这项工作部分是由中国国家基础研究计划 (2012CB825504)、中国国家自然科学基金 (91232720 和 9163210042)、中国科学院 (GJHZ201302 和 QYZDY-SSW-SMC015)、比尔和梅琳达盖茨支持的。基金会 (OPP1119434) 和 100-中国科学院人才培养计划。

1. 准备送料室
注意:染料标记喂养试验的加料室由两部分组成: 外容器 (作为盖子) 和内容器 (作为食物来源)。
<ol><li>从玻璃瓶中修改外部容器以培养果蝇(内径为31.8 毫米, 高度为80毫米) (图 1A, 1C)。用一层1% 琼脂糖 (5 毫升) 盖住容器的底部, 以保持喂食装置的适当湿润, 并充当苍蝇行走的软基板 (图 1B)。容器从而提供一个受控但自然主义的设置, 对苍蝇进行最小的应力或扰动。<ol>
<li>准备外面的容器。将0.5 克琼脂糖悬浮在50毫升的双蒸馏水中, 加热时经常搅拌, 然后煮沸, 完全溶解在一个带有热板的磁力搅拌器上。</li>
		<li>当1% 琼脂糖冷却到约60摄氏度, 转移5毫升1% 琼脂糖到一个空的外部容器 (图 1B), 并让容器保持开放, 直到冷却到室温, 以产生琼脂糖垫。</li>
	</ol>
</li>
	<li>准备染料标签食品。染料标签食品的组成根据不同的实验目的而变化。这里是一个例子, 准备的染料标签食品只含有蔗糖。<ol>
<li>添加0.5 克琼脂糖到50毫升的双蒸馏水, 热频繁搅拌, 并煮沸完全溶解在一个磁性搅拌器与热板。</li>
		<li>当上述1% 琼脂糖冷却到约60摄氏度, 添加1.71 克蔗糖的琼脂糖, 以获得 100 mM 蔗糖。</li>
		<li>添加0.25 克蓝色染料 (erioglaucine 二钠) 到琼脂糖溶液中获得 0.5% (瓦特/v) 染料标签食品。</li>
	</ol>
</li>
	<li>准备里面的容器。将空内容器放在平坦的水平面上, 然后将750µL 的蓝色染料标签食品添加到单独的食品容器中, 让它凝固至少5分钟。 注意:内部容器是一个小的塑料碟, 内径为13.6 毫米, 高度为7.5 毫米 (图 1A, 1C)。它充满了染料标签的食物, 然后定位在琼脂糖垫的外部容器的中心。</li>
	<li>将填充的内容器转移到准备好的外部容器中, 并将其放置在琼脂糖垫的中心。让喂食室在室温下保持40分钟的开放, 直到外部容器的壁没有凝结。 注意:干墙是必要的, 以防止苍蝇被困在墙上, 接触水滴。</li>
	<li>用无染料食物 (相同成分但不含染料) 为背景减法准备喂食室。</li>
</ol>2. 苍蝇的制备
注意:成年女性飞行5至10天后, 羽化通常用于喂养化验。建议对不同基因型的苍蝇进行制备, 包括基因控制, 并进行平行试验。20飞行 (从一个哺养的房间) 生成一个数据点。
<ol><li>考虑到饲养成年苍蝇的人口密度。通常, 根据菌株和培养瓶的尺寸来改变亲本的数量来控制人口密度。举个例子, 将广州 S 的15男性和30女性转移到一个文化瓶 (直径31.8 毫米和高度80毫米), 让雌性产卵一天, 然后删除成年苍蝇。</li>
	<li>准备苍蝇 thermogenetic 激活。<ol>
<li>使用 Gal4/UAS 系统38 , 通过跨越 UAS-dTrpA1 与不同 GAL4 驱动程序线 (图 2B), 在不同的神经元中表达 dTRPA133 。</li>
		<li>保持成人飞行在标准食物在22°c, 60% 相对湿度和 12 h/12 h 光黑暗的周期。</li>
		<li>在喂食试验前两天, 在一个2飞垫上, 在一个立体显微镜下, 将苍蝇放在一个新鲜的食物瓶中, 并保持每瓶20只苍蝇的密度。</li>
	</ol>
</li>
	<li>准备苍蝇 optogenetic 激活。<ol>
<li>使用 Gal4/UAS 系统38 , 通过与不同 GAL4 驱动程序线交叉的无人驾驶 CsChrimson 苍蝇, 在不同的神经元中表达 CsChrimson36 。</li>
		<li>收集新的 eclosed (24 小时内) 苍蝇和转移到一个小瓶与标准食品含有400µM 全反式视网膜。</li>
		<li>为了防止过早激活, 用铝箔包裹饲养瓶, 然后放入一个黑暗的盒子里, 保护苍蝇不受不必要的光刺激。保持苍蝇在25摄氏度, 60% 湿度在黑暗中 4-6 天。</li>
		<li>在喂食试验前两天, 在 CO2飞垫上用立体显微镜对雌性苍蝇进行排序。收集20只苍蝇到一个食品瓶, 并保持他们在黑暗的喂养测试之前。</li>
		<li>尽可能快地在昏暗的灯光下进行所有操作, 以避免环境光线造成的影响。使用放置在远离工作区域的 LED 灯, 工作区的强度为5µW/厘米2。 注意:使用饿死的广州 S 苍蝇作为喂养试验的积极控制. 剥夺这些苍蝇的食物和维持在1% 琼脂糖24小时之前的喂养测试。这些苍蝇的食物消耗水平有助于评估日常的波动。</li>
