意: 适当的抗体的例子: AlexaFluor 488 (1:250), anti-V5:DyLight 549 (1:200) 和 DyLight 647 (1:100)-共轭抗体. </li>
	<li> 在200和 #181 中洗脑3次1小时; 成人洗脑液, 在200和 #181 的最后一次洗涤, 在4和 #176; C 或1-2 小时; 在 pbs 的最后洗涤步骤删除任何痕迹的海卫一 X-100, 是必要的最佳染色效果。装在安装介质上的大脑, 在玻璃片上有一个防剂. </li>
</ol> 6。安装的大脑成像 <ol> <li> 使用剪刀将成像间隔剪裁到适当的大小。对于高分辨率显微镜, 三明治的标本和成像间隔两个玻璃片. 
	注: 成像垫片是薄 (0.12 毫米厚) 胶粘剂垫片, 剥离和坚持玻璃片或显微镜幻灯片, 允许安装的标本没有压缩. </li>
	<li> 使用镊子, 从一个表面上取下粘合剂衬垫, 然后在玻璃片 (22 mm x 60 mm) 的表面上应用垫片、胶粘剂侧。应用10和 #181; L 安装介质到新的玻璃片. </li>
	<li> 用 P10 吸管, 将大脑从200和 #181 中转移; L 管到片, 旁边的安装介质下降。与组织一起, 转移一些 PBS, 以保持大脑在解决方案. </li>
	<li> 用 P10 吸管将大脑从 pbs 移到安装介质的下降, 试图限制 pbs 的数量。将试样在片上的安装介质上传送到成像间隔。使用足够的安装介质来覆盖试样. </li>
	<li> 从垫片的上部取下其它粘合剂衬垫, 并在试样顶部加一个玻璃片。用镊子的尖端, 施加一个柔和的压力在胶粘剂区域密封。立即将安装的组织图像或存储在-20 和 #176 的幻灯片; C 用于以后的分析. </li>
</ol> 7。图像获取 <ol> <li> 使用 40X (na = 1.2, 浸水) 目标或 63X (na = 1.4, 油浸) 目标共聚焦显微镜获取共焦荧光图像的堆栈 (像素大小 = 1024 x 1024 在 xy 平面上;两个共焦部分之间的距离 = 0.5 和 #181; m)。在每个通道中调整探测器增益和偏移, 使图像中不存在饱和像素和零像素值;这样可以避免信息丢失, 并确保使用探测器的全部动态范围. 
	注: 由于3D 成像耗时, 因此可以通过在不同位置并行扫描多个样本来自动进行测量。为了避免蒸发与水浸泡的目的, 使用特殊的油浸泡介质与折射率 n = 1.33. </li>
	<li> 使用任何标准图像分析软件处理最大密度投影、通道色调变化以及亮度和对比度的调整 (请参阅 材料表 ). </li>
</ol>

