沙菲·萨米由内 (EY008117、EY010852)、NIDCD (DC007864、DC016278) 和 NIAID (1DP1AI124453) 提供资金支持。

1. 梯度测定设备的制造和装配设备
<ol><li>为单向梯度法制备铝化验板。<ol>
<li>用带锯和锋利的立式铣削, 将每个铝化验板 (图 1A) 从单一铝片中修剪和研磨, 其尺寸如下: 外层尺寸为 140 x 100 x 9 毫米, 内部尺寸为 130 x 90 x 8 毫米 (图 1B)。阳极处理用黑色颜料在每个化验盘内, 使幼虫更容易形象化, 防止生锈。 注: 用于单向梯度的铝化验板的制作是由一家机械厂完成的。阳极氧化是由商业供应商执行的。</li>
		<li>用13分界标记每个化验盘的上、下轮辋, 用一个永久性的标记隔开10毫米。将第一个和最后一个分界5毫米从检测板的内边缘 (图 1A, B) 放置。</li>
	</ol>
</li>
	<li>要制造双向梯度的铝检测板, 请用钣金剪刀切割出具有以下尺寸的铝板: 250 x 220 x 2 毫米阳极处理用黑色颜料测定钢板。 注: 用于双向渐变的铝检测板的制作是由一个机器车间完成的。阳极氧化是由商业供应商完成的。</li>
	<li>制造铝块。 注: 单向渐变需要两个块, 双向渐变需要三块。<ol>
<li>使用带锯 (尺寸为 50 x 255 x 14 毫米) 将铝块 (图 1C) 从单个铝片中切割 (图 1C、D)。</li>
		<li>使用垂直铣削和螺纹凹槽在一侧的开口端 (图 1D) 钻入块内的水通道 (直径7毫米)。用螺栓和螺纹密封胶带关闭槽孔, 形成 U 形水通道 (图 1C, D)。 注: 铝化验块的制作是由一个机器车间完成的。</li>
		<li>将铝块中的接头 (图 1D) 固定在一端的螺纹和螺纹密封胶带上。适合另一端与多个倒钩成硅管 (1/4 "ID x 3/8" OD x 1/16 "墙)。</li>
	</ol>
</li>
	<li>获得两个冷藏/加热循环浴缸。</li>
	<li>为梯度检测系统装配温度控制块。<ol>
<li>对于单向渐变, 将两个铝块连接到单独的水浴, 以便每个块的温度由独立的水循环系统控制 (图 2A)。使用硅胶管 (1/4 "ID x 3/8" OD x 1/16 "墙) 将水浴的出水口连接到铝块内侧的连接器 (图 2A)。另外, 使用油管将铝块的外侧接头连接到水浴的入口。</li>
		<li>对于双向渐变, 将三个块中的两个方块置于板的左右两侧, 并将它们连接到与油管相同的水浴中 (图 2B)。将第三个铝块连接到第二个水浴, 并放置在板的中心下面 (图 2B)。 注意: 只有当幼虫在一个边缘或另一方向梯度的区域中积聚时, 才需要这种仪器和化验。如果是这样, 重要的是要评估是否有一个边缘效应。为了测试这种可能性, 建立一个双向梯度, 在这种渐变中, 幼虫使用单向梯度 (例如18°c) 首选的边缘区域温度是双向渐变板块中间的温度 (图3E , F)。</li>
	</ol>
</li>
</ol>2. 幼虫同步
<ol><li>滋养苍蝇用于产卵。<ol>
<li>将酵母颗粒和蒸馏水与杵混合制成酵母膏。彻底研磨和搅拌, 直到糊状的稠度与花生酱相似。避免过份干糊, 当将苍蝇转移到新鲜的瓶子或湿糊时, 可能会剥落, 这会使苍蝇陷入陷阱。</li>
		<li>使用杵, 添加酵母膏接近标准果蝇瓶内壁, 就在食物表面之上。</li>
		<li>计数12-35 女性和多达一半的男性 (但不超过 10) 在一个 CO2垫, 并添加女性和男性每个酵母糊状瓶。</li>
		<li>为了保持苍蝇的食物与酵母膏湿润, 而苍蝇饲料, 结合这些瓶子在一个托盘, 持有多达100瓶, 20 开瓶含有蒸馏水只。把托盘放在一个透明的塑料袋和密封。</li>
		<li>在25摄氏度孵化器中孵化出48小时的托盘, 这样雌性就能得到充分的营养, 从而能够产卵大量的卵子。</li>
	</ol>
</li>
	<li>为产卵设置瓶。<ol>
<li>将营养的苍蝇转移到新的食品瓶中, 通过敲击它们来收集卵子。不要使用 CO2,这可能会导致苍蝇在下面的短时间窗口少产卵。 注: 标准果蝇食品瓶没有酵母膏用于收集卵子。</li>
		<li>允许苍蝇产卵3小时, 在25摄氏度, 以便所有的鸡蛋收集在一个相对狭窄的时间窗口。 注意: 这将使幼虫在同一发育阶段同步。</li>
		<li>把装有鸡蛋的瓶子放在装有水瓶的盘子里, 然后拧紧袋子。在12小时 light:12 h 暗循环下孵育25摄氏度。</li>
		<li>根据所需幼虫期, 在产卵 (AEL) 后的特定小时龄幼虫。 注:表 1是对 AEL 和幼虫期之间的关系的估计, 这取决于飞行存量、使用的食物、饲养温度等. AEL 与发育阶段的精确关系需要由每位调查员验证使用物理标准, 如嘴钩的形态学和气孔11。</li>
	</ol>
</li>
</ol>3. 温度梯度设置
<ol><li>准备单向渐变<ol>
<li>为了在化验过程中营造潮湿的环境, 请将两个铝块连接到两个水浴上, 用湿纸巾隔开10厘米 (图 2A)。</li>
		<li>打开两个水浴2小时之前开始的化验, 以允许足够的时间为铝块的温度平衡。 注: 建议进行模拟实验, 确定每个水浴的温度, 以达到所需的线性温度梯度。表 2列出了设置水浴的一些典型的温度对。但是, 请注意, 表面温度受周围气温的影响。油管的长度也会影响温度, 因为管子里有冷却。我们安装的油管长度是1.5 米。</li>
		<li>微波100毫升1% 琼脂糖在高功率设置在500毫升圆宽嘴瓶和倾吐25毫升/化验盘在水平台式。准备两个化验板, 可同时放置在铝块上 (图 2A)。</li>
		<li>琼脂糖凝固后 (10-20 分钟), 用标准的厨房海绵或三聚氰胺海绵轻轻擦拭每一个琼脂糖表面, 使琼脂糖表面略粗, 使水在琼脂糖凝胶上喷洒时, 会产生平滑的薄水膜,也不会形成水滴。</li>
		<li>用蒸馏水将盘子完全浸入容器中, 直到化验准备好, 以防止盘子变得干燥。</li>
	</ol></li>
	<li>准备双向渐变 (可选)<ol>
<li>为了在化验过程中营造潮湿的环境, 在湿纸巾上放置三个铝块8厘米 (图 2B)。</li>
		<li>打开两个水浴2小时之前开始的化验, 以允许足够的时间为铝块的温度平衡。 注: 建议进行模拟实验, 确定双向梯度水浴的温度。表 3列出了设置水浴的一些典型的温度对。但是, 请注意, 表面温度受周围气温的影响。油管的长度也会影响温度, 因为管子里有冷却。我们安装的油管长度是1.5 米。</li>
		<li>为了防止琼脂糖从盘子里溢出, 用贴标胶带将铝板的边缘包装成10毫米高的墙壁 (图 2B)。</li>
		<li>使用大功率设置, 微波200毫升1% 琼脂糖在500毫升圆形宽口瓶, 并在一个化验盘上倒入120毫升。</li>
		<li>琼脂糖凝固后 (约30分钟), 用标准的厨房海绵或三聚氰胺海绵轻轻擦拭每一个琼脂糖表面, 使琼脂糖表面略粗, 使水在琼脂糖凝胶上喷洒时, 产生平滑的薄水膜, 不水滴形成。</li>
		<li>用蒸馏水将化验盘完全浸入水中, 直至化验准备就绪, 以防止其干燥。</li>
	</ol></li>
	<li>设置单向渐变<ol>
<li>在梯度测定仪旁边的长凳上准备试剂和物品 (见材料表)。</li>
		<li>为了促进有效的温度传递, 在铝块和检测板之间的任何间隙, 通过喷洒水在板块之间的界面。</li>
		<li>使用手套, 从水容器中取出化验盘。如果水在琼脂糖凝胶和板材之间侵入, 导致表面突起, 则用 P1000 微去除水。</li>
