我们要感谢古尔本基安·德辛西亚研究所（IGC）为我们提供了获得本议定书中描述的部分实验设备的机会。这项工作得到了葡萄牙科学技术基金会（FCT）的支持，LISBOA-01-0145-FEDER-007660， PTDC/NEU-NMC/2459/2014、IF/00697/2014 和 La Caixa HR17-00595 到 PMD，以及澳大利亚研究理事会未来研究金 （FT170100259） 向 CKM 提供。

1. 准备蔗糖酵母 （SY） 饮食
<ol><li>称量所有干成分（阿加，酵母，蔗糖）的宏量营养素平衡和L3饲养饮食。准备1升食物所需的每种成分的克数在 图1B中注明。 注意：考虑到需要大约 13 mL 的食物来填充 60 毫米的培养皿。</li>
	<li>将所有成分溶解在无菌蒸馏水中（使用准备食物所需的总水量的约 50%），搅拌介质 5-10 分钟。</li>
	<li>自动滑轮50分钟。</li>
	<li>允许介质冷却后，在饮食中加入尼帕金和丙酸溶液，最终浓度（v/v）分别为3%和0.3%。在宏量营养素平衡饮食中，将蓝色食物染料添加到 1% 的最终浓度 （v/v） 中。用蒸馏水完成总体积。</li>
	<li>小心地将食物饮食倒入 60 毫米的培养皿中，使每个盘子中浇注的食物数量大致相同。用饮食的 P：C 比率标记盘子。 注意：在喂养检测当天准备宏量营养素平衡饮食。如果不可能，将准备好的饮食储存在4°C，密封容器中，最长持续3天。更长的储存期使饮食过于干燥和困难，幼虫无法钻进媒介。</li>
</ol>2. 父母线的遗传交叉
注：使用Gal4/UAS系统21 来设置遗传交叉。在此协议中，为了激活特定神经元群体的神经元功能，使用 UAS dTRPA1 线17 的女处女，并从 Janelia Gal4 线（图 2A）交叉到男性。使用的基因控制是 dTRPA1 线和"空 GAL4"线之间的交叉的后代，该线在用于生成 Rubin Gal4 集合的向量中携带 Gal4，但没有存在调节片段 （attP2）22。为了促进神经元抑制，可以使用 UAS 线编码 ShibireTS20， 而不是 dTRPA1。
<ol><li>设置60毫米胚胎收集笼与L3饲养饮食板，辅以一些活性酵母膏。</li>
	<li>将成年 UAS dTRPA1 女处女和 Janelia Gal4 雄性（5-8 天）转移到胚胎采集笼中，让交配在 25 °C、60% 湿度和 12：12 光暗周期（图 2A）下进行 24-48小时。对于60毫米胚胎采集笼，每个十字架使用约100个处女雌性和30个雄性。</li>
	<li>在交配期结束时，取出并丢弃遗传十字架中使用的L3饲养饮食板。用它们代替新鲜的L3饲养饮食板，以执行卵子产卵和幼虫分期。</li>
</ol>3. 准备三星幼虫 （L3）
<ol><li>将交美成人苍蝇转移到新鲜的L3饲养饮食板，并允许产卵发生3-4小时，在25°C（图2B）。确保所有的盘子都标有基因型、饮食的P：C比例和产卵日期。 注意：为了节省时间，将产卵直接放入 L3 饲养饮食中，从而避免对鸡蛋进行额外的处理。在小规模基因筛选的情况下，使用苹果汁阿加板可以优化产卵。</li>
	<li>在产卵期结束时，从笼子里取出盘子，用塑料盖盖住它们。如果酵母提取物用于补充L3饲养板，请务必在产卵结束时去除所有残留酵母。这对于避免幼虫生长过程中的不均匀喂养非常重要。 注：交美成人可以转移到新鲜的L3饲养饮食板，所以更多的蛋下进行，并可以获得更多的实验幼虫。在整个工作周内，可以与同一成年人一起连续产卵。</li>
	<li>估计每盘卵子的数量，并将幼虫密度保持在每盘最多200个胚胎。这种估计可以通过计算四分之一的盘子中的胚胎数量来完成。 注意：过度拥挤的盘子会延缓幼虫发育，影响幼虫喂养行为。</li>
	<li>在 18 °C（允许温度）、60% 湿度和 12：12 光暗循环下孵育 L3 饲养板，使幼虫生长 9 天（图 2B）。</li>
	<li>在产卵（AEL）后的第九天，从每个基因型（和复制品）中收集三组10L3进行测试。此外，收集 10 L3 组用于"零染料食品"控制。确保幼虫的收集是在一天中用于产卵的同等时间段内完成的（例如，如果卵子产卵发生在上午 10 点到下午 2 点之间，在 AEL 的同一时间段内收集幼虫），并且通过使用钳子#5或羽毛重量钳尽可能轻轻地进行。按照下一步（3.6）所示直接转移幼虫。 注："零染料食品"控制动物是幼虫，在喂养检测中给予食物没有蓝色染料。这种控制是必要的，以消除后吸收的幼虫提取物。</li>
	<li>将收集的实验幼虫转移到含有1mL水的塑料盘重量船上。请按照 图 3中给出的指示，确保收集 L3 而不是 L2。 注：收集L3到含有水或1倍磷酸盐缓冲盐水（PBS）的塑料船，对于在喂养检测开始前保持幼虫良好的水分非常重要。如果同时收集来自不同基因型的几个实验 L3 组，这一点尤其重要。跟踪每个组的收集顺序，因此将每个组食物匮乏时间的差异降至最低。在这一步中使用塑料船有利于可选的步骤4.3，因为它使幼虫直接漂浮到水浴。</li>