	</ol>
</li>
</ol>3. Thermogenetic 激活
<ol><li>在当天的同一时间进行所有喂养实验, 大约授时时间 4-631,32, 以最小化由于昼夜节律的变化。</li>
	<li>准备一个孵化器 (或一个有温度控制的加热器的小房间) 作为供暖环境。</li>
	<li>在行为实验之前, 平衡在实验温度 (30 °c) 上准备好的进料室, 2 小时, 以确保进料室的温度相对均匀。</li>
	<li>要开始喂食过程, 小心地将20飞从他们的家小瓶到一个预热的喂食室通过温和的攻丝。继续以30°c 为60分钟的喂养过程 (图 3A) . 注意:频繁的鼓动, 如打开和关闭孵化器门, 不利地影响喂养行为, 因此应尽量减少。</li>
	<li>在-80 摄氏度 (或-20 °c) 冻结喂食室, 停止喂养60分钟, 30 分钟。 注意:冷冻有助于在所有房间同时停止喂养。结冰的苍蝇在-80 °c 服务二个目的, 为 euthanizing 苍蝇和为存贮直到均匀化为一个星期。</li>
	<li>对于温度控制组, 转移20飞入室温进给室与染料食品, 开始喂养过程在22°c 60 分钟。</li>
	<li>在-80 摄氏度 (或-20 摄氏度) 的温度控制组中, 停止喂养30分钟。</li>
</ol>4. Optogenetic 激活
<ol><li>在当天的同一时间进行所有喂养实验, 大约授时时间 4-631,32, 以最小化由于昼夜节律的变化。</li>
	<li>小心地将20只苍蝇从一小瓶移入进料室, 然后轻轻敲击, 然后将腔侧放在照明设置上 (图 4A, 4B)。<ol>
<li>对于 optogenetic 操作, 使用橙色 led 阵列 (607 nm) 作为光源的刺激, 并固定在 led 上方的水平有机玻璃板, 以支持在照明期间的喂食室。将安装程序保持在25摄氏度的孵化器中, 60% 的湿度 (图 4A, 4B)。</li>
		<li>刺激遗传控制苍蝇平行与饕餮 > CsChrimson苍蝇, 但在不同的喂养室。</li>
	</ol>
</li>
	<li>继续喂养过程为60分钟与背面照明由橙色光。<ol>
<li>确定光遗传学实验36的最佳光照强度.对于饕餮 > CsChrimson飞行, 强度为2.2 兆瓦/毫米2会诱发瘫痪 (停止运动和失去体位控制), 因此, 使用中等强度0.1 兆瓦/毫米2来刺激饕餮> CsChrimson在喂食测试期间飞行。</li>
	</ol>
</li>
	<li>在-80 摄氏度 (或-20 摄氏度) 的情况下, 通过冷冻喂养室停止喂食30分钟。</li>
</ol>5. 食品消费的视觉估计
注意:视觉评分法提供了对食品消耗量的半定量估计。虽然该方法不像光学方法那样客观, 但它能够及时地通过大量的飞行菌株, 使其适合于第一轮的基因筛, 迅速缩小候选名单, 以便进行进一步的测试。
<ol><li>在喂食过程之后, 麻醉在一个立体显微镜下的 CO2飞垫上的苍蝇。根据其腹部的染料色食物的体积和强度, 为每只苍蝇分别评分 (图 2A)。 注意: 视觉估计的标准: 评分系统有4水平的食物摄取量对应于不同体积和强度的彩色食物在腹部。<ol>
<li>无可察觉的染料色的食物的比分飞行比分 0;给那些微弱的 (几乎不被发现的) 蓝色染料或用染料色的食物占领少于一个第三容量腹部 a 比分1。</li>
		<li>得分与强烈的染料色的食物占领腹部的一半的分数为2。</li>
		<li>分数为 3 (图 2A) 的强烈染色的内容占据超过一半的腹部得分。</li>
	</ol>
</li>
	<li>在每个实验条件下计算不同分数的苍蝇的比例 (图 2B)。</li>
</ol>6. 食品消耗量的比色量化
<ol><li>停止进食后, 将所有20只苍蝇从喂食室倒入一张称重纸上, 然后用软刷子将其转移到1.5 毫升管中。 注意:不要把所有的房间的-80 °c 存储同时, 以避免苍蝇粘在房间壁, 因为水凝结在寒冷的房间。</li>
	<li>添加500µL 的 PBST 管和融汇的苍蝇使用研磨机在60赫兹 5 s。<ol>
<li>当身体部位没有可检测的片段时, 通过观察确认充分的均匀化。</li>
	</ol>
</li>
	<li>旋转管与组织匀浆30分钟, 在1.3万转, 以清除碎片。</li>
	<li>离心后, 将100µL 的上清液转移到96井板的井中。</li>
	<li>在所有样品被装载了之后, 把板材放入一个板材读者测量样品的吸光度在630毫微米。</li>
	<li>要去除背景吸收, 减去上清液的吸光度, 从吃的苍蝇的食物没有染料从上清的吸收从蓝色食物喂养的苍蝇。 注意:这一步是可选的, 这取决于所用食物的成分。当苍蝇食品 (没有染料) 产生 630 nm 的吸光度, 有必要执行这一步骤, 以消除背景。</li>
</ol>