		<li>将化验盘放在铝块上, 使2厘米的分界与铝块的边缘完全匹配 (图 2A、C)。</li>
		<li>将水喷洒到板材表面 (覆盖琼脂糖表面的薄薄的水膜即可), 以防止琼脂糖凝胶干燥。确保水膜是连续的, 没有水滴, 因为幼虫会被困在水滴中。</li>
		<li>用纸板箱覆盖渐变系统, 以减少水的蒸发, 并有助于稳定凝胶表面的温度。等待5-10 分钟, 让温度平衡。</li>
		<li>检查板上12点的表面温度 (图 2C)。在每个区域内进行两次测量, 以确定区域内是否存在可变性。确保两点的温度在0.2 摄氏度以内。 注意: 区域内的可变性通常发生, 因为两个点的精确距离与铝块的边缘不相同。调整铝块板的位置, 以确保从任一边缘2厘米的分界与铝块的边缘完全匹配。</li>
		<li>如果测量的温度梯度偏离了所需的梯度, 增加或减少水浴温度设置, 并重新检查的温度后, 水浴 (s) 稳定 (s)。</li>
		<li>用纸板箱覆盖渐变系统, 直到开始化验。</li>
	</ol></li>
	<li>设置双向渐变 (可选)<ol>
<li>在梯度测定仪旁边的长凳上准备试剂和物品 (见材料表)。</li>
		<li>在三铝块表面喷洒水, 通过填充表面之间的空隙, 促进从铝块到化验板的有效温度传递。</li>
		<li>小心地从水容器中取出化验板, 放在铝块上。如果水在琼脂糖凝胶和盘子之间侵入, 这可能会引起表面上的颠簸, 用 P1000 微去除水。</li>
		<li>将化验盘放在铝块上, 以便从任一边缘的第一和最后一个区域的 midlines 与两侧铝块的边缘完全匹配 (图 2B、D)。</li>
		<li>将水喷洒到板材表面 (覆盖琼脂糖表面的薄薄的水膜即可), 以防止琼脂糖凝胶干燥。确保水膜是连续的, 没有水滴, 因为幼虫会被困在水滴中。</li>
		<li>用纸板箱覆盖渐变系统, 以减少水的蒸发, 并有助于稳定凝胶表面的温度。等待5-10 分钟, 使温度平衡。</li>
		<li>检查每个10区域的中线上两点的表面温度 (图 2D)。在每个区域内进行两次测量, 以确定区域内是否存在可变性。确保两点的温度在0.2 摄氏度以内。 注意: 区域内的可变性通常发生, 因为两个点的精确距离与铝块的边缘不相同。调整板在铝块上的位置, 以确保最接近每个边缘的第一和最后一个区域的 midlines 与两侧铝块的边缘完全匹配。</li>
		<li>如果测量的温度梯度偏离了所需的梯度, 调整水浴温度设置和重新检查的温度后, 水浴 (s) 稳定 (s)。</li>
		<li>用纸板箱盖住块直到化验开始。</li>
	</ol></li>
</ol>4. 幼虫的收集和洗涤
<ol><li>分离干净的幼虫 (选项 1)<ol>
<li>添加〜40毫升18% 蔗糖溶液到50毫升试管。用 scoopula 将食物瓶中的幼虫舀出来, 转到18% 蔗糖溶液中。</li>
		<li>彻底混合, 但轻轻地使用 scoopula 将幼虫与食物碎片分开。等待30-60 秒直到幼虫漂浮到管子的顶层。将含有幼虫 (~ 10 毫升) 的顶层倒入另50毫升管, 用新鲜的18% 蔗糖溶液填充管。等待30-60 秒, 直到幼虫再次浮到顶层。 注意: 大的食物颗粒有时会与幼虫一起转移, 如果不被移除, 可能会影响幼虫 thermotaxis。如果大的食品颗粒留在顶层, 重复步骤 4.1. 1-4. 1.2, 或手动删除它们使用 scoopula。</li>
		<li>将顶层的幼虫 (~ 10 毫升) 转移到另外两个50毫升的管子上, 用蒸馏水填满两个管子, 以减少蔗糖浓度, 使幼虫迅速沉入水中。等30-60 秒, 直到幼虫沉到水底。尽快执行以下步骤, 以防止幼虫溺水。</li>
		<li>通过轻轻地倾斜管子, 尽可能地丢弃更多的水。将幼虫与剩余的水一起倒入一管, 用蒸馏水填满管子。</li>
		<li>等待30-60 秒, 直到所有幼虫下沉和去除尽可能多的水, 通过轻轻倾斜管。重复这洗涤步骤2-4 次, 以完全去除蔗糖和所有可见的食物。</li>
		<li>丢弃尽可能多的水, 并将幼虫转移到一个空的35毫米培养皿醒酒。用水喷洒管子, 用小刷子帮助转移幼虫。</li>
		<li>使用 P1000 微去除培养皿中的多余水分。留出0.5 毫升的水, 防止幼虫脱水。</li>
		<li>把盖子放在盘子上, 以防幼虫逃跑。把盖子倒过来, 以减少幼虫通过盖子和盘子之间的小缝隙逃生的能力。允许幼虫恢复10-20 分钟。</li>
	</ol>
</li>
	<li>分离干净的幼虫 (选项 2) 注意: 这种清洁幼虫的替代方法可减轻洗涤过程中幼虫窒息的可能性。要使用此方法, 请在执行上述步骤 4.1. 1-4. 1.2 之后, 继续执行以下步骤。<ol>
<li>放置一个细胞过滤器 (300 µm, 保留幼虫72小时 AEL 或更老) 在50毫升管之上。</li>
		<li>在确认蔗糖溶液表面没有成人身体或食物碎片后, 通过过滤器将含有幼虫的顶层倒入滤网筛上的所有幼虫。用蒸馏水彻底冲洗幼虫, 直到50毫升管被填满。</li>
		<li>将细胞过滤器与幼虫一起取出, 并从管中处理使用过的水。将含有幼虫的过滤器放在空管上方, 再用蒸馏水洗涤幼虫, 直到50毫升管被填满。</li>
		<li>将过滤器倒置在一个空的35毫米培养皿上, 然后从细胞过滤器顶部喷出水, 将幼虫转移到培养皿中。使用一个小的油漆刷转移剩余的幼虫。</li>
		<li>在执行步骤5之前, 继续执行上述步骤4.1.7。</li>
	</ol>
</li>
</ol>5. 化验和计算
<ol><li>取出纸板盒, 在将幼虫转移到盘子前, 立即检查凝胶表面温度。尽量减少纸板箱打开的时间, 以防破坏温度平衡。如果表面干燥, 喷洒少量的水在表面上。</li>
	<li>在每个板块中心附近分布 150 50 幼虫 (介于3和4之间的6区), 用于单向梯度 (释放区;图 2C)。 注意: 对于双向梯度, 在每一半的中间区域分布200-400 幼虫 (图 2D)。</li>
	<li>在每个化验盘上放置一个微板块盖子, 以防止幼虫爬行。用纸板盒盖住安装板, 防止光线暴露, 这可能会影响琼脂糖凝胶上的幼虫选择。 注: 对于双向渐变, 不应用盖子盖板, 因为板块较大, 幼虫的首选温度在中间区域。因此, 很少有幼虫在边缘积聚, 有机会爬出来。</li>
	<li>允许化验以10-30 分钟为单向梯度, 并为双向梯度15-35 分钟, 这取决于幼虫的年龄 (表 4)。</li>
	<li>取下纸板箱和微板块盖。使用数码相机拍摄上面的车牌。取两张化验盘的照片, 以便调查员可以选择比较好的对比度和亮度进行分析。</li>
	<li>取出所有的幼虫从化验板和任何地方的盘子外的愿望。</li>
	<li>用蒸馏水彻底清洁化验板、管子和细胞过滤器。重复使用当天准备的化验盘, 除非琼脂糖的表面受损。</li>
	<li>计算每个区域幼虫的百分比分布。<ol>
<li>使用允许向图像添加标记的任何软件打开检测结果的照片图像。为了表明化验区, 每2厘米绘制垂直线根据分界在化验板上。</li>
		<li>计算每个区域中幼虫的数量并记录数字。不要在0.5 厘米的区域内从任何墙壁上计数幼虫。凝胶在壁附近较厚, 在这些区域表面温度不是线性的。</li>
		<li>对于单向渐变, 计算每个区域中的百分比分布, 如下所示: (在给定的2厘米区域中的幼虫数量)/(6 个区域中的幼虫总数) x 100。</li>
		<li>对于双向渐变, 计算每个区域中的百分比分布, 如下所示: (在给定的2厘米区域的幼虫数量)/(在梯度的每边5个区域幼虫的总数量) x 100。</li>
	</ol>
</li>
</ol>