</ol>4. 恒温遗传活化和无选择喂养检测
注意：建议在一天中大致相同的时间执行喂养检测，以尽量减少与昼夜节律相关的可能变化。此外，始终运行控制实验（"空Gal4"线的后代交叉到UAS dTRPA1和"零染料食物"幼虫），与兴趣的基因型平行。
<ol><li>设置一个孵化器到30°C（不允许的温度），并保持高湿度（至少65%）避免在检测过程中幼虫脱水。</li>
	<li>在开始进食检测之前，通过在 30 °C 加热板 30 分钟来平衡检测板的温度。</li>
	<li>（可选）在37°C的水浴中加热实验幼虫2分钟。执行这一步骤与动物在塑料重量船包含一些水。 注：此步骤的目的是通过促进自喂养检测开始以来神经元的启动来加强神经元激活。</li>
	<li>将多个定时器设置为1小时。使用的定时器数量取决于正在测试的实验组的数量和实验者处理幼虫的熟练程度。 注意：使用多个定时器对于保持所有基因型的检测持续时间一致至关重要。</li>
	<li>小心地从塑料船上排出水，用湿润的软刷，轻轻地将 L3 组从船上转移到检测板的中心。将板盖放回原处，启动每个板（或一组板）的限时器，以保持准确的 1 小时进餐会话。</li>
	<li>允许幼虫在黑暗中以30°C（图2C）为食1小时。 注意：在黑暗中检测的性能对于控制不同饮食中视觉暗示的差异非常重要，因为饮食的色调会有所不同，即使它们含有相同的染料浓度。</li>
	<li>通过将盘子转移到冰浴中来停止喂食检测。尽可能压下冰层，为板块提供稳定的表面。 注意：寒冷的温度会通过抑制挖洞和挖掘行为来促进喂养的结束。大多数幼虫会在几分钟后浮出水面，促进它们在以下步骤中的恢复。</li>
</ol>5. 食品染料提取
<ol><li>为每组 10 个 L3 测试准备 2 mL 微管，其中含有大约相同数量的 0.5 mm 玻璃珠（足以填充微管底部）和 300 μL 的冰冷甲醇。使用长凳冷却器将微管保持在寒冷中。 注意：甲醇具有高度易燃性和毒性。遵循所有安全程序建议处理这种试剂，包括在通风良好的区域工作，戴氮化物手套。 注意：使用甲醇对于修复幼虫样本和避免基质层中的黑色素化反应非常重要。</li>
	<li>使用#5或羽毛重量力，小心地从进食检测板中回收 10 L3 组，并将它们转移到含有一些水的检测板的盖子上。冲洗幼虫，以清除他们身上的任何食物碎片，同时轻轻地处理幼虫，以避免任何伤害。记录每个复制品的幼虫数量，以便量化每种幼虫的平均食物摄入量。 注意：受伤的幼虫应该丢弃，因为它们会有黑色素化角质层，不适合着色定量。</li>
	<li>将 L3 组转移到 5.1 中准备的 2 mL 微管。</li>
	<li>使用在第 5.1 步添加的组织裂解器和玻璃珠，用机械裂解方法将幼虫组织酶从肠道中提取食物染料。（如果组织解酒器不可用，请使用同质化的害虫）。优先执行此步骤在 4 °C （图 2D）。 注：此步骤的持续时间将取决于所使用的设备。使用传统的组织解压器，1分钟的提取就足够了。如果时间限制，协议可以在本步骤结束时暂停，然后继续。将样品储存在-20°C。</li>
	<li>通过将 2 mL 微管直接倒置到新的 1.5 mL 微管上，将提取物转移到清洁 1.5 mL 微管中。如果轻轻执行，大部分玻璃珠将停留在2毫升微管的底部。</li>
	<li>通过离心提取物（最高速度为 10 分钟）在 4 °C 下清除细胞碎片。</li>
	<li>收集超自然人清洁1.5 mL微管。如果细胞碎片在超自然体中仍然可见，则重复步骤 5.6 和 5.7。</li>
</ol>6. 食品消费的彩色定量
<ol><li>准备标准解决方案，通过在开始的蓝色染料溶液的甲醇中执行串行 1：2 稀释，生成校准曲线。作为空白，仅使用甲醇。标准的浓度取决于动物的食物摄入水平。 注意：在此处显示的试验屏幕中，由于幼虫提取物获得的染料浓度在 0.02 至 1.93 μL/mL 之间，因此使用通过测量甲醇中 2 μL/mL 蓝色染料溶液 8 个连续稀释的吸收量而获得的标准曲线。如有必要，根据实验样品的染料浓度增加或降低这些溶液的浓度。</li>
	<li>将实验样品的 100 μL（第 5.7 步获得）、标准和空白（步骤 6.1）转移到 96 井微板的井中，并使用板阅读器（图 2E）测量 600 nm 的吸收量。要去除背景吸收，测量从幼虫饲料中获得的提取物的吸收量，而不使用蓝色染料作为幼虫提取物的"零"（"零染料食物控制"）。</li>
	<li>生成标准曲线，并将每个实验幼虫组样品的吸收值与食物摄入量（mL 中的体积）相关联。通过考虑步骤 5.2 中每组采集的幼虫数量，查找每只幼虫的平均食物消耗量</li>
</ol>