<ol>
	<li>Gao, Q., Horvath, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurobiology+of+feeding+and+energy+expenditure.">Neurobiology of feeding and energy expenditure.</a> Annu Rev Neurosci. 30, 367-398 (2007).</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>Edgecomb, R. S., Harth, C. E., Schneiderman, A. M. <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+feeding+behavior+in+adult+Drosophila+melanogaster+varies+with+feeding+regime+and+nutritional+state.">Regulation of feeding behavior in adult Drosophila melanogaster varies with feeding regime and nutritional state.</a> J Exp Biol. 197, 215-235 (1994).</li><li>Miyamoto, T., Slone, J., Song, X., Amrein, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fructose+receptor+functions+as+a+nutrient+sensor+in+the+Drosophila+brain.">A fructose receptor functions as a nutrient sensor in the Drosophila brain.</a> Cell. 151 (5), 1113-1125 (2012).</li><li>Morton, G. J., Cummings, D. E., Baskin, D. G., Barsh, G. S., Schwartz, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+nervous+system+control+of+food+intake+and+body+weight.">Central nervous system control of food intake and body weight.</a> Nature. 443 (7109), 289-295 (2006).</li><li>Pool, A. H., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feeding+regulation+in+Drosophila.">Feeding regulation in Drosophila.</a> Curr Opin Neurobiol. 29, 57-63 (2014).</li><li>Soderberg, J. A., Carlsson, M. A., Nassel, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Insulin-Producing+Cells+in+the+Drosophila+Brain+also+Express+Satiety-Inducing+Cholecystokinin-Like+Peptide,+Drosulfakinin.">Insulin-Producing Cells in the Drosophila Brain also Express Satiety-Inducing Cholecystokinin-Like Peptide, Drosulfakinin.</a> Front Endocrinol (Lausanne). 3, 109 (2012).</li><li>Stafford, J. W., Lynd, K. M., Jung, A. Y., Gordon, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integration+of+taste+and+calorie+sensing+in+Drosophila.">Integration of taste and calorie sensing in Drosophila.</a> J Neurosci. 32 (42), 14767-14774 (2012).</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> Proc Natl Acad Sci U S A. 102 (37), 13289-13294 (2005).</li><li>Itskov, P. 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=Automated+monitoring+and+quantitative+analysis+of+feeding+behaviour+in+Drosophila.">Automated monitoring and quantitative analysis of feeding behaviour in Drosophila.</a> Nat Commun. 5, 4560 (2014).</li><li>Mair, W., 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=Calories+do+not+explain+extension+of+life+span+by+dietary+restriction+in+Drosophila.">Calories do not explain extension of life span by dietary restriction in Drosophila.</a> PLoS Biol. 3 (7), e223 (2005).</li><li>Diegelmann, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+CApillary+FEeder+Assay+Measures+Food+Intake+in+Drosophila+melanogaster.">The CApillary FEeder Assay Measures Food Intake in Drosophila melanogaster.</a> J Vis Exp. (121), (2017).</li><li>Ja, W. W., 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=Prandiology+of+Drosophila+and+the+CAFE+assay.">Prandiology of Drosophila and the CAFE assay.</a> Proc Natl Acad Sci U S A. 104 (20), 8253-8256 (2007).</li><li>Shiraiwa, T., Carlson, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proboscis+extension+response+(PER)+assay+in+Drosophila.">Proboscis extension response (PER) assay in Drosophila.</a> J Vis Exp. (3), e193 (2007).</li><li>Qi, W., 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+quantitative+feeding+assay+in+adult+Drosophila+reveals+rapid+modulation+of+food+ingestion+by+its+nutritional+value.">A quantitative feeding assay in adult Drosophila reveals rapid modulation of food ingestion by its nutritional value.</a> Mol Brain. 8, 87 (2015).</li><li>Ja, W. W., 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=Water-+and+nutrient-dependent+effects+of+dietary+restriction+on+Drosophila+lifespan.">Water- and nutrient-dependent effects of dietary restriction on Drosophila lifespan.</a> Proc Natl Acad Sci U S A. 106 (44), 18633-18637 (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> Nat Methods. 11 (5), 535-540 (2014).</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=Acidic+Food+pH+Increases+Palatability+and+Consumption+and+Extends+Drosophila+Lifespan.">Acidic Food pH Increases Palatability and Consumption and Extends Drosophila Lifespan.</a> J Nutr. 145 (12), 2789-2796 (2015).</li><li>Dus, M., Min, S., Keene, A. C., Lee, G. Y., Suh, G. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taste-independent+detection+of+the+caloric+content+of+sugar+in+Drosophila.">Taste-independent detection of the caloric content of sugar in Drosophila.</a> Proc Natl Acad Sci U S A. 108 (28), 11644-11649 (2011).</li><li>Shen, P., Cai, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+neuropeptide+F+mediates+integration+of+chemosensory+stimulation+and+conditioning+of+the+nervous+system+by+food.">Drosophila neuropeptide F mediates integration of chemosensory stimulation and conditioning of the nervous system by food.</a> J Neurobiol. 47 (1), 16-25 (2001).