<ol>
	<li>Fowler, M. A., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drosophila+TRP+channels+and+animal+behavior.">Drosophila TRP channels and animal behavior.</a> Life Sci. 92, 394-403 (2013).</li><li>Palkar, R., Lippoldt, E. K., McKemy, D. 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+molecular+and+cellular+basis+of+thermosensation+in+mammals.">The molecular and cellular basis of thermosensation in mammals.</a> Curr Opin Neurobiol. 34, 14-19 (2015).</li><li>Vriens, J., Nilius, B., Voets, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peripheral+thermosensation+in+mammals.">Peripheral thermosensation in mammals.</a> Nat Rev Neurosci. 15 (9), 573-589 (2014).</li><li>Rosenzweig, 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+Drosophila+ortholog+of+vertebrate+TRPA1+regulates+thermotaxis.">The Drosophila ortholog of vertebrate TRPA1 regulates thermotaxis.</a> Genes Dev. 19, 419-424 (2005).</li><li>Kwon, Y., Shim, H. S., Wang, X., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+thermotactic+behavior+via+coupling+of+a+TRP+channel+to+a+phospholipase+C+signaling+cascade.">Control of thermotactic behavior via coupling of a TRP channel to a phospholipase C signaling cascade.</a> Nat Neurosci. 11, 871-873 (2008).</li><li>Kwon, Y., Shen, W. L., Shim, H. S., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fine+thermotactic+discrimination+between+the+optimal+and+slightly+cooler+temperatures+via+a+TRPV+channel+in+chordotonal+neurons.">Fine thermotactic discrimination between the optimal and slightly cooler temperatures via a TRPV channel in chordotonal neurons.</a> J Neurosci. 30 (31), 10465-10471 (2010).</li><li>Shen, W. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+rhodopsin+in+temperature+discrimination+in+Drosophila.">Function of rhodopsin in temperature discrimination in Drosophila.</a> Science. 331 (6022), 1333-1336 (2011).</li><li>Sokabe, T., Chen, H. S., Luo, J., Montell, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+switch+in+thermal+preference+in+Drosophila+larvae+depends+on+multiple+rhodopsins.">A switch in thermal preference in Drosophila larvae depends on multiple rhodopsins.</a> Cell Rep. 17, 336-344 (2016).</li><li>Ni, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Ionotropic+Receptors+IR21a+and+IR25a+mediate+cool+sensing+in+Drosophila.">The Ionotropic Receptors IR21a and IR25a mediate cool sensing in Drosophila.</a> Elife. 5, 13254 (2016).</li><li>Luo, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Navigational+decision+making+in+Drosophila+thermotaxis.">Navigational decision making in Drosophila thermotaxis.</a> J Neurosci. 30 (12), 4261-4272 (2010).</li><li>Ashburner, M., Golic, K. G., Hawley, R. 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> Drosophila: a laboratory handbook. , (2005).</li></ol>