<ol>
	<li>Raubenheimer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Nature of nutrition - a unifying framework from animal adaptation to human. , (2012).</li><li>Carvahlo, M. J. a., Mirth, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Coordinating+morphology+with+behavior+during+development:+an+integrative+approach+from+a+fly+perspective.">Coordinating morphology with behavior during development: an integrative approach from a fly perspective.</a> Frontiers in Ecology and Evolution. , (2015).</li><li>Steck, 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=Internal+amino+acid+state+modulates+yeast+taste+neurons+to+support+protein+homeostasis+in+Drosophila.">Internal amino acid state modulates yeast taste neurons to support protein homeostasis in Drosophila.</a> Elife. 7, 31625 (2018).</li><li>Itskov, P. M., Ribeiro, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dilemmas+of+the+gourmet+fly:+the+molecular+and+neuronal+mechanisms+of+feeding+and+nutrient+decision+making+in+Drosophila.">The dilemmas of the gourmet fly: the molecular and neuronal mechanisms of feeding and nutrient decision making in Drosophila.</a> Frontiers in Neuroscience. 7, 12 (2013).</li><li>Bjordal, M., Arquier, N., Kniazeff, J., Pin, J. P., Leopold, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensing+of+amino+acids+in+a+dopaminergic+circuitry+promotes+rejection+of+an+incomplete+diet+in+Drosophila.">Sensing of amino acids in a dopaminergic circuitry promotes rejection of an incomplete diet in Drosophila.</a> Cell. 156 (3), 510-521 (2014).</li><li>Grandison, R. C., Piper, M. D., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amino-acid+imbalance+explains+extension+of+lifespan+by+dietary+restriction+in+Drosophila.">Amino-acid imbalance explains extension of lifespan by dietary restriction in Drosophila.</a> Nature. 462 (7276), 1061-1064 (2009).</li><li>Lee, K. P., 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=Lifespan+and+reproduction+in+Drosophila:+New+insights+from+nutritional+geometry.">Lifespan and reproduction in Drosophila: New insights from nutritional geometry.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (7), 2498-2503 (2008).</li><li>Levine, M. E., 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=Low+protein+intake+is+associated+with+a+major+reduction+in+IGF-1,+cancer,+and+overall+mortality+in+the+65+and+younger+but+not+older+population.">Low protein intake is associated with a major reduction in IGF-1, cancer, and overall mortality in the 65 and younger but not older population.</a> Cell Metabolism. 19 (3), 407-417 (2014).</li><li>Solon-Biet, S. 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=The+ratio+of+macronutrients,+not+caloric+intake,+dictates+cardiometabolic+health,+aging,+and+longevity+in+ad+libitum-fed+mice.">The ratio of macronutrients, not caloric intake, dictates cardiometabolic health, aging, and longevity in ad libitum-fed mice.</a> Cell Metabolism. 19 (3), 418-430 (2014).</li><li>Piper, M. 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=A+holidic+medium+for+Drosophila+melanogaster.">A holidic medium for Drosophila melanogaster.</a> Nature Methods. 11 (1), 100-105 (2014).</li><li>Jones, W. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+expanding+reach+of+the+GAL4/UAS+system+into+the+behavioral+neurobiology+of+Drosophila.">The expanding reach of the GAL4/UAS system into the behavioral neurobiology of Drosophila.</a> BMB Reports. 42 (11), 705-712 (2009).</li><li>Deshpande, S. 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=Quantifying+Drosophila+food+intake:+comparative+analysis+of+current+methodology.">Quantifying Drosophila food intake: comparative analysis of current methodology.</a> Nature Methods. 11 (5), 535-540 (2014).</li><li>Neckameyer, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+trophic+role+for+serotonin+in+the+development+of+a+simple+feeding+circuit.">A trophic role for serotonin in the development of a simple feeding circuit.</a> Developmental Neuroscience. 32 (3), 217-237 (2010).</li><li>Gasque, G., Conway, S., Huang, J., Rao, Y., Vosshall, L. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Small+molecule+drug+screening+in+Drosophila+identifies+the+5HT2A+receptor+as+a+feeding+modulation+target.">Small molecule drug screening in Drosophila identifies the 5HT2A receptor as a feeding modulation target.</a> Scientific Reports. 3, (2013).</li><li>Schoofs, 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=Selection+of+motor+programs+for+suppressing+food+intake+and+inducing+locomotion+in+the+Drosophila+brain.">Selection of motor programs for suppressing food intake and inducing locomotion in the Drosophila brain.</a> PLoS Biology. 12 (6), 1001893 (2014).</li><li>Pocas, G. M., Crosbie, A. E., Mirth, C. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=When+does+diet+matter?+The+roles+of+larval+and+adult+nutrition+in+regulating+adult+size+traits+in+Drosophila+melanogaster.">When does diet matter? The roles of larval and adult nutrition in regulating adult size traits in Drosophila melanogaster.</a> Journal of Insect Physiology. , 104051 (2020).