<ol>
	<li>Freeman, M. R., Doherty, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+cell+biology+in+Drosophila+and+vertebrates.">Glial cell biology in Drosophila and vertebrates.</a> Trends Neurosci. 29 (2), 82-90 (2006).</li><li>Stork, T., Bernardos, R., Freeman, M. R. <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+glial+cell+development+and+function+in+Drosophila.">Analysis of glial cell development and function in Drosophila.</a> Cold Spring Harb Protoc. 2012 (1), 1-17 (2012).</li><li>Corty, M. M., Freeman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+in+neuroscience:+Architects+in+neural+circuit+design:+Glia+control+neuron+numbers+and+connectivity.">Cell biology in neuroscience: Architects in neural circuit design: Glia control neuron numbers and connectivity.</a> J Cell Biol. 203 (3), 395-405 (2013).</li><li>Hidalgo, A., Kato, K., Sutcliffe, B., McIlroy, G., Bishop, S., Alahmed, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+neuron-glia+interactions+and+cell+number+adjustments+in+the+fruit+fly.">Trophic neuron-glia interactions and cell number adjustments in the fruit fly.</a> Glia. 59 (9), 1296-1303 (2011).</li><li>Liu, J., Speder, P., Brand, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+brain+development+and+homeostasis+by+local+and+systemic+insulin+signalling.">Control of brain development and homeostasis by local and systemic insulin signalling.</a> Diabetes Obes Metab. 16, 16-20 (2014).</li><li>Kurant, E., Axelrod, S., Leaman, D., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six-microns-under+acts+upstream+of+Draper+in+the+glial+phagocytosis+of+apoptotic+neurons.">Six-microns-under acts upstream of Draper in the glial phagocytosis of apoptotic neurons.</a> Cell. 133 (3), 498-509 (2008).</li><li>Barres, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mystery+and+magic+of+glia:+a+perspective+on+their+roles+in+health+and+disease.">The mystery and magic of glia: a perspective on their roles in health and disease.</a> Neuron. 60 (3), 430-440 (2008).</li><li>Edenfeld, G., Stork, T., Klambt, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-glia+interaction+in+the+insect+nervous+system.">Neuron-glia interaction in the insect nervous system.</a> Curr Opin Neurobiol. 15 (1), 34-39 (2005).</li><li>Oland, L. A., Tolbert, L. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Key+interactions+between+neurons+and+glial+cells+during+neural+development+in+insects.">Key interactions between neurons and glial cells during neural development in insects.</a> Annu Rev Entomol. 48, 89-110 (2003).</li><li>Stork, T., Sheehan, A., Tasdemir-Yilmaz, O. E., Freeman, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuron-glia+interactions+through+the+Heartless+FGF+receptor+signaling+pathway+mediate+morphogenesis+of+Drosophila+astrocytes.">Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes.</a> Neuron. 83 (2), 388-403 (2014).</li><li>Zwarts, L., Van Eijs, F., Callaerts, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glia+in+Drosophila+behavior.">Glia in Drosophila behavior.</a> J Comp Physiol A Neuroethol Sens Neural Behav Physiol. 201 (9), 879-893 (2015).</li><li>Nern, A., Pfeiffer, B. D., Svoboda, K., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+new+site-specific+recombinases+for+use+in+manipulating+animal+genomes.">Multiple new site-specific recombinases for use in manipulating animal genomes.</a> Proc Natl Acad Sci U S A. 108 (34), 14198-14203 (2011).</li><li>Nern, A., Pfeiffer, B. D., Rubin, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimized+tools+for+multicolor+stochastic+labeling+reveal+diverse+stereotyped+cell+arrangements+in+the+fly+visual+system.">Optimized tools for multicolor stochastic labeling reveal diverse stereotyped cell arrangements in the fly visual system.</a> Proc Natl Acad Sci U S A. 112 (22), E2967-E2976 (2015).</li><li>Viswanathan, S., Williams, M. E., Bloss, E. B., Stasevich, T. J., Speer, C. M., Nern, A., Pfeiffer, B. D., Hooks, B. M., Li, W. P., English, B. P., Tian, T., Henry, G. L., Macklin, J. J., Patel, R., Gerfen, C. R., Zhuang, X., Wang, Y., Rubin, G. M., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-performance+probes+for+light+and+electron+microscopy.">High-performance probes for light and electron microscopy.</a> Nat Methods. 12 (6), 568-576 (2015).</li><li>Livet, J., Weissman, T. A., Kang, H., Draft, R. W., Lu, J., Bennis, R. A., Sanes, J. R., Lichtman, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450 (7166), 56-62 (2007).</li><li>Hampel, S., Chung, P., McKellar, C. E., Hall, D., Looger, L. L., Simpson, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+Brainbow:+A+recombinase-based+fluorescence+labeling+technique+to+subdivide+neural+expression+patterns.">Drosophila Brainbow: A recombinase-based fluorescence labeling technique to subdivide neural expression patterns.</a> Nat Methods. 8 (3), 253-259 (2011).</li><li>Hadjieconomou, D., Rotkopf, S., Alexandre, C., Bell, D. M., Dickson, B. J., Salecker, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Flybow:+Genetic+multicolor+cell+labeling+for+neural+circuit+analysis+in+Drosophila+melanogaster.">Flybow: Genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster.</a> Nat Methods. 8 (3), 260-266 (2011).</li><li>Kremer, M. C., Jung, C., Batelli, S., Rubin, G. M., Gaul, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+glia+of+the+adult+Drosophila+nervous+system.">The glia of the adult Drosophila nervous system.</a> Glia. 65 (4), 606-638 (2017).</li></ol>

没有一个作者有竞争或相互冲突的利益。

该协议描述了一个简单和有效的方法来研究在高分辨率的兴趣组织内的不同细胞类型的形态学。采用 MCFO 技术, 多种不同表位标记的记者组合使用, 用于多色随机标记 (图 2)。与其他方法类似, 如 Brainbow/Flybow15,16,17, MCFO 通过标记共增加了标签的多样性, 从而使细胞边界的可视化成为细胞间相互作用的研究。例如, 有三记者, 七潜在的标记组合是可能的。与以前的标记方法相比, MCFO 技术显示了一个更精确和易于控制的细胞标记密度17: Flybow 1.0 苍蝇表达一个默认的标记, 使单细胞标签更困难;Flybow 2.0 苍蝇需要两个不同的 Flp recombinases 的表达, 使系统更加复杂。
根据所使用的特定 GAL4 驱动程序, MCFO 技术可以适应于在任何发展阶段研究任何组织。在这里, 该技术已被用来表征的形态学的一般神经胶质亚型在成年果蝇脑高分辨率。单个细胞的多色标记允许不同边界的可视化, 并有助于理解, 例如, 两个相邻细胞之间的空间相互作用;除了 PNG 和干细胞, 这意味着形成上皮细胞, 所有其他神经子亚型显示瓷砖, 这是他们尽量减少接触与他们的胶质邻居, 同时最大限度地接触与包络的神经元室 (细胞体, 轴突, 树突, 和突触)。他们都发送精细的 lamellipodial 或 filopodial 进程不仅进入他们的本地邻居, 但也进入遥远的环境18。
为了使这项协议成功, 在18° c 后方飞行是至关重要的, 以避免系统中的泄漏。在解剖和整个染色过程中, 重要的是要避免任何损害组织的最佳染色效果。最后, 需要考虑两个技术方面: 热休克时间定义了标记表达的细胞数量。短暂的热休克将导致一些细胞的标记, 而长期的热休克将导致在许多细胞的标签表达的极端情况下, 所有的首次登记税停止登记的磁带是从记者, 在所有的细胞。在这种情况下, 只有一种颜色 (三表位标签的合并) 将存在, 防止单一细胞形态的可视化。热冲击时间因 Flp 和 GAL4 驱动的插入位置而异, 因此可能需要初始优化步骤。标记表达式是随机的, 不能针对由 GAL4 驱动程序驱动的特定的单元格亚群。在一个细胞内, 可以表达不同的标记, 使相邻细胞的抗体染色具有不同的颜色。但是, 偶尔会在一个单元格中表达多个标记, 从而导致两种颜色的重叠。