作者没有什么可透露的。

为确保本议定书的成功, 必须采取步骤, 获得足够数量的幼虫进行实验。这些包括预先喂养的苍蝇在酵母膏含瓶 2-3 d, 以改善产卵。瓶子需要放在装有水瓶的托盘中, 并封闭在一个透明的塑料袋中, 这样可以保持食物的水分, 并促进幼虫有效喂养, 同时允许接触正常的光-暗循环。然而, 酵母糊不应该是如此柔软, 苍蝇成为陷阱。每瓶雌性的数量取决于基因型。在w1118的情况下, 通常有足够的12雌性和6雄蛋产卵3小时。两个小瓶通常提供足够的幼虫 (100-200 幼虫) 放置在一个盘子上的单向检测。如果苍蝇的数量比野生型或孵化的幼虫比例减少, 增加额外的雌性 (多达 30-35/瓶) 和雄性。
在执行这些化验时, 有许多意想不到的变异原因。发育阶段影响幼虫的温度偏好。因此, 必须仔细使用已定义阶段的幼虫。要做到这一点, 收集幼虫在一个狭窄的时间框架 (如3小时)。由于养殖条件 (温度、湿度、光-暗循环和食物类型) 和某些突变影响了发育速度, 不严格依赖卵产卵后的时间分析可比较老化的幼虫。同样重要的是检查身体性状, 如口钩的大小和气孔7 , 以确定不同基因型的幼虫是否处于同一发育阶段。1st, 2 和 3rd幼虫开发 (表 1) 的时间是基于玉米膳食为基础的食物和孵化在25摄氏度之下在 12 h light:12 h 黑暗的周期.以糖蜜为基础的食物幼虫生长缓慢。适当的水化和食物新鲜度也会影响幼虫的生长。食物中的水量应使食物不太干燥, 不能从瓶子中剥离, 也不能由于过量的水而太松。理想情况下, 靠近食物表面的 ~ 5 毫米层是由生长的幼虫调节的, 当瓶子倾斜或被挖掘时, 它就容易移动。这种情况可以使用新鲜的食物与50-100 幼虫。
在开始试验前, 必须建立稳定的温度梯度。因此, 在开始化验之前, 打开水浴1-2 小时, 因为水浴产生热量, 并可能改变室温, 这反过来又有可能影响梯度。1-2 小时后, 大多数空调房间的环境温度 equilibrates。但是, 必须在每个环境中确定这一点。此外, 当产生一个具有较大温度范围 (> 3.0 °c/厘米) 的渐变时, 很难获得稳定的梯度。一个微小的运动, 检测板更显著地改变温度梯度时, 范围是大 (例如> 3.0 °c/厘米)。我们发现, 1-2 摄氏度/厘米梯度产生最稳定的梯度。
有几个额外的考虑, 需要控制, 以限制变异的 thermotaxis 化验。洗涤幼虫是至关重要的, 因为存在的食物颗粒或蔗糖对幼虫可能影响化验。洗涤步骤必须彻底, 但很快, 因为限制他们的氧气供应过量, 而他们被淹没在水中可能会影响他们的健康。因此, 我们建议使用细胞过滤器 (选项 2) 来清洁幼虫。300µm 孔径的滤网在 early-3rd龄期 (72 小时 AEL) 或较旧的幼虫中工作良好。此外, 重要的是, 在同一时间进行实验, 如从授时时间 (ZT) ZT4 到 ZT8 (ZT=0 是当灯打开), 以限制变异性由于影响的昼夜节律的温度选择。琼脂糖板上的水分水平也会导致结果的变异性。虽然盘子的表面需要湿润, 水滴可以诱捕幼虫, 因此需要避免。
在测试中限制可变性将使得在不进行大量独立实验的情况下获得可靠的结果成为可能。通常, 3-8 独立实验足以获得可靠的结果。正确的制表结果对于 thermotaxis 化验的成功也是至关重要的。不要计数在区域分布的幼虫在 0.5 cm 之内从铝墙壁, 因为温度不是线性的在这些区域。一些幼虫将被定位在两个区域之间的边界, 在试验结束的时候。如果超过50% 的身体长度驻留在一个区域, 然后将幼虫列入该区域的制表。如果幼虫在两个区域的每一个精确地是 50%, 然后计数动物作为0.5 幼虫在每个区域。
虽然梯度测定允许幼虫辨别温度的不同, 但对较低温度和上温的限制是可以有效测试的。测试温度 < 10 摄氏度是不可行的, 因为在较低温度下幼虫的流动性大大降低。虽然梯度与上部温度达到42°c 可以建立, 几乎没有控制幼虫停留在任何区域 > 28 °c。因此, 连续梯度测定法不能用于区分不同区域 > 28 摄氏度之间的温度偏好。然而, 梯度测定在这些高温下可能被用来表征突变体, 如果他们有高度转移的温暖温度偏好。
如果突变体有运动缺陷, 可能有必要进行额外的实验, 以确保表观温度偏爱不准确, 由于移动损伤。为了弥补这个潜在的问题, 可能需要建立更长的化验时间, 让动物有更多的时间移动到他们想要的区域。双向梯度法也可以用来测试动物是否选择相同的首选区域, 不管他们最初放置在板块。
总之, 这里描述的梯度测定比其他 thermotaxis 测定有优势。虽然双向选择分析是有用的, 因为它们可以产生健壮的结果, 特别是当两个区域之间的温度差异较大时, 它们并不能有效地揭示一个实验中幼虫首选的理想温度。为此, 需要执行多种双向选择组合, 以确定最喜欢的温度5、6、7。相比之下, 梯度分析为动物提供了一个在一个连续的温度景观环境中选择其首选温度区的机会。因此, 不需要设计一个大组合的双向选择, 以确定首选的热环境。使用圆形培养皿研究 thermotaxis, 以评估温度对幼虫10运动动力学的影响。但是, 在区分不同温度之间的偏好方面, 这一点用处不大, 因为每个区域的大小不同。最后, 由于实验的简便性, 以及它在鉴别小温度偏好的方法中的效用, 它可以用来筛查许多潜在参与幼虫温度偏好的候选基因, 这可能否则被忽略使用其他化验