</li><li>Hamada, F. N., 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=An+internal+thermal+sensor+controlling+temperature+preference+in+Drosophila.">An internal thermal sensor controlling temperature preference in Drosophila.</a> Nature. 454 (7201), 217-220 (2008).</li><li>Pfeiffer, B. 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=Tools+for+neuroanatomy+and+neurogenetics+in+Drosophila.">Tools for neuroanatomy and neurogenetics in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (28), 9715-9720 (2008).</li><li>Jenett, 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=A+GAL4-driver+line+resource+for+Drosophila+neurobiology.">A GAL4-driver line resource for Drosophila neurobiology.</a> Cell Reports. 2 (4), 991-1001 (2012).</li><li>Kitamoto, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conditional+modification+of+behavior+in+Drosophila+by+targeted+expression+of+a+temperature-sensitive+shibire+allele+in+defined+neurons.">Conditional modification of behavior in Drosophila by targeted expression of a temperature-sensitive shibire allele in defined neurons.</a> Journal of Neurobiology. 47 (2), 81-92 (2001).</li><li>Brand, A. H., Perrimon, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Targeted+gene+expression+as+a+means+of+altering+cell+fates+and+generating+dominant+phenotypes.">Targeted gene expression as a means of altering cell fates and generating dominant phenotypes.</a> Development. 118 (2), 401-415 (1993).</li><li>Shirangi, T. R., Stern, D. L., Truman, 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=Motor+control+of+Drosophila+courtship+song.">Motor control of Drosophila courtship song.</a> Cell Reports. 5 (3), 678-686 (2013).</li><li>Mirth, C. 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=Food+intake+and+food+choice+are+altered+by+the+developmental+transition+at+critical+weight+in+Drosophila+melanogaster.">Food intake and food choice are altered by the developmental transition at critical weight in Drosophila melanogaster.</a> Animal Behaviour. 126, 195-208 (2017).</li><li>Simpson, S. J., Raubenheimer, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obesity:+the+protein+leverage+hypothesis.">Obesity: the protein leverage hypothesis.</a> Obesity Reviews. 6 (2), 133-142 (2005).</li><li>Raubenheimer, D., Simpson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrative+models+of+nutrient+balancing:+application+to+insects+and+vertebrates.">Integrative models of nutrient balancing: application to insects and vertebrates.</a> Nutrition Research Reviews. 10 (1), 151-179 (1997).</li><li>Li, H. 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=A+GAL4+driver+resource+for+developmental+and+behavioral+studies+on+the+larval+CNS+of+Drosophila.">A GAL4 driver resource for developmental and behavioral studies on the larval CNS of Drosophila.</a> Cell Reports. 8 (3), 897-908 (2014).</li><li>Bhatt, P. K., Neckameyer, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+analysis+of+the+larval+feeding+circuit+in+Drosophila.">Functional analysis of the larval feeding circuit in Drosophila.</a> Journal of Visualized Experiments. (81), e51062 (2013).</li><li>Wong, R., Piper, M. D. W., Blanc, E., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pitfalls+of+measuring+feeding+rate+in+the+fruit+fly+Drosophila+melanogaster.">Pitfalls of measuring feeding rate in the fruit fly Drosophila melanogaster.</a> Nature Methods. 5 (3), 214-215 (2008).</li><li>Almeida-Carvalho, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ol1mpiad:+concordance+of+behavioural+faculties+of+stage+1+and+stage+3+Drosophila+larvae.">The Ol1mpiad: concordance of behavioural faculties of stage 1 and stage 3 Drosophila larvae.</a> Journal of Experimental Biology. 220, 2452-2475 (2017).</li><li>Rodrigues, M. 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=Drosophila+melanogaster+larvae+make+nutritional+choices+that+minimize+developmental+time.">Drosophila melanogaster larvae make nutritional choices that minimize developmental time.</a> Journal of Insect Physiology. 81, 69-80 (2015).</li><li>Wong, R., Piper, M. D., Wertheim, B., Partridge, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+food+intake+in+Drosophila.">Quantification of food intake in Drosophila.</a> PLoS One. 4 (6), 6063 (2009).</li><li>Wu, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+control+of+foraging+and+social+behavior+by+the+Drosophila+neuropeptide+Y-like+system.">Developmental control of foraging and social behavior by the Drosophila neuropeptide Y-like system.</a> Neuron. 39 (1), 147-161 (2003).</li><li>Wu, Q., Zhang, Y., Xu, J., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+hunger-driven+behaviors+by+neural+ribosomal+S6+kinase+in+Drosophila.">Regulation of hunger-driven behaviors by neural ribosomal S6 kinase in Drosophila.</a> Proceedings of the National Academy of Sciences of the United States of America. 102 (37), 13289-13294 (2005).</li><li>Lingo, P. R., Zhao, Z., Shen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-regulation+of+cold-resistant+food+acquisition+by+insulin-+and+neuropeptide+Y-like+systems+in+Drosophila+melanogaster.">Co-regulation of cold-resistant food acquisition by insulin- and neuropeptide Y-like systems in Drosophila melanogaster.</a> Neuroscience. 148 (2), 371-374 (2007).</li></ol>