</li><li>Yang, Z., 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=Octopamine+mediates+starvation-induced+hyperactivity+in+adult+Drosophila.">Octopamine mediates starvation-induced hyperactivity in adult Drosophila.</a> Proc Natl Acad Sci U S A. 112 (16), 5219-5224 (2015).</li><li>Ramdya, P., Schneider, J., Levine, J. 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+neurogenetics+of+group+behavior+in+Drosophila+melanogaster.">The neurogenetics of group behavior in Drosophila melanogaster.</a> J Exp Biol. 220 (Pt 1), 35-41 (2017).</li><li>Sanchez-Alcaniz, J. A., Zappia, G., Marion-Poll, F., Benton, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+mechanosensory+receptor+required+for+food+texture+detection+in+Drosophila.">A mechanosensory receptor required for food texture detection in Drosophila.</a> Nat Commun. 8, 14192 (2017).</li><li>Zhan, Y. P., Liu, L., Zhu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Taotie+neurons+regulate+appetite+in+Drosophila.">Taotie neurons regulate appetite in Drosophila.</a> Nat Commun. 7, 13633 (2016).</li><li>Yu, Y., 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=Regulation+of+starvation-induced+hyperactivity+by+insulin+and+glucagon+signaling+in+adult+Drosophila.">Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila.</a> Elife. 5, (2016).</li><li>Wood, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sirtuin+activators+mimic+caloric+restriction+and+delay+ageing+in+metazoans.">Sirtuin activators mimic caloric restriction and delay ageing in metazoans.</a> Nature. 430 (7000), 686-689 (2004).</li><li>Inagaki, H. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+Neuromodulation+In+Vivo:+TANGO-Mapping+of+Dopamine+Signaling+Reveals+Appetite+Control+of+Sugar+Sensing.">Visualizing Neuromodulation In Vivo: TANGO-Mapping of Dopamine Signaling Reveals Appetite Control of Sugar Sensing.</a> Cell. 148 (3), 583-595 (2012).</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), e6063 (2009).</li><li>Sen, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moonwalker+Descending+Neurons+Mediate+Visually+Evoked+Retreat+in+Drosophila.">Moonwalker Descending Neurons Mediate Visually Evoked Retreat in Drosophila.</a> Curr Biol. 27 (5), 766-771 (2017).</li><li>Ewing, L. S., Ewing, A. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Courtship+of+Drosophila+melanogaster+in+large+observation+chambers:+the+influence+of+female+reproductive+state.">Courtship of Drosophila melanogaster in large observation chambers: the influence of female reproductive state.</a> Behaviour. 101 (1), 243-252 (1987).</li><li>Chatterjee, A., Tanoue, S., Houl, J. H., Hardin, P. E. <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+gustatory+physiology+and+appetitive+behavior+by+the+Drosophila+circadian+clock.">Regulation of gustatory physiology and appetitive behavior by the Drosophila circadian clock.</a> Curr Biol. 20 (4), 300-309 (2010).</li><li>Xu, K., Zheng, X., Sehgal, A. <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+feeding+and+metabolism+by+neuronal+and+peripheral+clocks+in+Drosophila.">Regulation of feeding and metabolism by neuronal and peripheral clocks in Drosophila.</a> Cell Metab. 8 (4), 289-300 (2008).</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), 217-255 (2008).</li><li>Viswanath, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion+channels+-+Opposite+thermosensor+in+fruitfly+and+mouse.">Ion channels - Opposite thermosensor in fruitfly and mouse.</a> Nature. 423 (6942), 822-823 (2003).</li><li>Yu, Y., 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=Regulation+of+starvation-induced+hyperactivity+by+insulin+and+glucagon+signaling+in+adult+Drosophila.">Regulation of starvation-induced hyperactivity by insulin and glucagon signaling in adult Drosophila.</a> Elife. 5, (2016).</li><li>Klapoetke, N. C., 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nat Methods. 11 (3), 338-346 (2014).</li><li>Viswanath, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Opposite+thermosensor+in+fruitfly+and+mouse.">Opposite thermosensor in fruitfly and mouse.</a> Nature. 423 (6942), 822-823 (2003).</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>Lee, K. S., You, K. H., Choo, J. K., Han, Y. M., Yu, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+short+neuropeptide+F+regulates+food+intake+and+body+size.">Drosophila short neuropeptide F regulates food intake and body size.</a> J Biol Chem. 279 (49), 50781-50789 (2004).</li><li>Marella, S., Mann, K., Scott, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopaminergic+Modulation+of+Sucrose+Acceptance+Behavior+in+Drosophila.">Dopaminergic Modulation of Sucrose Acceptance Behavior in Drosophila.</a> Neuron. 73 (5), 941-950 (2012).</li><li>Albin, S. 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+Subset+of+Serotonergic+Neurons+Evokes+Hunger+in+Adult+Drosophila.">A Subset of Serotonergic Neurons Evokes Hunger in Adult Drosophila.</a> Current Biology. 25 (18), 2435-2440 (2015).</li><li>Ro, 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=Serotonin+signaling+mediates+protein+valuation+and+aging.">Serotonin signaling mediates protein valuation and aging.</a> eLife. 5, e16843 (2016).</li></ol>