Thermotaxis 是由移动的动物使用的选择环境以最有利情况1,2,3。如果气候过热或寒冷, 这种行为对生存至关重要。此外, 许多动物对舒适范围内的温度差异很小, 并以理想的温度寻找周围环境。这是特别重要的 poikilothermic 生物, 如果蝇, 平衡他们的体温与环境。监测幼虫 thermotaxis 的检测在识别和澄清诸如果蝇瞬态受体电位 (激进党) 通道4、5、6等分子传感器的作用方面起到了一定的帮助,rhodopsins7,8和离子型受体受体 (IRs)9, 赋予这些动物与温度敏感性在不同的温度范围。
双向选择测试提供了一种研究幼虫6,7的热偏好的方法。该化验需要建立两个不同的温度区, 并允许动物选择一侧的另一边。双向选择测试的结果可以是健壮的, 特别是当两个选项之间的温度差异较大时。此外, 由于每种化验都只涉及对两组进行制表, 所以数据可以表示为简单的偏好指数。双向选择方法的简便和简单性也适合于基因筛。然而, 一个主要的限制是需要许多实验来建立野生型或变种动物的首选温度。
梯度分析为在单一化验8中建立首选温度提供了机会。此外, 与双向选择试验不同, 它允许对一组动物的分布进行评估, 同时面对持续的温度范围。一种梯度测定法使用培养皿和单动物, 非常适合于描述个别动物的详细行为10。然而, 由于培养皿是圆的, 温度区的大小变化, 并逐渐变小取决于距离从中心。因此, 这种设置并不适合监测动物种群的温度选择。
一种连续的热梯度装置, 它非常适合于评估幼虫群的温度偏好, 这里有一个长方形的竞技场, 这里描述。该装置结构简单, 装配方便。此外, 梯度是线性的, 是灵活的, 因为它可以用来评估 thermotaxis 在大温度范围从10°c 到42°c。该检测是快速和直接的执行和产生可重现的数据。除了报告幼虫的偏爱温度外, 它还在单个实验中揭示了动物种群在整个线性范围内的偏好。由于这些优势, 这是一个很好的选择, 以确定基因所需的 thermotaxis。

<table><tbody><tr><td>&lt;strong&gt;Gradient assay apparatus&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>PolyScience 9106, Refrigerated/Heated 6L Circulating Bath</td><td>Thomas Scientific</td><td>9106</td><td>This model is discontinued. Updated replacement models include: 1186R00 and 1197U04 for 120 V, 60 Hz, or 1184L08 and 1197U04 for 240 V, 50 Hz.</td></tr><tr><td>Aluminum assay plate (for single directional gradient)</td><td></td><td></td><td>Outer size: 14 x 10.1 x 0.9 cm, inner size: 12.9 x 8.7 x 0.8 cm, black anodized.</td></tr><tr><td>Aluminum plate (for bidirectional gradient)</td><td></td><td></td><td>25 x 22 x 0.2 cm, black anodized.</td></tr><tr><td>Aluminum block</td><td></td><td></td><td>Outer size: 25.5 x 5 x 1.4 cm, parameters of inner channels are shown in Figure 1D.</td></tr><tr><td>Connector for aluminum blocks and tubing</td><td>McMaster-Carr</td><td>91355K82</td><td></td></tr><tr><td>Tygon Sanitary Silicone Tubing</td><td>Tygon</td><td>57296</td><td>1/4&quot; ID x 3/8&quot; OD x 1/16&quot; wall</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Items and reagents for assay&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Pestle</td><td>USA Scientific</td><td>17361</td><td>Pestle for 1.5 mL microcentrifuge tubes</td></tr><tr><td>Thermometer</td><td>Fluke</td><td>51II</td><td></td></tr><tr><td>Thermocouple</td><td>Fluke</td><td>K type</td><td></td></tr><tr><td>Universal microplate lid</td><td>Corning</td><td>6980A77</td><td></td></tr><tr><td>35 mm dish</td><td>Corning</td><td>9380D40</td><td></td></tr><tr><td>Labeling tape (for bidirectional gradient)</td><td>Fisher Scientific</td><td>15-951</td><td>Fisherbrand labeling tape 2 in x 14 yds</td></tr><tr><td>Agarose</td><td>Invitrogen</td><td>16500500</td><td>Prepare 1% solution</td></tr><tr><td>Sucrose</td><td>Sigma</td><td>S0389-5KG</td><td>Prepare 18% solution right before starting assay</td></tr><tr><td>Paint brush</td><td>Fisher Scientific</td><td>11860</td><td></td></tr><tr><td>50 mL centrifuge tubes</td><td>Denville</td><td>C1062-P</td><td></td></tr><tr><td>Scoopula</td><td>Fisher Scientific</td><td>14-357Q</td><td></td></tr><tr><td>500 mL round wide-mouth bottle</td><td>Pyrex</td><td>1395-500</td><td></td></tr><tr><td>Cell strainer (300 mm pore)</td><td>PluriSelect</td><td>43-50300</td><td>Optional item for larvae washing</td></tr><tr><td>Cardboard box (vial tray)</td><td>Genesee Scientific</td><td>FS32-124</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;&lt;em&gt;Drosophila&lt;/em&gt;&lt;/strong&gt;&lt;strong&gt; food&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Distilled water</td><td></td><td></td><td>22,400 mL</td></tr><tr><td>Cornmeal, yellow (extra fine mesh,flocked) 20 kg</td><td>LabScientific Inc.</td><td>NC0535320</td><td>1,609 g</td></tr><tr><td>Brewers yeast 100 lbs</td><td>MP Biomedicals</td><td>ICN90331280</td><td>379 g</td></tr><tr><td>NutriSoy&amp;reg; Soy Flour (10 kg/unit)</td><td>Genesee Scientific</td><td>62-115</td><td>221 g</td></tr><tr><td>&lt;em&gt;Drosophila&lt;/em&gt; Agar, Type II (5 kg)</td><td>Genesee Scientific</td><td>66-103</td><td>190 g</td></tr><tr><td>Karo light corn syrup</td><td>Karo</td><td></td><td>1,700 mL</td></tr><tr><td>Methyl 4-hydroxybenzoate (suspend in 200 proof ethanol)</td><td>Sigma Aldrich</td><td>H5501-5KG</td><td>72 g/240 mL</td></tr><tr><td>Propionic acid puriss. p.a.,&gt;99.5% (GC)</td><td>Sigma Aldrich</td><td>81910-1 L</td><td>108 mL</td></tr><tr><td>Phosphoric acid&amp;nbsp;ACS reagent, &amp;ge;85 wt. % in H&lt;sub&gt;2&lt;/sub&gt;O</td><td>Sigma Aldrich</td><td>438081-500 mL</td><td>8.5 mL</td></tr></tbody></table>

a temperature gradient assay to determine thermal preferences of drosophila larvae