作者没有什么可透露的。

通过此协议，当接触不同P：C成分的饮食时，可以测试幼虫在特定神经元种群的恒温激活下调节蛋白质和碳水化合物（两种主要宏量营养素）的摄入水平的能力。这种方法是在幼虫初步筛选的背景下测试的，目的是确定与控制不同宏量营养素质量的饮食中的食物摄入量相关的神经元人群。这项工作还有助于证明 ，果蝇 幼虫是研究与营养平衡相关的喂养行为的神经元基础的有价值的动物模型...

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61323/quantification-macronutrients-intake-thermogenetic-neuronal-screen">Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae</a>. A figure was updated.
Figure 1&#160;was updated from:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61323/61323fig1.jpg" /> 
Figure 1: The sucrose-yeast (SY) diets used in our protocol. (A) The blue dots represent the isocaloric (248 calories/L) macronutrient balancing diets used in the feeding assay, which differ in the protein to carbohydrate (P:C) ratios: 1:1, 1:4 and 1:16. The beige dot represents the diet used to rear the experimental third-instar larvae (L3), which contained a P:C ratio of 1:2 and a caloric density of 495 calories/L. (B) Detailed composition and nutritional information of the sucrose-yeast (SY) based diets. The components are the same for all the diets: agar, sucrose and yeast. The amount in grams of the components needed to prepare 1 L of diet is shown. Note that 1% (v/v) of blue dye must be added to the macronutrient balancing diets and to the L3 rearing diet nipagin and propionic acid solutions must be added to a final concentration (v/v) of 3% and 0.3%, respectively. <a href="https://www.jove.com/files/ftp_upload/61323/61323fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
to:
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61323/61323fig1v2.jpg" /> 
Figure 1: The sucrose-yeast (SY) diets used in our protocol. (A) The blue dots represent the isocaloric (248 calories/L) macronutrient balancing diets used in the feeding assay, which differ in the protein to carbohydrate (P:C) ratios: 1:1, 1:4 and 1:16. The beige dot represents the diet used to rear the experimental third-instar larvae (L3), which contained a P:C ratio of 1:2 and a caloric density of 495 calories/L. (B) Detailed composition and nutritional information of the sucrose-yeast (SY) based diets. The components are the same for all the diets: agar, sucrose and yeast. The amount in grams of the components needed to prepare 1 L of diet is shown. Note that 1% (v/v) of blue dye must be added to the macronutrient balancing diets and to the L3 rearing diet nipagin and propionic acid solutions must be added to a final concentration (v/v) of 3% and 0.3%, respectively. <a href="https://www.jove.com/files/ftp_upload/61323/61323fig1v2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

An erratum was issued for:&#160;<a href="https://www.jove.com/t/61323/quantification-macronutrients-intake-thermogenetic-neuronal-screen">Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae</a>. A figure was updated.