神经胶质是神经系统的神经细胞细胞, 长期以来被认为为神经元提供了一个静止的框架, 因此没有得到详细的研究。然而, 在人类中, 胶质细胞在 NS (约 90%) 中占绝大多数, 并分成几个不同的类别, 包括星状细胞、突、小胶质细胞和雪旺电池。在果蝇中, 胶质细胞在 NS 中占10% 左右。有趣的是, 它们的形态和功能与脊椎动物的1,2中发现的非常相似。它们的形态包括血脑屏障 (BBB) 形成上皮细胞、鞘层和星形星状胞。
果蝇中枢神经系统 (CNS) 包括以下主要结构: 包含神经元细胞体的皮层区域;neuropils 那海港突触连接;连接不同 neuropiles 的小的和大的轴突束;与中枢神经系统连接感觉器官和肌肉的外周神经 (图 1)。胶质细胞被发现与所有这些解剖结构相关: 皮质胶质细胞 (CG) 在皮层区, 星形胶质细胞和鞘胶质细胞 (如) 在 neuropile 区, 鞘胶质细胞也与中央轴突和周边神经 (地名), 最后, 两个片状胶质细胞, 膜胶质细胞 (PG) 和 subperineurial (), 其中一起形成一个连续的层覆盖整个 NS (图 2)。
以往的研究表明, 胶质细胞在 NS 的发展中起着重要的作用。它们通过对系统循环的胰岛素样肽进行反应来监测神经细胞的数量, 为神经元提供营养支持, 如星形胶质细胞-神经元乳酸梭, 并通过吞噬3,4来消除死亡神经元,5,6. 在成熟的 ns 中, 胶质细胞维持血脑屏障, 接受神经递质并维持离子稳态, 作为主要的免疫单元在 ns, 因为巨噬细胞不能破坏 bbb, 并调节突触活动以及动物行为6,7,8,9,10,11。
不同的胶质亚型是否具有特殊功能仍然是一个重要的开放性问题。然而, 对胶质细胞进行系统的全基因组分析, 特别是在成人身上, 由于缺乏适当的基因工具来进行操作而受到阻碍。在这里, 一个方法, 使细胞形状的有效和容易的表征研究复杂的细胞间的相互作用提出。这项技术已被用于表征不同的胶质亚型在成人果蝇脑中的形态学, 但是, 根据具体的 GAL4 驱动程序, 它可以适应研究神经元12,13, 任何类型的混合细胞, 原则上任何发展阶段的任何组织。