作者声明没有竞争的财政利益。

本报告的重点是在 thermogenetic 和 optogenetic 激活的背景下, 对食品消费进行染料标记喂养测定的技术过程, 以操纵神经元控制喂养。这一简单而可靠的协议将有助于阐明候选神经元在喂养控制中的作用, 测量苍蝇的食物偏好, 并通过喂食的遗传屏幕24识别喂养控制回路中的新玩家。
染料标记策略是研究短期喂养行为的可行方法。染料, erioglaucine 钠, 溶入和摄取的?...

量化摄入的食物量对于评估大脑在响应内部需求 (如饥饿状态) 和外部因素 (如食品质量和适口性) 方面的多种喂养控制方面非常重要1,2,3,4,5,6,7,8,9. 近年来, 在果蝇中破译喂养控制的神经基质的努力导致了多种化验的发展, 直接量化摄取的食物量或作为喂养动机的指标。10,11,12,13,14,15,16。
毛细管进纸器 (咖啡馆) 检测12,13被开发用来测量玻璃 microcapillary 中液态食物的消耗量。咖啡馆化验是高度敏感和可重复的17 , 并简化了食品消耗量的测量, 特别是用于定量长期喂养18。然而, 这种化验要求苍蝇爬上 microcapillary 的尖端和饲料倒置, 这是不适合所有菌株。此外, 由于使用咖啡馆化验试验的苍蝇必须饲养在液态食品上, 这些饲养条件对新陈代谢状况或潜在营养不良的影响仍有待确定。
喙扩展响应 (每) 检测11,14计数的喙延伸反应的频率, 以温和的接触食物滴。每种化验结果证明, 作为评估个人飞行和驴子喂养动机的一个很好的方法食物适口性和食品含量的影响18,19。然而, 它不是直接定量的摄入量。
最近, 研制了一种半自动方法--人工喂养法 (MAFE)15。在 MAFE, 一只固定的苍蝇用含有食物的 microcapillary 人工喂养。鉴于可同时监测喙的延伸反应和食物消耗量, MAFE 适合于评估营养价值和药理操作的效果。然而, 固定一只苍蝇可能会对其行为表现产生负面影响, 包括进食。
此外, 还开发了飞行喙和活动检测器 (FlyPAD)10 , 以自动量化喂养行为。使用机器视觉方法, FlyPAD 记录苍蝇和食物之间的物理相互作用, 以量化喙延长的频率和持续时间, 作为喂养动机的指示器。FlyPAD 提供了一种高通量的方法来监视自由移动苍蝇的喂养行为, 尽管该系统的灵敏度和鲁棒性仍有待更多研究12进一步确认。
标签策略经常被用来估计苍蝇的食物摄取量。用化学示踪剂标记食物是常见的, 在进食后, 测量摄取的示踪物量来计算食物摄取量。放射性示踪剂16,17,20,21,22,23,24,25允许通过角质层检测不均匀的苍蝇。该方法提供了显著的低变异性和高灵敏度的18, 对食品的长期摄入量的研究是可行的。然而, 在使用这种化验时, 应考虑到可用的放射性同位素和不同的吸光度和排泄率。
标签和追踪食物摄取与无毒的食物颜色是一个更安全和更简单的选择2,3,26,27,28。在食用含有可溶性和不可吸收染料的食物后, 苍蝇是均匀的, 摄取的染料的用量后来用分光光度计3,24,28,29.标签策略易于执行, 并提供了高效率, 但有一个警告。从摄取染料中估计的食物摄入量比实际体积小, 因为在苍蝇开始喂养17之后, 排泄开始早于15分钟。此外, 该检测通常在60分钟内评估食物摄取量, 仅适用于短期喂养行为的调查24,28。此外, 多个内部和外部因素,如基因型17、性别17、交配状态17、饲养密度30、昼夜节律3132和食品质量3,8,16, 影响食物摄取量。因此, 喂养持续时间可能需要根据特定的实验条件进行调整。除了促进食品摄取量的量化, 食品颜色还用于评估食物选择2,19,27, 并可视化的半月板在咖啡馆化验12microcapillary。
在这里, 我们介绍了一个协议的结合操作的神经元活动与染料标记方法。这一策略已被证明是有益的, 在我们的遗传性研究的饲养控制在成年果蝇24。视觉评分法允许快速估计食物消耗量;因此, 它是有用的筛选通过大量的菌株及时的方式。然后用比色法对屏幕上的候选者进行详细分析, 以提供客观和精确的量化。
除了喂食化验, 我们还描述了 thermogenetic27,33,34,35和 optogenetic36在果蝇中强行激活目标神经元的方法。通过 thermogenetic 操作激活神经元是简单和方便的果蝇瞬态受体电位锚蛋白 1 (dTRPA1), 这是一个温度和电压门控阳离子通道, 增加神经元的兴奋性时, 环境温度升高23摄氏度以上33,37;然而, 在高温下测试动物可能会对行为产生不良影响。激活果蝇中神经元的另一种有效方法是使用光遗传学与 CsChrimson 36, 这是 channelrhodopsin 的一个红色移位变体, 它增加了当暴露于光时神经元的兴奋性.光遗传学提供更高的时间分辨率和较小的干扰行为比 thermogenetics。将食物摄取量与神经元活性的调控相结合, 是研究喂养神经机制的有效途径。
我们详细描述了喂养室的准备和要测试的苍蝇。使用Taotie-Gal4作为模型24, 我们描述了通过 thermogenetics 和光遗传学激活神经元。该议定书还介绍了两种用染料标记食品定量测定食品消耗量的试验。

