我们要感谢 Xiaolan Ye，因为该协议是根据她最初所做的观察而制定的。我们感谢 Petrascheck 实验室的所有过去和现在的成员，感谢他们在开发和优化该方案方面提供的帮助。这项工作由 R21 AG080376（至 MP）资助。一些菌株由 CGC 提供，该 CGC 由 NIH 研究基础设施计划办公室 （P40 OD010440） 资助。

<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/66422/66422fig01.jpg"> 图 1：细菌清除测定示意图。方案主要部分的图形概述（从上到下）：准备细菌，同步蠕虫种群，在 96 孔板中接种，对蠕虫进行消毒，添加药物，在第 1 天和第 4 天测量 OD600。每个描述的左侧显示了这些特定步骤的详细说明。缩写：OD = 光密度;FUdR = 氟脱氧尿嘧啶。<a href="https://www.jove.com/files/ftp_upload/66422/66422fig01large.jpg" target="_blank">请单击此处查看此图的较大版本。</a>
1. 菌种制备
注意：本节详细介绍了该测定中使用的饲养细菌的制备。检测中使用的特定 大肠杆菌 菌株称为 OP50。OP50 是饲养 秀丽隐杆线虫的最广泛使用的菌株。使用的 OP50 菌株对羧青霉素/氨苄青霉素具有抗性，可防止蠕虫培养物与其他细菌的交叉污染9。
<ol><li>提前 4-5 天准备 OP50，并在使用前一天进行 X 射线照射。确保所有遇到 OP50 的材料都是无菌的29,30。</li>
	<li>第 -8 天：星期三（第 1 周）：<ol><li>通过接种 5 mL LB 和 100 μg/mL 氨苄青霉素和 0.1 μg/mL 两性霉素 B 和单个 OP50 菌落来制备预接种物，并在 37 °C 下在细菌振荡器中孵育约 6 小时。尽早接种，让培养物充分生长，使培养物变得浑浊。</li>
		<li>一旦培养物浑浊，在 250 mL 含有 100 μg/mL 氨苄青霉素的 TB 中以 1：2,000 稀释 OP50 的孕前培养物。将培养物在细菌振荡器中于 37 °C 孵育过夜 17-19 小时。 注意：不应让培养物生长超过 19 小时。</li>
	</ol></li>
	<li>第 -7 天：星期四（第 1 周）<ol><li>孵育 17-19 小时后，将 OP50 液体培养物转移到无菌离心管中，并在 4 °C 的台式离心机中以 3,100 × g 离心 15 分钟。</li>
		<li>弃去上清液，用无菌水重悬 OP50 沉淀，然后重新离心。重复此洗涤 2 次。</li>
		<li>第二次洗涤后，弃去上清液，将 OP50 沉淀重悬于无菌水中至体积为 50 mL，然后将其转移到预先称重的 50 mL 锥形管中。通过在 4 °C 的台式离心机中以 3,100 × g 离心 20 分钟来沉淀 OP50。</li>
		<li>第三次洗涤后，小心去除所有剩余的上清液，确保试管中没有水残留。通过从带有沉淀的离心管的重量中减去空离心管的重量来计算沉淀的重量。 注：颗粒重量通常在 3-3.5 g 之间。</li>
		<li>将 OP50 沉淀在 S-完全缓冲液中彻底重悬至浓度为 100 mg/mL，确保不存在结块。</li>
		<li>确保 100 mg/mL 的 OP50 浓度对应于 2 × 1010 个细菌/mL。如果光密度与每 mL 细菌数之间的关系已知，则通过分光光度法确定每 mL 的细菌浓度。如有必要，使用 S-完全缓冲液将 OP50 补料溶液的浓度调节至 2 × 1010 个细菌/mL。</li>
		<li>在琼脂平板上测试 OP50 以确定其是否受到污染。</li>
		<li>将悬浮在 4 °C 的 S-完全缓冲液中的 OP50 储存。</li>