<table>
	<tbody>
		<tr>
			<td>Water bath</td>
			<td>Grant</td>
			<td>GD100</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR tubes</td>
			<td>Sarstedt</td>
			<td>72.737.002</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Dumont</td>
			<td>11251-20</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissecting dish 30 mm x 12 mmm</td>
			<td>Electron Microscopy Sciences</td>
			<td>70543-30</td>
			<td>Glass dissection dish</td>
		</tr>
		<tr>
			<td>Pyrex 3 Depression Glass Spot Plate</td>
			<td>Corning</td>
			<td>7223-34</td>
			<td>Glass dissection plates</td>
		</tr>
		<tr>
			<td>Sylgard Black</td>
			<td>SYLGARD, Sigma-Aldrich</td>
			<td>805998</td>
			<td>home made with charcoal</td>
		</tr>
		<tr>
			<td>ExpressFive S2 cell culture medium</td>
			<td>Invitrogen</td>
			<td>10486-025</td>
			<td></td>
		</tr>
		<tr>
			<td>20% PFA</td>
			<td>Electron Microscopy Sciences</td>
			<td>15713</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Roth</td>
			<td>3051.3</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Jackson Laboratories</td>
			<td>005-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal donkey serum</td>
			<td>Jackson Laboratories</td>
			<td>017-000-121</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A9647</td>
			<td></td>
		</tr>
		<tr>
			<td>Rabbit HA-tag</td>
			<td>Cell Signaling</td>
			<td>C29F4</td>
			<td>Primary antibody, dilution 1:500</td>
		</tr>
		<tr>
			<td>Rat FLAG-tag</td>
			<td>Novus Biologicals</td>
			<td>NBP1-06712</td>
			<td>Primary antibody, dilution 1:100</td>
		</tr>
		<tr>
			<td>Mouse V5-tag:DyLight 549</td>
			<td>AdSerotec</td>
			<td>0411</td>
			<td>Conjugated antibody, dilution 1:200</td>
		</tr>
		<tr>
			<td>anti-rabbit AlexaFluor 488</td>
			<td>Invitrogen</td>
			<td>A11034</td>
			<td>Secondary antibody, dilution 1:250</td>
		</tr>
		<tr>
			<td>anti-rat DyLight 647</td>
			<td>Jackson Laboratories</td>
			<td>712-605-153</td>
			<td>Secondary antibody, dilution 1:100</td>
		</tr>
		<tr>
			<td>Vecta Shield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>SlowFate Gold</td>
			<td>Invitrogen</td>
			<td>S36937</td>
			<td></td>
		</tr>
		<tr>
			<td>Secure Seal Spacer</td>
			<td>Grace Biolabs</td>
			<td></td>
			<td>Contact company for ordering</td>
		</tr>
		<tr>
			<td>Microscope cover glass 22 X 60 mm</td>
			<td>Marienfeld</td>
			<td>101152</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope cover glass 22 x 22 mm</td>
			<td>Roth</td>
			<td>H874</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo Microscope, Leica MZ6</td>
			<td>Leica</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope LSM710</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Immersol</td>
			<td>Zeiss</td>
			<td>518 F</td>
			<td>Immersion oil for fluorescence-microscopy, halogen free</td>
		</tr>
		<tr>
			<td>Immersol</td>
			<td>Zeiss</td>
			<td>W 2010</td>
			<td>Immersion fluid for water-immersion objectives, halogen free</td>
		</tr>
		<tr>
			<td>R56F03-GAL4 (EG)</td>
			<td>Bloomington Stock Center</td>
			<td>39157</td>
			<td>GAL4 driver</td>
		</tr>
		<tr>
			<td>R86E01-GAL4 (ALG)</td>
			<td>Bloomington Stock Center</td>
			<td>45914</td>
			<td>GAL4 driver</td>
		</tr>
		<tr>
			<td>hspFlpPestOpt; UAS-FRT-stop-FRT-myr-smGFP-HA, UAS-FRT-stop-FRT-myr-smGFP-FLAG, UAS-FRT-stop-FRT-myr-smGFP-V5</td>
			<td>Bloomington Stock Center</td>
			<td>64085</td>
			<td>UAS reporter (https://bdscweb.webtest.iu.edu/stock/misc/mcfo.php)</td>
		</tr>
		<tr>
			<td>Fiji (Image J)</td>
			<td></td>
			<td></td>
			<td>Image analysis software</td>
		</tr>
		<tr>
			<td>Multi Time Macro</td>
			<td>Zeiss</td>
			<td></td>
			<td>Software for automated scanning</td>
		</tr>
	</tbody>
</table>

application of multicolor flpout technique to study high resolution single cell morphologies and cell interactions of glia in drosophila

细胞表现出不同的形态和复杂的解剖关系。细胞如何与邻居互动？单元格类型之间的交互是否不同, 甚至在给定类型中也有所不同？他们遵循什么样的空间规则？到目前为止, 由于缺乏用于高分辨率单细胞标记的工具, 这些基本问题的答案在体内受到了阻碍。在这里, 提供了一个详细的协议目标单细胞与多色 FlpOut (MCFO) 技术。这种方法依赖于三不同标记的记者 (HA, 旗帜和 V5) 在无人控制, 保持沉默的转录终结者两侧的两个首次登记税网站 (首次登记-停止-首次登记税)。热休克脉冲诱发热休克诱导的 Flp 重组的表达, 它在单个单元格中随机删除了首次登记停用的卡带: 表达仅发生在同时表达 GAL4 驱动程序的单元格中。这将导致一个给定单元格类型的不同颜色的单元格数组, 它允许在高分辨率下可视化单个细胞形态。作为一个例子, MCFO 技术可以结合特定的胶质 GAL4 驱动, 以可视化的形态, 不同的胶质亚型在成年果蝇脑。