Erratum: Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

所有动物都依靠均衡的饮食来获得生存、生长和繁殖所需的营养物质。选择吃什么和吃多少受许多相互作用的因素的影响，相关的动物的内部状态，如饱腹程度和环境条件，如食物质量2，3，4，5。蛋白质和碳水化合物是两种主要的宏量营养素，其均衡摄入对维持动物的生理过程至关重要。因此，对控制喂养行为和维持这些宏量营养素均衡摄入的神经机制的理解是极其相关的。这是因为生命史特征，如寿命，胎儿和代谢健康直接影响蛋白质摄入量水平6，7，8，9，10。
使用更简单、更易操作的生物体，表现出与复杂动物（包括哺乳动物）一起进化保存的喂养习惯，对于这类研究至关重要。重要的是，这些更简单的动物模型提供了一个很好的机会，在昂贵，道德和技术更有效的环境中剖析复杂的生物问题。在过去的几十年里 ，Drosophila以其强大的遗传工具包、复杂而陈规定型的行为以及保存了哺乳动物的外围和营养感应机制结构，成为行为神经生物学家11的富有成效的模型。归根结底，希望通过了解这种动物的食物摄入是如何调节的，有了更简单的神经系统，我们就可以开始解开人类饮食失调背后的神经元故障。
对喂养行为的神经元基材的研究在很大程度上取决于能否同时测量动物的食物摄入量，同时操纵动物的神经元活动。由于摄入的食物数量最少，量化苍蝇吃的食物量极具挑战性，目前所有可用的方法都存在重大限制。因此，金本位制是采用互补方法12的组合。从历史上看，成年苍蝇一直被看好为遗传和行为模型。然而，果蝇幼虫，也提供了机会，以研究神经元基质编码喂养行为。幼虫中枢神经系统（CNS）约有12，000个神经元，比包含约150，000个神经元的成人要复杂得多。这种较低的复杂性不仅在数字上，而且在功能上都是如此，因为幼虫行为依赖于更简单的机车功能和感官系统。尽管幼虫的神经系统明显简单，但幼虫仍然表现出完整的喂养行为，一些方法可以量化果蝇幼虫的食物摄入量。通过与神经元活动的操纵配对，果蝇幼虫可以构成一个高度可牵引的模型，用于理解食物摄入的神经调节。
提供这里是一个详细的协议，量化食物摄入量在幼虫暴露在不同宏量营养素品质的饮食。饮食，所谓的宏量营养素平衡饮食，在蛋白质和碳水化合物含量上有所不同，特别是关于蛋白质与碳水化合物（P：C）的比例：1：1（富含蛋白质的饮食），1：4（中间饮食）和1：16（蛋白质贫乏的饮食），如 图1A所示。简言之，使用这三种异种糖-酵母（SY）为基础的饮食用蓝色食物染料染色，建立了定量无选择喂养检测。由于酵母提取物和蔗糖被用作蛋白质和碳水化合物来源，并且都含有碳水化合物，因此通过改变这两个成分的平衡（如图16 和 图1B所示）获得了P：C比率的变化。 图 2中提供了协议的示意图概述，其中显示了主要的实验步骤。
该协议的建立旨在调查特定神经元人群在调节幼虫喂养水平在不同P：C比率的饮食中以及在恒温遗传神经元屏幕中的作用。一个特征良好的神经遗传学工具被使用从瞬态受体潜力（TRP）家族：嗜异性瞬态受体潜力通道（dTRPA1），这是一个温度和电压门的晶体通道，允许在环境温度上升到25°C17以上时激发行动潜力。为了表达dTRPA1转基因，我们利用了Gal4线基于从罗宾实验室建立的d罗索菲拉基因组的cis-监管区域，在Janelia研究校园18，19的FlyLight项目的背景下。
虽然协议，这里描述，已经建立在激活屏幕的背景下，它可以很容易地适应实验者的其他特定需求或兴趣，即执行抑制屏幕使用温度敏感神经元消音器ShibireTS20，替代dTRPA1。此适应和其他适应性在协议和讨论部分进行讨论。