包括果蝇在内的许多动物都能够辨别环境温度的细微差异, 这使他们能够找到他们喜欢的热景观。为了定义幼虫在定义的线性范围内的温度偏好, 我们开发了一种使用温度梯度的检测方法。为了建立单向梯度, 两个铝块连接到独立的水浴, 每一个控制温度的个别区块。两个块设置渐变的下限和上限。温度梯度是通过将琼脂糖涂层的铝板放在两个水控块上, 使板块跨越它们之间的距离而建立的。在水块顶部设置的铝板的两端定义了最小和最大温度, 两块之间的区域形成线性温度梯度。梯度法可应用于不同年龄的幼虫, 可用于识别表现表型的突变体, 如那些有影响基因的突变的 opsins, 这是温度歧视所必需的。

为了建立一个18°c-28 °c 的单向梯度, 我们把两个水浴的温度设置为16.8 摄氏度和31摄氏度。我们通过测量在所有6个区域的上下部分的26个位置的温度, 在区域之间的边界线, 以及琼脂糖凝胶表面的极端末端 (图 2C, 2E), 获得温度在13点。沿梯度的温度分布是近线性的 (Y = 0.9672 * X + 16.19, R2 = 0.9961) (图 2E)。
我们测定了不同年龄的控制 (w1118) 幼虫的温度偏好。1st (24 1.5 h AEL), 2 和 (48 @ 1.5 h AEL) 和 early-3rd幼虫 (72 @ 1.5 h) 在24°c 区域显示了峰值 (图 3A, B)。在 3路龄幼虫发育过程中, 温度偏好发生变化。在18°c 区 (图 3A, B) 累积的 mid-3rd龄 (96 1.5 h AEL) 的最大百分比, 随着年龄的增长, 这种偏倚增加。在 late-3rd幼虫 (前攀登; 120 1.5 h AEL), 50% 聚在18°c 区域, 并且这个温度的选择是 ~ 4 倍高于毗邻20°c 区域 (18 °c 区域, 50.2%; 20 °c 区域, 15.1%;图 3A, B)。在w1118 的18°c 区积累的倾向不是由于边缘效应, 因为 late-3rd龄幼虫仍然积累在18°c 区域, 使用双向温度梯度 (图 3E, F).
我们还测试了 late-3rd龄幼虫 (120 1.5 h AEL), 基因突变, 在舒适范围内的温度差异判别。这些包括trpA1, 在18°c-24 °c 范围5,7,8要求正常温度选择。幼虫以零变异在trpA1 (trpA11) 均匀地分布在整个18°c-28 °c 梯度 (图 3C)。幼虫只缺 A 和 B 亚型 (trpA1-ABG4), 或者 C 和 D 亚型 (trpA1-CDG4) 也显示严重的损伤 (图 3C)。苍蝇编码磷脂酶 Cβ (PLC21C 和 NORPA) 的两个亚型, 和影响NORPA (NORPAP24) 但不PLC21C (PLC21CP319) 的突变也扰乱18°c 范围内的积累 (图 3D)。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57963/57963fig1.jpg"> 图1。用于执行单向温度梯度测定的仪器。(A)用于测定幼虫 thermotaxis 行为的铝测试板。在顶部和底部的13条黑线标定12个区域 (每个10毫米)。板的底部是电化铝与黑色油漆, 使它更容易形象化的幼虫。(B)铝板的尺寸 (以 mm 表示)。铝板的外层尺寸为 140 x 100 x 9 毫米。铝板的内部尺寸为 130 x 90 x 8 毫米。分界用10毫米隔开。第一和最后分界是5毫米从板的内部区域的边缘。(C)用于控制渐变温度的铝块的顶部和侧面视图。该块有两个连接器用于连接到一个水浴的硅管。(D)铝块的尺寸。铝块的外层尺寸为 255 x 50 x 14 毫米。内水径为7毫米。两个30毫米连接器在左边连接与延伸到水浴的硅管材。<a href="https://www.jove.com/files/ftp_upload/57963/57963fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57963/57963fig2.jpg"> 图2。单和双向梯度检测装置。(A)在两个铝块上有两个铝板的单向渐变设置。铝块的温度由两个水浴的循环水控制。(B)为双向渐变安排三个铝块、水浴和铝板 (250 x 220 毫米)。左和右块连接到相同的水浴, 中间块连接到其他水浴。铝化验板用胶带包裹, 形成10毫米壁, 含有1% 琼脂糖。(C)检查温度的位置 (用点表示), 并在盘子上释放幼虫。在开始实验之前, 检查每个区域内两点的温度, 以确认所需的线性温度梯度是建立的。幼虫在中线附近的指定区域内释放。幼虫计数在每个 2 cm 区域之内。(D)在双向梯度上检查温度 (由点表示) 和幼虫释放区的位置。在双向梯度的每一半的中线上释放出相同数量的幼虫。幼虫的数量在每 10 (2 cm) 区域计数。一个典型的温度集 (18 °c-26 °c) 在琼脂糖表面被表明。(E)沿边界线和每个区域的 midlines 的温度测量的单方向梯度样品。数据代表平均温度 n=8 化验 (150 50 幼虫/化验)。这个数字的部分是从 Sokabe等复制的。8轻微的修改。<a href="https://www.jove.com/files/ftp_upload/57963/57963fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57963/57963fig3.jpg"> 图3。使用单向和双向梯度测定的代表性结果.(A、B)单方向梯度上6区幼虫的平均百分率。数据包括 3rd龄幼虫在指定的小时后产卵 (AEL)。n=6-7。表示±SEM 中的误差线. (C) 攀登前 late-3rd幼虫控制 (w1118) 和trpA1突变体在单向梯度上的热分布。n=3-4。误差线表示±SEM. (D) late-3路龄幼虫控制 (w1118) 和 PLCβ突变体在单向梯度上的热分布。n=4-6。误差线表示±SEM. (E) 在双向梯度上, 有代表性地分布了攀登前、late-3rd龄幼虫 (w1118)。左和右检测区域由虚线指示, 并由中心 (阴影区域) 中的无计数区域分隔。(F) 沿热梯度的每个区域的攀登前 late-3rd龄幼虫 (w1118) 的百分比。幼虫被放置在左边和正确的释放区。检测区由中心内的3厘米无计数区隔开, 并独立计算分布。误差线表示中小企业。n=3 化验 (200-400 幼虫/化验)。这个数字的部分是从 Sokabe等复制的。8轻微的修改。<a href="https://www.jove.com/files/ftp_upload/57963/57963fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>小时 AEL</td>
			<td colspan="2">幼虫阶段</td>
		</tr>
<tr>
<td>24</td>
			<td colspan="2">1st龄</td>
		</tr>
<tr>
<td>48</td>
			<td colspan="2">2和龄</td>
		</tr>
<tr>
<td>72</td>
			<td colspan="2">Early-3路龄</td>
		</tr>
<tr>
<td>96</td>
			<td colspan="2">Mid-3路龄</td>
		</tr>
<tr>
<td>120</td>
			<td colspan="2">Late-3rd龄, 就在攀登阶段前</td>
		</tr>
</tbody></table>表1。产卵后小时 (AEL) 与幼虫期的关系。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>琼脂糖板 (坡) 温度梯度</td>
			<td>水浴温度</td>
			<td>铝块温度</td>
		</tr>
<tr>
<td>10.0-25.0°C (1.5°C/厘米)</td>
			<td>〜 6.5-7°C 28.5°C</td>
			<td>~ 8.5°C 26.8°C</td>
		</tr>
<tr>
<td>18.0-28.0°C (1°C/厘米)</td>
			<td>~ 16.8°C 31.0°C</td>
			<td>~ 17.8°C 29.7°C</td>
		</tr>
<tr>
<td>14.0-34.0°C (%/厘米)</td>
			<td>~ 10.0°C 40.0°C</td>
			<td>~ 11.8°C 36.8°C</td>
		</tr>
<tr>
<td>12.5-42.0°C (2.95°C/厘米)</td>
			<td>~ 7.0°C 55.0°C</td>
			<td>~ 9.4°C 49.4°C</td>
		</tr>
</tbody></table>表2。典型的温度梯度和水浴和铝块的对应的温度为单向梯度。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>琼脂糖板的温度梯度</td>
			<td>水浴温度</td>
			<td>铝块温度</td>
		</tr>
<tr>
<td>22-10-22°C (1.5°C/厘米)</td>
			<td>~ 5.0°C 25.0°C</td>
			<td>~ 7.5°C 24.0°C</td>
		</tr>
<tr>
<td>26-18-26°C (1°C/厘米)</td>
			<td>~ 15.8°C 30.6°C</td>
			<td>~ 16.9°C 28.4°C</td>
		</tr>
<tr>
<td>30-14-30°C (或厘米)</td>
			<td>~ 8.5°C 36.4°C</td>
			<td>~ 10.9°C 32.8°C</td>
		</tr>
<tr>
<td>36-12.5-36°C (2.95°C/厘米)</td>
			<td>~ 5.0°C 47.2°C</td>
			<td>~ 7.9°C 40.9°C</td>
		</tr>
</tbody></table>表3。典型的温度梯度和水浴和铝块的对应的温度为双向梯度。
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody>
<tr>
<td>幼虫年龄 (AEL)</td>
			<td>检测时间 (单方向)</td>
			<td>检测时间 (双向)</td>
		</tr>
<tr>
<td>24小时</td>
			<td>30分钟</td>
			<td>35分钟</td>
		</tr>
<tr>
<td>48小时</td>
			<td>22分钟</td>
			<td>27分钟</td>
		</tr>
<tr>
<td>72小时</td>
			<td>16分钟</td>
			<td>21分钟</td>
		</tr>
<tr>
<td>96小时</td>
			<td>13分钟</td>
			<td>18分钟</td>
		</tr>
<tr>
<td>120小时</td>
			<td>10分钟</td>
			<td>15分钟</td>
		</tr>
</tbody></table>表4。不同幼虫年龄 (AEL) 和相应的化验时间。