本节说明了在成人果蝇脑中使用 MCFO 技术可以获得的结果的示例。 图 3显示了该方法的示意图。三不同的膜标记的记者 (myr-smGFP-HA, myr-smGFP-旗和 myr-smGFP-V5) 在无人控制系统下, 由两个首次登记税网站 (首次登记-停止-首次登记税) 两侧的转录终结者保持沉默。热休克脉冲诱导 Flp 重组的表达, 它在单个单元格中随机删除了首次登记停用的卡带。这将导致给定单元格类型的不同颜色的单元格数组, 由所使用的 GAL4 驱动程序指定。
总的来说, 七种颜色是可能的。图 4和图 5分别显示了果蝇触角叶 (AL) 中的 EG 和细胞的单一形态。随着热休克时间的增加, 标记细胞的数量增加, 这取决于 GAL4 驱动程序的类型, 因此需要对热休克协议进行初始优化。图 6显示了在果蝇光学叶 (OL) 中使用单 MCFO 记者的标记。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56177/56177fig1.jpg"> 
图 1: 成人果蝇NS 的解剖.皮质区域 (斑点灰色区域) 包含所有神经元和多数胶质细胞身体, 而 neuropile 区域 (蓝色区域) 包含突触连接。道区 (深蓝) 连接不同的 neuropiles。此图已从克莱默et al.中修改(2017)18.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56177/56177fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56177/56177fig2.jpg"> 
图 2:果蝇CNS 中的一般神经胶质亚型.由特定的 GAL4 驱动程序驱动的膜标记 GFP (UAS-mCD8-GFP) 报告器允许五种通用的神经胶质亚型的可视化 (PG: 膜胶质,: subperineurial 胶质, CG: 皮层胶质细胞, 如: 星形胶质细胞, 如鞘胶质细胞)。使用前标记 NC82 (BRUCHPILOT) 检测到神经区域 (洋红区域)。在底部, 也显示了不同的胶质亚型的细胞计数。缩放栏 = 100 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56177/56177fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56177/56177fig3.jpg"> 
图 3: MCFO 技术的交叉方案.MCFO 技术示意图。Homozygousvirgins 携带 MCFO 盒和 Flp (hsp-Flp; 10 xuas-首次登记税-停止-首次登记税-myr-smGFP-房委会, 10 xuas-首次登记税-停止-首次登记税-myr-smGFP-旗帜, 10 xuas-首次登记税-停止-首次登记, myr-smGFP-V5--与特定的纯合 GAL4 驱动F1 的一代, 然后热震惊。根据从记者随机删除的首次登记税-停止-首次登记的录音带的数目, 有七种颜色是可能的。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56177/56177fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56177/56177fig4.jpg"> 
图 4: 鞘胶质细胞 (EG) 在触角瓣 (AL) 中的形态学.wholemount 免疫后的果蝇的共焦 z 栈。(A) 用前标记 NC82 (BRUCHPILOT) 检测到的 AL 的形态学。(B-D)热休克诱发 EGcells 的随机标记次数: 8 分钟 (B)、8.5 分钟 (C) 和10分钟 (D)。在 ensheath 的表面上, 有许多不同的形状和尺寸. 相邻的 eg 细胞覆盖不同的区域, 但在接触区域部分 interdigitate。刻度栏 = 20 µm. HA: 人类流感血。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56177/56177fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 5" class="xfigimg" src="/files/ftp_upload/56177/56177fig5.jpg"> 
图 5: 触角瓣 (AL) 中星形胶质细胞状胶质细胞的形态学.wholemount 免疫后的果蝇的共焦 z 栈。(A) 用前标记 NC82 (BRUCHPILOT) 检测到的 AL 的形态学。(B-D)热休克诱发细胞的随机标记时间: 8 分钟 (B)、10分钟 (C) 和 15 min (D)。显示可变大小和形态, 但主要覆盖不重叠区域。刻度栏 = 20 µm. HA: 人类流感血。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56177/56177fig5large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 6" class="xfigimg" src="/files/ftp_upload/56177/56177fig6.jpg"> 
图 6: 在视叶 (OL) 中鞘胶质细胞的形态学.wholemount 免疫后的果蝇OL 的共焦 z 栈。(A-c)MCFO 标记与三停止卡式记者与 HA、旗子和 V5 myr-smGFPs。Flp 重组是由37° c 的10分钟热休克诱导的。(A-c)将显示单独的 MCFO 记者和合并 (D)。刻度栏 = 20 µm. HA: 人类流感血。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56177/56177fig6large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila at JoVE.com

Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

细胞表现出不同的形态, 并与它们的邻居建立了各种相互作用。该协议描述了如何揭示单细胞的形态学, 并利用已建立的 Gal4/UAS 表达系统来研究细胞间的相互作用。

多色 FlpOut 技术在果蝇中的应用研究胶质细胞的高分辨率单细胞形态学

Cells display different morphologies and complex anatomical relationships. How do cells interact with their neighbors? Do the interactions differ between ...