<table border="0" cellpadding="0" cellspacing="0" style="width:2112px;" width="2113">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">1.5 mL microtubes</td>
			<td style="width:271px;">Sarstedt AG &#38; Co.</td>
			<td style="width:247px;">72.690.001</td>
			<td style="width:1272px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">10xPBS</td>
			<td style="width:271px;">Nytech</td>
			<td style="width:247px;">MB18201</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">2.0 mL microtubes</td>
			<td style="width:271px;">Sarstedt AG &#38; Co.</td>
			<td style="width:247px;">72.695.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">60 mm petri dishes</td>
			<td style="width:271px;">Greiner Bio-one, Austria</td>
			<td style="width:247px;">628161</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">96 well microplates</td>
			<td style="width:271px;">Santa Cruz Biotechnology</td>
			<td style="width:247px;">SC-204453</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Agar</td>
			<td style="width:271px;">Pr&#243;-vida, Portugal</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Bench cooler</td>
			<td style="width:271px;">Nalgene, USA</td>
			<td style="width:247px;">Labtop Cooler 5115-0032</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Blue food dye</td>
			<td style="width:271px;">Rayner, Billingshurst, UK</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Cell disruption media</td>
			<td style="width:271px;">Scientific Industries, Inc.</td>
			<td style="width:247px;">888-850-6208</td>
			<td>(0.5 mm glass beads)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Dish weight boats</td>
			<td style="width:271px;">Santa Cruz Biotechnology</td>
			<td style="width:247px;">SC-201606</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Embryo collection cage for 60 mm petri dishes</td>
			<td style="width:271px;">Flystuff, Scientific Laboratory Supplies, UK</td>
			<td style="width:247px;">FLY1212 (59-100)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Featherweight forceps</td>
			<td style="width:271px;">BioQuip Products, USA</td>
			<td style="width:247px;">4750</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Fly food for stocks maintenance</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>1 L food contains: 10 g Agar, 100 g Yeast Extract, 50 g Sucrose, 30 mL Nipagin, 3 mL propionic acid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Forceps #5</td>
			<td style="width:271px;">Dumont</td>
			<td style="width:247px;">0108-5-PS</td>
			<td>Standard tips, INOX, 11cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Incubator</td>
			<td style="width:271px;">LMS Ltd, UK</td>
			<td style="width:247px;">Series 2, Model 230</td>
			<td>For thermogenetic feeding assay (30&#8728;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Incubator</td>
			<td style="width:271px;">Percival Scientific, USA</td>
			<td style="width:247px;">DR36NL</td>
			<td>To stage larvae (19&#8728;C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Janelia lines</td>
			<td style="width:271px;">Janelia Research Campus</td>
			<td style="width:247px;"></td>
			<td>Detailed information in Table 2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Macronutrient balancing diets</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>Composition and nutritional information in Figure 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Methanol</td>
			<td style="width:271px;">VWR</td>
			<td style="width:247px;">CAS number: 67-56-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nipagin (Methyl 4-hydroxybenzoate)</td>
			<td style="width:271px;">Sigma-Aldrich</td>
			<td style="width:247px;">H5501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Nitrile gloves</td>
			<td style="width:271px;">VWR, USA</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Refrigerated centrifuge</td>
			<td style="width:271px;">Eppendorf, Germany</td>
			<td style="width:247px;">5804 R / Serial number: 5805CI364293</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Rubin Gal4 ines</td>
			<td style="width:271px;">Janelia Research Campus</td>
			<td style="width:247px;"></td>
			<td>Stoks available at Bloomington Drosophila Stock Center</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">ShibireTS UAS line</td>
			<td style="width:271px;">Bloomington Drosophila Stock Center</td>
			<td style="width:247px;">BDSC number: 66600</td>
			<td>Provided by Carlos Ribeiro Group</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Soft brushes</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>For sorting anaesthetised fruit flies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Spectrophotometer plate reader</td>
			<td style="width:271px;">Thermo Fisher Scientific</td>
			<td style="width:247px;">Multiskan Go 51119300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Stereo microscope</td>
			<td style="width:271px;">Nikon</td>
			<td style="width:247px;">1016625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Sucrose</td>
			<td style="width:271px;">Sidul, Portugal</td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Third-instar larvae (L3) rearing diet</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td>Composition and nutritional information in Figure 1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Timer</td>
			<td style="width:271px;"></td>
			<td style="width:247px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tissue lyzer / bead beater</td>
			<td style="width:271px;">MP Biomedicals, USA</td>
			<td style="width:247px;">FastPrep-24 6004500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">TRPA1 UAS line</td>
			<td style="width:271px;">Bloomington Drosophila Stock Center</td>
			<td style="width:247px;">BDSC number: 26264</td>
			<td>Expresses TrpA1 under UAS control; may be used to activate neurons experimentally at 25 &#8728;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Water bath</td>
			<td style="width:271px;">Sheldon Manufacturing Inc., USA</td>
			<td style="width:247px;">W20M-2 / 03068308 / 9021195</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Yeast extract</td>
			<td style="width:271px;">Pr&#243;-vida, Portugal</td>
			<td style="width:247px;"></td>
			<td>51% Protein, 15% Carbohydrate</td>
		</tr>
	</tbody>
</table>

quantification of macronutrients intake in a thermogenetic neuronal screen using drosophila larvae

觅食和喂养行为使动物能够获得对其发育、健康和健身至关重要的能量和营养来源。研究这些行为的神经元调节对于理解营养平衡背后的生理和分子机制至关重要。使用遗传可牵引的动物模型，如蠕虫、苍蝇和鱼，极大地促进了这些类型的研究。在过去的十年里，果蝇果蝇褪黑素被神经生物学家用作一种强大的动物模型，研究喂养和觅食行为的神经元控制。虽然毫无疑问是有价值的，但大多数研究都对成年苍蝇进行了研究。在这里，我们描述了一个协议，利用更简单的幼虫神经系统来研究神经元基板控制喂养行为时，幼虫暴露在饮食不同的蛋白质和碳水化合物含量。我们的方法基于定量色度无选择喂养检测，在神经元恒温激活屏幕的背景下执行。作为读出，幼虫在1小时间隔内食用的食物量被用于暴露在三种染料标签饮食之一，这些饮食的蛋白质与碳水化合物（P：C）的比例不同。通过识别调节不同宏量营养素质量饮食中食用食物量的候选神经元人群，在幼虫果蝇的神经遗传学筛查中证明了此协议的有效性。我们还能够将测试的基因型分类并归类为表型类。除了简要回顾文献中目前可用的方法外，还讨论了这些方法的优点和局限性，并就如何使该方案适应其他具体实验提出了一些建议。