<table><tbody><tr><td>UAS-CsChrimson</td><td>Bloomintoon</td><td>55135</td><td></td></tr><tr><td>UAS-dTrpA1</td><td>Bloomintoon</td><td>26263</td><td></td></tr><tr><td>TDC1-GAL4&amp;nbsp;</td><td>Bloomintoon</td><td>9312</td><td></td></tr><tr><td>TDC2-GAL4</td><td>Bloomintoon</td><td>9313</td><td></td></tr><tr><td>sNPF-GAL4</td><td></td><td></td><td>Provided by Z. Zhao</td></tr><tr><td>NPF-GAL4</td><td></td><td></td><td>Provided by Y. Rao</td></tr><tr><td>TH-GAL4</td><td></td><td></td><td>Provided by Y. Rao</td></tr><tr><td>5-HT-GAL4</td><td></td><td></td><td>Provided by Y. Rao</td></tr><tr><td>AKH-GAL4</td><td></td><td></td><td>Provided by Y. Rao</td></tr><tr><td>dip2-GAL4</td><td></td><td></td><td>Provided by Y. Rao</td></tr><tr><td>Taotie-GAL4</td><td></td><td></td><td>Provided by J. Carlson</td></tr><tr><td>Agarose</td><td>Biowest</td><td>G-10</td><td></td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S7903</td><td></td></tr><tr><td>Erioglaucine disodium salt</td><td>Sigma</td><td>861146</td><td></td></tr><tr><td>all-trans-retinal&amp;nbsp;</td><td>Sigma&amp;nbsp;</td><td>R2500</td><td>stored in darkness</td></tr><tr><td>Triton X-100</td><td>Amresco</td><td>9002-93-01</td><td></td></tr><tr><td>Fly food</td><td></td><td>1 L food contains: 77.7 g corn meal, 32.19 g yeast, 5 g agar, 0.726 g CaCl&lt;sub&gt;2&lt;/sub&gt;, 31.62 g sucrose, 63.2 g glucose, 2 g potassium sorbate, pH&amp;nbsp;&amp;nbsp;&amp;nbsp;</td><td></td></tr><tr><td>&amp;nbsp;1x PBS buffer&amp;nbsp;</td><td></td><td>1 L 1X PBS contains: 8 g Nacl, 0.2 g Kcl, 1.44 g Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;, 0.24 g KH&lt;sub&gt;2&lt;/sub&gt;PO&lt;sub&gt;4&lt;/sub&gt;, pH 7.4</td><td></td></tr><tr><td>PBST buffer</td><td></td><td>1X PBS with 1% Triton X-100</td><td></td></tr><tr><td>&amp;nbsp;Grinding mill</td><td>Shang Hai Jing Xin</td><td>Tissuelyser-24</td><td></td></tr><tr><td>Incubator</td><td>Ning Bo Jiang Nan</td><td>HWS-80</td><td></td></tr><tr><td>Magnetic stirrer with a heat plate</td><td>Chang Zhou Bo Yuan</td><td>CJJ 78-1</td><td></td></tr><tr><td>Spectrometer</td><td>Thorlabs</td><td>CCS200/M</td><td></td></tr><tr><td>Microplate Spectrophotometer</td><td>Thermo Scientific&amp;nbsp;</td><td>Multiskan GO Type: 1510, REF 51119200</td><td></td></tr><tr><td>Fluorescence stereo microscope&amp;nbsp;</td><td>Leica</td><td>&amp;nbsp;M205FA</td><td></td></tr><tr><td>Stereo microscope</td><td>Leica&amp;nbsp;</td><td>S6E</td><td></td></tr><tr><td>Outside container</td><td>Jiang Su Hai Men</td><td>glass vial with a diameter of 31.8 mm and a height of 80 mm (inside dimension)</td><td></td></tr><tr><td>Inside container&amp;nbsp;</td><td>Beijing Yi Ran machinery factory</td><td>plastic dish with a diameter of 13.6 mm and a height of 7.5 mm (inside dimension)</td><td></td></tr><tr><td>1.5 mL Eppendorf tubes</td><td>Hai Men Ning Mong</td><td></td><td></td></tr><tr><td>&amp;nbsp;96 well plate</td><td>Corning Incorporated</td><td>&amp;nbsp;Costar 3599</td><td></td></tr><tr><td>LEDs</td><td>Xin Xing Yuan Guangdian</td><td>607 nm, 3W&amp;nbsp;</td><td>https://item.taobao.com/item.htm?id=20158878058</td></tr></tbody></table>