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Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

8.1K 次观看.  Ludwig-Maximilians-University Munich. 细胞表现出不同的形态, 并与它们的邻居建立了各种相互作用。该协议描述了如何揭示单细胞的形态学, 并利用已建立的 Gal4/UAS 表达系统来研究细胞间的相互作用。

视频: Application of MultiColor FlpOut Technique to Study High Resolution Single Cell Morphologies and Cell Interactions of Glia in Drosophila

Assessment and Evaluation of the High Risk Neonate: The NICU Network Neurobehavioral Scale

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant. The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. These are infants who are at increased risk for poor developmental outcome because of insults during prenatal development, such as substance exposure or prematurity or factors such as poverty, poor nutrition or lack of prenatal care that can have adverse effects on the intrauterine environment and affect the developing fetus. The NNNS assesses the full range of infant neurobehavioral performance including neurological integrity, behavioral functioning, and signs of stress/abstinence. The NNNS is a noninvasive neonatal assessment tool with demonstrated validity as a predictor, not only of medical outcomes such as cerebral palsy diagnosis, neurological abnormalities, and diseases with risks to the brain, but also of developmental outcomes such as mental and motor functioning, behavior problems, school readiness, and IQ. The NNNS can identify infants at high risk for abnormal developmental outcome and is an important clinical tool that enables medical researchers and health practitioners to identify these infants and develop intervention programs to optimize the development of these infants as early as possible. The video shows the NNNS procedures, shows examples of normal and abnormal performance and the various clinical populations in which the exam can be used.

The NICU Network Neurobehavioral Scale (NNNS) was developed as an assessment for the at-risk infant. The purpose of this article is to describe the NNNS, provide video examples of the NNNS procedures and discuss the ways in which the exam has been used.

评估和高危新生儿的评价:新生儿重症监护病房的网络神经行为量表

There has been a long-standing interest in the assessment of the neurobehavioral integrity of the newborn infant.  The NICU Network Neurobehavioral Scale ...

科研

行为学

A Procedure to Study the Effect of Prolonged Food Restriction on Heroin Seeking in Abstinent Rats

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during abstinence. Moreover, various environmental challenges can exacerbate this effect, as well as increase ongoing drug intake.
The procedure we describe here highlights the impact of a common environmental challenge, food restriction, on drug craving that is expressed as an augmentation of drug seeking in abstinent rats.
Rats are implanted with chronic intravenous i.v. catheters, and then trained to press a lever for i.v. heroin over a period of 10-12 days. Following the heroin self-administration phase the rats are removed from the operant conditioning chambers and housed in the animal care facility for a period of at least 14 days. While one group is maintained under unrestricted access to food (sated group), a second group (FDR group) is exposed to a mild food restriction regimen that results in their body weights maintained at 90% of their nonrestricted body weight. On day 14 of food restriction the rats are transferred back to the drug-training environment, and a drug-seeking test is run under extinction conditions (i.e.&#160;lever presses do not result in heroin delivery).
The procedure presented here results in a highly robust augmentation of heroin seeking on test day in the food restricted rats. In addition, compared to the acute food deprivation manipulations we have used before, the current procedure is a more clinically relevant model for the impact of caloric restriction on drug seeking. Moreover, it might be closer to the human condition as the rats are not required to go through an extinction-training phase before the drug-seeking test, which is an integral component of the popular reinstatement procedure.

A procedure that allows a demonstration of robust augmentation of drug seeking in food-restricted rats is described. Following heroin self-administration training, rats go through an abstinence period, in a drug-free environment, during which they are mildly food restricted. Drug seeking is then tested in the drug-associated environment.

一个过程来研究延长食品的限制对海洛因寻求戒断大鼠的影响

In human drug addicts, exposure to drug-associated cues or environments that were previously associated with drug taking can trigger relapse during ...

Study Motor Skill Learning by Single-pellet Reaching Tasks in Mice

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and retrieve food rewards positioned at a specific location, their motor performance (defined as accuracy and speed) improves progressively over time, and plateaus after persistent training. Once such reaching skill is mastered, its further maintenance does not require constant practice. Here we introduce a single-pellet reaching task to study the acquisition and maintenance of skilled forelimb movements in mice. In this video, we first describe the behaviors of mice that are commonly encountered in this learning and memory paradigm, and then discuss how to categorize these behaviors and quantify the observed results. Combined with mouse genetics, this paradigm can be utilized as a behavioral platform to explore the anatomical underpinnings, physiological properties, and molecular mechanisms of learning and memory.

Persistent practice improves the precision of coordinated movements.&#160;Here we introduce a single-pellet reaching task, which is designed to assess the learning and memory of forelimb skill in mice.&#160;

学习运动技能通过单颗粒到达任务小鼠学习

Reaching for and retrieving objects require precise and coordinated motor movements in the forelimb. When mice are repeatedly trained to grasp and ...