果蝇幼虫以摄入过量碳水化合物23（图2E中的示意图图）为代价调节其蛋白质摄入量。事实上，这种蛋白质摄入的优先顺序已经观察到在许多其他动物，被称为蛋白质杠杆24，25。
利用这种强大的喂养行为反应，设计了一个基于行为的屏幕，旨在识别参与宏量营养素平衡的神经元群体。?...

Watch this Scientific Journal Video about Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae at JoVE.com

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

这里描述的是一个协议，使颜色定量在定义的时间间隔内食用的食物量，由 德罗索菲拉梅拉诺加斯特 幼虫暴露在不同宏量营养素品质的饮食。这些检测是在神经元恒温屏幕的背景下进行的。

使用 德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

quantification-macronutrients-intake-thermogenetic-neuronal-screen

Research

JoVE Journal

Neuroscience

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

3.6K 次观看.  Monash University. 这里描述的是一个协议，使颜色定量在定义的时间间隔内食用的食物量，由 德罗索菲拉梅拉诺加斯特 幼虫暴露在不同宏量营养素品质的饮食。这些检测是在神经元恒温屏幕的背景下进行的。

视频: Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

科研

神经科学

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

欲求联想嗅觉学习<em&gt;果蝇</em&gt;幼虫

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

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

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

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

谷氨酸和GABA快速微离子导入：一个有用​​的工具，调查突触整合

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

Inducing Plasticity of Astrocytic Receptors by Manipulation of Neuronal Firing Rates

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

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

诱导星形胶质细胞受体的可塑性由神经元的放电频率操作

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

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of the right type and quantity of food therefore has a major influence on quality of life. Research on feeding behavior focuses on the underlying processes that ensure actual feeding and unravels the role of factors regulating internal energy homeostasis and the neuronal bases of decision-making. The model organism Drosophila melanogaster, with its great variety of genetically traceable tools for labeling and manipulating single neurons, allows mapping of neuronal networks and identification of molecular signaling cascades involved in the regulation of food intake. This report demonstrates the CApillary FEeder assay (CAFE) and shows how to measure food intake in a group of flies for time spans ranging from hours to days. This easy-to-use assay consists of glass capillaries filled with liquid food that flies can freely access and feed on. Food consumption in the assay is accurately determined using simple measurement tools. Herein we describe step-by-step the method from setup to successful execution of the CAFE assay, and provide practical examples to analyze the food intake of a group of flies under controlled conditions. The reader is guided through possible limitations of the assay, and advantages and disadvantages of the method compared to other feeding assays in D. melanogaster are evaluated.

The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster

The CApillary FEeder (CAFE) assay is a simple, budget-friendly, highly reliable method for investigating mechanisms underlying food intake. Used with the highly versatile genetic model organism Drosophila melanogaster, it provides a powerful means of gaining new insights into regulatory mechanisms of food intake.

毛细管馈线分析测量的摄食<em&gt;果蝇</em

For most animals, feeding is an essential behavior for securing survival, and it influences development, locomotion, health and reproduction. Ingestion of ...

Quantification of Drosophila Grooming Behavior

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a grooming assay protocol and novel chamber design that is cost-efficient and scalable for either small or large-scale studies of Drosophila grooming. Flies are dusted all over their body with Brilliant Yellow dye and given time to remove the dye from their bodies within the chamber. Flies are then deposited in a set volume of ethanol to solubilize the dye. The relative spectral absorbance of dye-ethanol samples for groomed versus ungroomed animals are measured and recorded. The protocol yields quantitative data of dye accumulation for individual flies, which can be easily averaged and compared across samples. This allows experimental designs to easily evaluate grooming ability for mutant animal studies or circuit manipulations. This efficient procedure is both versatile and scalable. We show work-flow of the protocol and comparative data between WT animals and mutant animals for the Drosophila type I Dopamine Receptor (DopR).

This protocol describes a scalable individual grooming assay technique in Drosophila that yields robust, quantitative data to measure grooming behavior. The method is based on comparing the difference in dye accumulation on the bodies of un-groomed versus groomed animals over a set period of time.

果蝇美容行为的量化

Drosophila grooming behavior is a complex multi-step locomotor program that requires coordinated movement of both forelegs and hindlegs. Here we present a ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

新的分析冷痛觉在<em&gt;果蝇</em&gt;幼虫

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

嗜德维人神经元树突状树突状复杂度的定量分析

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

使用 德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

使用德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化