Watch this Scientific Journal Video about A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae at JoVE.com

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

在这里, 我们提出了一个协议, 以确定的首选环境温度的果蝇幼虫使用连续的热梯度。

温度梯度法测定果蝇幼虫的热偏好

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

a-temperature-gradient-assay-to-determine-thermal-preferences

Nanyang Technological University

Research

JoVE Journal

Neuroscience

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

7.5K 次观看.  University of California, Santa Barbara. 在这里, 我们提出了一个协议, 以确定的首选环境温度的果蝇幼虫使用连续的热梯度。

视频: A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

局部和全局的方法评估热痛觉<em&gt;果蝇</em&gt;幼虫

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

科研

神经科学

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

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

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

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

一种自动测定果蝇在空间和时间温度变化中的性能的方法

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

行为学

<meta charset="utf8"></head><body>使用 DAM2 系统对 果蝇 进行高通量耐热性测量

Heat Tolerance Assays Using the Drosophila Activity Monitor System: A Guide to an Executable Application for Data Analysis

The study of heat tolerance in&#8239;Drosophila melanogaster has been of particular interest to researchers for decades, with a common approach to assessing heat tolerance being to monitor the time to knockdown (TKD) after exposure to an elevated temperature. Classically, flies are housed in individual vials and placed inside a heated water bath. TKD is then monitored manually by researchers. While very well-established, there remain problems of subjectivity and consistent application of a tangible definition of cessation of all movement, including muscular spasms, when implementing these manual assays. We have developed a high-throughput method for automating heat tolerance assays using the TriKinetics Drosophila Activity Monitors (DAM2). To accompany the DAM2 system, we have written a program and created an easy-use executable to automatically read the last time of movement from the activity data generated. This script then writes to a .csv file the time to heat paralysis (TKD) for each fly. Our data show that this automated DAM2 method is consistent and reliable. Meanwhile, activity profiles created from the activity count data are of interest. These activity profiles can be compiled and have the potential to expand heat tolerance assays to include the relatively unstudied behavioral components of heat tolerance. This protocol will describe in detail how to use the DAM2 system and the HoTDAM! software to estimate heat tolerance in D. melanogaster.

<meta charset="utf8"></head><body>使用 果蝇 活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

This report describes a method for measuring adult Drosophila melanogaster time to knockdown using a Drosophila Activity Monitor (DAM2) in response to an air conduction heat stressor within an incubator chamber. The DAM2 measures activity by recording individual fly movements as they cross an infrared beam. Data analysis is facilitated by a novel executable file created by the authors.

使用 果蝇 活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

The study of heat tolerance in&#8239;Drosophila melanogaster has been of particular interest to researchers for decades, with a common approach to ...

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 ...