Uncovering Beat Deafness: Detecting Rhythm Disorders with Synchronized Finger Tapping and Perceptual Timing Tasks

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here with the goal of uncovering rhythm disorders, such as beat deafness. Beat deafness is characterized by poor performance in perceiving durations in auditory rhythmic patterns or poor synchronization of movement with auditory rhythms (e.g., with musical beats). These tasks include the synchronization of finger tapping to the beat of simple and complex auditory stimuli and the detection of rhythmic irregularities (anisochrony detection task) embedded in the same stimuli. These tests, which are easy to administer, include an assessment of both perceptual and sensorimotor timing abilities under different conditions (e.g., beat rates and types of auditory material) and are based on the same auditory stimuli, ranging from a simple metronome to a complex musical excerpt. The analysis of synchronized tapping data is performed with circular statistics, which provide reliable measures of synchronization accuracy (e.g., the difference between the timing of the taps and the timing of the pacing stimuli) and consistency. Circular statistics on tapping data are particularly well-suited for detecting individual differences in the general population. Synchronized tapping and anisochrony detection are sensitive measures for identifying profiles of rhythm disorders and have been used with success to uncover cases of poor synchronization with spared perceptual timing. This systematic assessment of perceptual and sensorimotor timing can be extended to populations of patients with brain damage, neurodegenerative diseases (e.g., Parkinson&#8217;s disease), and developmental disorders (e.g., Attention Deficit Hyperactivity Disorder).

Behavioral tasks that allow for the assessment of perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) are presented. Synchronization of finger tapping to the beat of an auditory stimuli and detecting rhythmic irregularities provides a means of uncovering rhythm disorders.

揭开节拍耳聋:检测节律紊乱与同步手指敲击和感知定时任务

A set of behavioral tasks for assessing perceptual and sensorimotor timing abilities in the general population (i.e., non-musicians) is presented here ...

A Novel Approach to Assess Motor Outcome of Deep Brain Stimulation Effects in the Hemiparkinsonian Rat: Staircase and Cylinder Test

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to screen different neurostimulation patterns in hemiparkinsonian (unilateral lesioned) rats. It consists of creating a unilateral Parkinson&#39;s lesion by injecting 6-hydroxydopamine (6-OHDA) into the right medial forebrain bundle, implanting chronic stimulation electrodes into the subthalamic nucleus and evaluating motor outcomes at the end of 24 hr periods of cable-bound external neurostimulation. The stimulation was conducted with constant current stimulation. The amplitude was set 20% below the individual threshold for side effects. The motor outcome evaluation was done by the assessment of spontaneous paw use in the cylinder test according to Shallert and by the assessment of skilled reaching in the staircase test according to Montoya. This protocol describes in detail the training in the staircase box, the cylinder test, as well as the use of both in hemiparkinsonian rats. The use of both tests is necessary, because the staircase test seems to be more sensitive for fine motor skill impairment and exhibits greater sensitivity to change during neurostimulation. The combination of the unilateral Parkinson model and the two behavioral tests allows the assessment of different stimulation parameters in a standardized way.

Deep brain stimulation (DBS) is an effective treatment option for Parkinson's disease. We established a study design to screen novel stimulation paradigms in rats. The protocol describes the use of the staircase test and cylinder test for motor outcome assessment in DBS treated hemiparkinsonian rats.

一种新的方法，以评估在帕金森病大鼠脑深部电刺激效应的运动结果:楼梯和缸测试

Deep brain stimulation of the subthalamic nucleus is an effective treatment option for Parkinson&#39;s disease. In our lab we established a protocol to ...

Reversible Cooling-induced Deactivations to Study Cortical Contributions to Obstacle Memory in the Walking Cat

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For example, in the cat, visual information about an impending obstacle can modulate stepping for avoidance. Locomotor adaptation can also occur independent of vision, as sudden tactile inputs to the leg by an expected obstacle can modify the stepping of all four legs for avoidance. Such complex locomotor coordination involves supraspinal structures, such as the parietal cortex. This protocol describes the use of reversible, cooling-induced cortical deactivation to assess parietal cortex contributions to memory-guided obstacle locomotion in the cat. Small cooling loops, known as cryoloops, are specially shaped to deactivate discrete regions of interest to assess their contributions to an overt behavior. Such methods have been used to elucidate the role of parietal area 5 in memory-guided obstacle avoidance in the cat.

Complex locomotion in naturalistic environments requiring careful coordination of the limbs involves regions of the parietal cortex. The following protocol describes the use of reversible cooling-induced deactivation to demonstrate the role of parietal area 5 in memory-guided obstacle avoidance in the walking cat.

可逆冷却诱导 Deactivations 对行走猫的障碍记忆的皮层贡献研究

On complex, naturalistic terrain, sensory information about an environmental obstacle can be used to rapidly adjust locomotor movements for avoidance. For ...

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, and issues commands to start or stop feeding. Deciphering the processes underlying the decision-making of timely and moderate feeding carries major implications in our understanding of physiological and psychological disorders related to feeding control. Simple, quantitative, and robust methods are required to measure the food ingestion of animals after experimental manipulation, such as forcibly increasing the activities of certain target neurons. Here, we introduced dye-labeling-based feeding assays to facilitate the neurogenetic study of feeding control in adult fruit flies. We review available feeding assays, and then describe our methods step-by-step from setup to analysis, which combine thermogenetic and optogenetic manipulation of neurons controlling feeding motivation with dye-labeled food intake assay. We also discuss the advantages and limitations of our methods, compared with other feeding assays, to help readers choose an appropriate assay.

Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila

Quantitative food-intake assays with dyed food provide a robust and high-throughput means to evaluate feeding motivation. Combining the food consumption assay with thermogenetic and optogenetic screens is a powerful approach to investigate the neural circuits underlying appetite in adult Drosophila melanogaster.

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

Food consumption is under the tight control of the brain, which integrates the physiological status, palatability, and nutritional contents of the food, ...

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific responses to changing temperatures according to their physiological tolerance and adaptability. Drosophila flies also possess a temperature sensing system that has become fundamental to understanding the neural basis of temperature processing in ectotherms. We present here a temperature-controlled arena that permits fast and precise temperature changes with temporal and spatial control to explore the response of individual flies to changing temperatures. Individual flies are placed in the arena and exposed to pre-programmed temperature challenges, such as uniform gradual increases in temperature to determine reaction norms or spatially distributed temperatures at the same time to determine preferences. Individuals are automatically tracked, allowing the quantification of speed or location preference. This method can be used to rapidly quantify the response over a large range of temperatures to determine temperature performance curves in Drosophila or other insects of similar size. In addition, it can be used for genetic studies to quantify temperature preferences and reactions of mutants or wild-type flies. This method can help uncover the basis of thermal speciation and adaptation, as well as the neural mechanisms behind temperature processing.

An Automated Method to Determine the Performance of Drosophila in Response to Temperature Changes in Space and Time

Here we present a protocol to automatically determine the locomotor performance of Drosophila at changing temperatures using a programmable temperature-controlled arena that produces fast and accurate temperature changes in time and space.

一种自动测定果蝇在空间和时间温度变化中的性能的方法

Temperature is a ubiquitous environmental factor that affects how species distribute and behave. Different species of Drosophila fruit flies have specific ...

Assessing the Coherence of Parents' Short Narratives Regarding their Child Using the Five-Minute Speech Sample Procedure

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with parents. This paper focuses on such a method, namely Five-Minute Speech Sample-Coherence (FMSS-Coherence). In this method, a parent is asked to speak for five uninterrupted minutes about her/his child and their relationship. The resulted narrative is coded for coherence, namely the extent to which the parent provides&#160;in the narrative&#160;a clear, consistent, multidimensional and well-supported portrayal of the child. FMSS-Coherence is based on attachment research that shows that the coherence of parents&#8217; narratives is indicative of the quality of the parent-child relationship and child adjustment. It overcomes the limitations of attachment narrative measures which are typically labor intensive. FMSS-Coherence is less nuanced than extant attachment narrative measures. Yet, studies of families from different cultural backgrounds, across different child ages and in the context of typically developing children as well as children with special needs suggest that coherence can be reliably evaluated using the FMSS procedure. Furthermore, parents&#8217; FMSS-Coherence is associated with parenting quality and children&#8217;s socio-emotional adjustment. Thus, it holds promise for researchers and practitioners who seek a relatively time- and cost-effective method for assessing the coherence of parents&#8217; narratives regarding their child.

This paper introduces a method for assessing the coherence of parents' short narratives regarding their child and their relationship using the five-minute speech sample procedure.

使用五分钟言语样本程序评估家长对孩子的简短叙述的一致性

Valid and efficient measures for assessing the quality of parent-child relationships are needed to facilitate research and evidence-based practice with ...

Radiotracer Administration for High Temporal Resolution Positron Emission Tomography of the Human Brain: Application to FDG-fPET

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose analogue, 18F-fluordeoxyglucose (FDG-fPET), it is now possible to measure the dynamics of glucose metabolism with temporal resolutions approaching those of functional magnetic resonance imaging (fMRI). This direct measure of glucose uptake has enormous potential for understanding normal and abnormal brain function and probing the effects of metabolic and neurodegenerative diseases. Further, new advances in hybrid MR-PET hardware make it possible to capture fluctuations in glucose and blood oxygenation simultaneously using fMRI and FDG-fPET.
The temporal resolution and signal-to-noise of the FDG-fPET images is critically dependent upon the administration of the radiotracer. This work presents two alternative continuous infusion protocols and compares them to a traditional bolus approach. It presents a method for acquiring blood samples, time-locking PET, MRI, experimental stimulus, and administering the non-traditional tracer delivery. Using a visual stimulus, the protocol results show cortical maps of the glucose-response to external stimuli on an individual level with a temporal resolution of 16 s.

This manuscript describes two radiotracer administration protocols for FDG-PET (constant infusion and bolus plus infusion) and compares them to bolus administration. Temporal resolutions of 16 s are achievable using these protocols.

人脑高时态分辨率正态发射断层扫描的放射跟踪器管理:在FDG-fPET中的应用

Functional positron emission tomography (fPET) provides a method to track molecular targets in the human brain. With a radioactively-labelled glucose ...