combining quantitative food-intake assays and forcibly activating neurons to study appetite in drosophila

食品消费受到大脑的严密控制, 它整合了食物的生理状态、适口性和营养成分, 并发出启动或停止进食的命令。破译及时和适度喂养决策的过程, 对我们理解与喂养控制有关的生理和心理障碍有重大影响。在实验操作后, 需要简单、定量和健壮的方法来测量动物的食物摄取量, 例如强行增加某些靶神经元的活性。在这里, 我们介绍了基于染料标记的饲料化验, 以促进遗传性研究的饲养控制在成年果蝇。我们回顾了现有的喂养方法, 然后从设置到分析, 逐步描述我们的方式, 这结合 thermogenetic 和 optogenetic 操作的神经元控制喂养动机与染料标签食品摄入量测定。我们还讨论了我们的方法的优势和局限性, 与其他喂养化验相比, 以帮助读者选择适当的化验。

Thermogenetic 屏幕。
食欲异常增加会导致食物摄入量升高, 而不考虑生理需求。我们利用这个方案设计了一个高通量行为屏幕, 以获得与饥饿和饱足状态相关的神经元的遗传处理 (图 1)。屏幕产生Taotie-Gal424。当Taotie-Gal4神经元被强行激活为30摄氏度时,饕餮 > TrpA1苍蝇摄取?...

Watch this Scientific Journal Video about Combining Quantitative Food-intake Assays and Forcibly Activating Neurons to Study Appetite in Drosophila at JoVE.com

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

通过染色食品进行定量的食品摄入量测定, 为评价饲用动机提供了一种强有力的、高吞吐量的方法。将食品消费检测与 thermogenetic 和 optogenetic 筛结合起来, 是研究成人果蝇的食欲的神经回路的有力方法.

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, ...

combining-quantitative-food-intake-assays-forcibly-activating-neurons

Research

JoVE Journal

Behavior

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

8.2K 次观看.  University of Science and Technology of China. 通过染色食品进行定量的食品摄入量测定, 为评价饲用动机提供了一种强有力的、高吞吐量的方法。将食品消费检测与 thermogenetic 和 optogenetic 筛结合起来, 是研究成人果蝇的食欲的神经回路的有力方法.

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

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

科研

神经科学

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 ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental factors, and the toxicological and pharmacological effects of numerous substances. Few methods currently implemented are amenable to high throughput measurement. The Microplate Feeder Assay (MFA) was developed for quantifying the consumption of liquid food for individual flies using absorbance. In this assay, flies consume liquid food medium from select wells of a 1536-well microplate. By incorporating a dilute tracer dye into the liquid food medium and loading a known volume into each well, absorbance measurements of the well acquired before and after consumption reflect the resulting change in volume (i.e., volume consumed). To enable high throughput analysis with this method, a 3D-printed coupler was designed that allows flies to be sorted individually into 96-well microplates. This device precisely orients 96- and 1536-well microplates to give each fly access to up to 4 wells for consumption, thus enabling food preference quantification in addition to regular consumption. Furthermore, the device has barrier strips that toggle between open and closed positions to allow for controlled containment and release of a column of samples at a time. This method enables high throughput measurements of consumption of aqueous solutions by many flies simultaneously. It also has the potential to be adapted to other insects and to screen consumption of nutrients, toxins, or pharmaceuticals.

A High Throughput Microplate Feeder Assay for Quantification of Consumption in Drosophila

The microplate feeder assay offers an economical, high throughput method for quantifying liquid food consumption in Drosophila. A 3D-printed device connects a 96-well microplate in which flies are housed to a 1536-well microplate from which flies consume a feeding solution with a tracer dye. The solution volume decline is measured spectrophotometrically.