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a novel Drosophila circadian output, called the temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night 3. Surprisingly, the TPR and locomotor activity are controlled through distinct circadian neurons3. Drosophila locomotor activity is a well&#160;known circadian behavioral output and has provided strong contributions to the discovery of many conserved mammalian circadian clock genes and mechanisms4. Therefore, understanding TPR will lead to the identification of hitherto unknown molecular and cellular circadian mechanisms. Here, we describe how to perform and analyze the TPR assay. This technique not only allows for dissecting the molecular and neural mechanisms of TPR, but also provides new insights into the fundamental mechanisms of the brain functions that integrate different environmental signals and regulate animal behaviors. Furthermore, our recently published data suggest that the fly TPR shares features with the mammalian BTR3. Drosophila are ectotherms, in which the body temperature is typically behaviorally regulated. Therefore, TPR is a strategy used to generate a rhythmic body temperature in these flies5-8. We believe that further exploration of Drosophila TPR will facilitate the characterization of the mechanisms underlying body temperature control in animals.

Design and Analysis of Temperature Preference Behavior and its Circadian Rhythm in Drosophila

We recently identified a novel Drosophila circadian output, temperature preference rhythm (TPR), in which the preferred temperature in flies rises during the day and falls during the night. TPR is regulated independently from another circadian output, locomotor activity. Here&#160;we describe the design and analysis of TPR in Drosophila.

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

The circadian clock regulates many aspects of life, including sleep, locomotor activity, and body temperature (BTR) rhythms1,2. We recently identified a ...

生物学

Measurement of Larval Activity in the Drosophila Activity Monitor

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This protocol repurposes an instrument often used to measure adult activity, the TriKinetics Drosophila activity monitor (MB5 Multi-Beam Activity Monitor) to study larval activity. The instrument can monitor the movements of animals in 16 individual 8 cm glass assay tubes, using 17 infrared detection beams per tube. Logging software automatically saves data to a computer, recording parameters such as number of moves, times sensors were triggered, and animals&#8217; positions within the tubes. The data can then be analyzed to represent overall locomotion and/or position preference as well as other measurements. All data are easily accessible and compatible with basic graphing and data manipulation software. This protocol will discuss how to use the apparatus, how to operate the software and how to run a larval activity assay from start to finish.

Measurement of Larval Activity in the Drosophila Activity Monitor

This report describes a method for measuring Drosophila larval activity using the TriKinetics Drosophila Activity Monitor. The device employs infrared beams to detect movements of up to 16 individual animals. Data can be analyzed to represent motion parameters including rates and the positions of the animals within the assay chambers.

幼虫活动的测量<em&gt;果蝇</em&gt;活动监视器

Drosophila larvae are used in many behavioral studies, yet a simple device for measuring basic parameters of larval activity has not been available. This ...

High-Throughput Assays of Critical Thermal Limits in Insects

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are essential for predicting responses to climate change. This work describes two high-throughput protocols for measuring insect thermal limits: one for assessing critical thermal minima (CTmin), and the other for assessing heat knock down time (KDT) in response to a static heat stressor. In the CTmin assay, individuals are placed in an acrylic-jacketed column, subjected to a decreasing temperature ramp, and counted as they fall from their perches using an infrared sensor. In the heat KDT assay, individuals are contained in a 96 well plate, placed in an incubator set to a stressful, hot temperature, and video recorded to determine the time at which they can no longer remain upright and move. These protocols offer advantages over commonly used techniques. Both assays are low cost and can be completed relatively quickly (~2 h). The CTmin assay reduces experimenter error and can measure a large number of individuals at once. The heat KDT protocol generates a video record of each assay and thus removes experimenter bias and the need to continuously monitor individuals in real time.

Thermal limits can predict the environments organisms tolerate, which is valuable information in the face of rapid climate change. Described here are high-throughput protocols to assess critical thermal minima and heat knockdown time in insects. Both protocols maximize the throughput and minimize the cost of the assays.

昆虫关键热极限的高通量测定

Upper and lower thermal limits of plants and animals are important predictors of their performance, survival, and geographic distributions, and are ...

Determining Temperature Preference of Mosquitoes and Other Ectotherms

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on their fitness, as well as other characteristics. Consequently, many such ectotherms seek out their optimal temperature range. Although temperature preferences of mosquitoes and other insects have been well studied, the traditional experimental setup is performed using a temperature gradient on an aluminum surface in a highly enclosed space. In some cases, this equipment restricts many natural behaviors, such as flying, which may be important in preference selection.
The objective of this study is to observe insect preference for air temperature by using a two-chamber apparatus with sufficient room for flight. The two chambers consist of independent temperature-controlled incubators, each with a large aperture. The incubators are connected by these apertures using a short acrylic bridge. Inside the incubators are two netted cages, linked via the apertures and bridge, allowing the insects to freely fly between the different conditions. The acrylic bridge also acts as a temperature gradient between the two incubators.
Due to the spacious area in the cage and easy construction, this method can be used to study any small ectotherm and/or any manipulation which may alter temperature preference including sensory organ manipulation, diet, gut flora, and endosymbiont presence at biosafety levels 1 or 2 (BSL 1 or 2). Additionally, the apparatus can be used for the study of pathogen infection using further containment (e.g., inside of a biosafety cabinet) at BSL 3.

Insects have an optimal environmental temperature range which they seek to remain within, and many external and internal factors can alter this preference. Here, we describe a cost-effective and simple method to study temperature choice, which allows insects to freely exhibit their natural behaviors.

确定蚊子和其他变温动物的温度偏好

Most insects and other ectotherms have a relatively narrow optimal temperature window, and deviation from their optima can have significant effects on ...

温度梯度法测定果蝇幼虫的热偏好

摘要

探索更多视频

此视频中的章节

局部和全局的方法评估热痛觉

一种自动测定果蝇在空间和时间温度变化中的性能的方法

使用果蝇活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

使用果蝇活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

使用果蝇活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

新的分析冷痛觉在

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

幼虫活动的测量

昆虫关键热极限的高通量测定

确定蚊子和其他变温动物的温度偏好

温度梯度法测定果蝇幼虫的热偏好

摘要

探索更多视频

此视频中的章节

局部和全局的方法评估热痛觉

一种自动测定果蝇在空间和时间温度变化中的性能的方法

使用 果蝇 活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

使用 果蝇 活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

使用 果蝇 活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

新的分析冷痛觉在

温度偏好行为及其在德罗索菲拉的昼夜节律的设计与分析

幼虫活动的测量

昆虫关键热极限的高通量测定

确定蚊子和其他变温动物的温度偏好

使用果蝇活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

使用果蝇活动监测系统进行耐热性测定:数据分析的可执行应用程序指南

使用果蝇活动监测系统进行耐热性测定:数据分析的可执行应用程序指南