用于果蝇消费量化的高吞吐量微板馈线分析

Quantifying food intake in Drosophila is used to study the genetic and physiological underpinnings of consumption-associated traits, their environmental ...

行为学

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

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 Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

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

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

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 ...

A Rapid Food-Preference Assay in Drosophila

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate their food environment. The fruit fly, Drosophila melanogaster, is a genetically tractable model organism that is widely used to decipher the molecular, cellular, and neural underpinnings of food preference. To analyze fly food preference, a robust feeding method is needed. Described here is a two-choice feeding assay, which is rigorous, cost-saving, and fast. The assay is Petri-dish-based and involves the addition of two different foods supplemented with blue or red dye to the two halves of the dish. Then, ~70 prestarved, 2-4-day-old flies are placed in the dish and&#160;allowed to choose between blue and red foods in the dark for about 90 min. Examination of the abdomen of each fly is followed by the calculation of the preference index. In contrast to multiwell plates, each Petri dish takes only ~20 s to fill and saves time and effort. This feeding assay can be employed to quickly determine whether flies like or dislike a particular food.

A Rapid Food-Preference Assay in Drosophila

We present a protocol for a two-choice feeding assay for flies. This feeding assay is fast and easy to run and is suitable not only for small-scale laboratory research, but also for high-throughput behavioral screens in flies.

果蝇的快速食物偏好分析

To select food with nutritional value while avoiding the consumption of harmful agents, animals need a sophisticated and robust taste system to evaluate ...

In Vivo Calcium Imaging of Taste-Induced Neural Responses in Adult Drosophila

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model organism, Drosophila melanogaster. A key strength of this methodology is its ability to record taste-induced neural responses in awake animals without the need for anesthesia. This approach employs binary expression systems (e.g., Gal4-UAS) to express the calcium indicator GCaMP in specific neurons of interest. This protocol describes a procedure in which flies expressing GCaMP are mounted with the labellum securely positioned, enabling fluorescence in the brain to be recorded at millisecond resolution under a confocal microscope while a solution is applied to the labellum, stimulating all labellar taste sensilla. The examples provided focus on calcium responses in primary gustatory receptor neurons of D. melanogaster. However, this approach can be adapted to record from other neurons of interest within the brain of Drosophilids or other insect species. This imaging method enables researchers to simultaneously record collective calcium responses from groups of gustatory neurons across the labellum, complementing electrophysiological tip recordings that quantify action potentials from individual neurons. The in vivo calcium imaging technique outlined here has been instrumental in uncovering molecular and cellular mechanisms of chemosensation, identifying unique temporal response patterns in primary taste neurons, investigating mechanisms of gustatory modulation, and exploring taste processing in downstream circuits.

A calcium imaging approach enables the recording of taste-induced responses in the brains of awake flies while a solution is applied to the labellum. Primary gustatory responses in Drosophila melanogaster are used as an example, but this protocol can be adapted to study downstream neurons or other species.

体内研究成年果蝇味觉诱导神经反应的钙成像

For nearly two decades, in vivo calcium imaging has been an effective method for measuring cellular responses to taste stimuli in the fruit fly model ...

Taste Preference Assay: A Method for Measuring Feeding Behavior in Drosophila

Source: Bantel, A. P. and Tessier, C. R. <a href="https://www.jove.com/video/54403/" target="_blank">Taste Preference Assay for Adult Drosophila</a>. J. Vis. Exp. (2016).
This video describes the taste preference assay, a behavioral method used to measure attraction or avoidance towards colored solutions that taste differently by assessing the fly&#39;s abdominal coloration after ingestion of the preferred substance. The featured protocol demonstrates the procedure used to measure flies&#39; preference towards solutions of varying sucrose concentrations.

味觉偏好测定:一种测量果蝇摄食行为的方法

Source: Bantel, A. P. and Tessier, C. R. Taste Preference Assay for Adult Drosophila. J. Vis. Exp. (2016).
This video describes the taste preference ...

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

摘要

探索更多视频

此视频中的章节

从查明的神经元和取食行为的钙信号的同时记录果蝇

欲求联想嗅觉学习

单苍蝇检测方法的觅食行为的

用于果蝇消费量化的高吞吐量微板馈线分析

一个简单的方法来衡量在改建悬赏寻求行为使用

毛细管馈线分析测量的摄食

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

果蝇的快速食物偏好分析

体内研究成年果蝇味觉诱导神经反应的钙成像

味觉偏好测定:一种测量果蝇摄食行为的方法

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

摘要

探索更多视频

此视频中的章节

从查明的神经元和取食行为的钙信号的同时记录果蝇

欲求联想嗅觉学习

单苍蝇检测方法的觅食行为的

用于果蝇消费量化的高吞吐量微板馈线分析

一个简单的方法来衡量在改建悬赏寻求行为使用

毛细管馈线分析测量的摄食

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

果蝇的快速食物偏好分析

体内研究成年果蝇味觉诱导神经反应的钙成像

味觉偏好测定:一种测量果蝇摄食行为的方法

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