	</ol></li>
</ol>
<ol start="4"><li>第 -3 天： 周一（第 2 周）<ol><li>通过 X 射线照射杀死细菌，以防止细菌在检测过程中生长。</li>
	</ol></li>
</ol>2. 同步蠕虫培养物制备
注意：本节讨论同步 Worm 种群的准备工作。所有与蠕虫种群接触的材料都是无菌的。除非另有说明，否则所有板均保持在 20 °C。
<ol><li>第 -6 天：周五（第 1 周），下午 4：00<ol><li>将动物转移到新鲜的盘子中。<ol><li>取一个 NGM 板，其中大部分蠕虫种群由饥饿的 L1 幼虫组成。 注意：混合总体也会产生结果。使用长期饥饿的 L1 蠕虫可能会影响当前或未来几代的摄食行为，因为饥饿会留下至少三代的表观遗传标记。</li>
			<li>用不超过 1 mL 的无菌水洗掉蠕虫。将 ~300 μL 洗去的蠕虫种群分装到接种有浓 OP50 （~300 mg/mL） 的 NGM 平板上。让板在板干燥器或本生灯下干燥。在 20°C 下孵育板，直到大部分种群包含妊娠成虫（星期一）。 注：此时间范围针对野生型 N2 蠕虫种群进行了优化，可能因菌株而异。应考虑发育迟缓的蠕虫，并尽早进行分块和/或尽早播种，以便所有测试的菌株都能同时达到成年。</li>
		</ol></li>
	</ol></li>
	<li>第 -3 天：周一（第 2 周）上午 10：00<ol><li>建立同步填充 谨慎！漂白剂对皮肤和眼睛有腐蚀性。处理它需要手套和安全眼镜。<ol><li>用最多 10 mL 的无菌水将蠕虫从板中清洗干净，以收集蠕虫。将此蠕虫/水溶液转移到 15 mL 锥形管中。</li>
			<li>通过在工作台上通过重力沉降水器洗涤蠕虫约 4 分钟（相反，在 310 × g 的台式离心机中离心洗涤 2 分钟。</li>
			<li>使用小吸头抽吸弃去上清液，并加入最多 15 mL 的无菌水。重复 3 次。</li>
			<li>去除上清液，加入 5 mL 新鲜制备的漂白剂/NaOH 溶液（1.8 mL 家用漂白剂、0.5 mL 10 N NaOH、7.7 mL dH2O）。</li>
			<li>在室温下孵育 5 分钟或直到蠕虫破裂。每分钟轻轻涡旋至少 10 秒。在解剖显微镜下监测进度。 注意：打开蠕虫所需的时间可能因菌株而异。将蠕虫留在漂白剂溶液中太久可能会导致虫卵无法存活。</li>
			<li>一旦所有成体裂开或溶解（最终体积为 15 mL）并以 1,300 × g 离心 2 分钟，加入 M9 缓冲液以中和反应。</li>
			<li>用 13 mL M9 缓冲液清洗鸡蛋 3 次。</li>
			<li>用 10 mL 的 S-完全缓冲液洗涤鸡蛋一次，以 1,300 × g 离心 2 分钟。</li>
			<li>吸出上清液，加入 10 mL S-complete，然后将悬浮液转移至新的 15 mL 锥形管中。在室温下在章动器或类似设备上轻轻旋转管子过夜。</li>
			<li>可选：如果在 S 完全缓冲液中最终离心后存在成体胴体，则通过 40 μm 细胞过滤器过滤蠕虫溶液。</li>
		</ol></li>
	</ol></li>
	<li>第 -2 天：星期二（第 2 周），下午 1：00<ol><li>将动物播种到板中<ol><li>使用解剖镜检查蠕虫是否已孵化。通过使用解剖镜计数 10 μL 液滴中的蠕虫种群，确定 S-完全缓冲液中蠕虫的浓度;每个样品计数 5-10 滴。如果每 10 μL 液滴中的蠕虫浓度超过每滴 30 条蠕虫，则用 S-complete 将总储备液稀释至每滴较低的蠕虫密度，以便于计数。</li>
			<li>将液体蠕虫混合物以 120 μL 的体积接种到 96 孔板的每个孔中，体积如下（终浓度）：60 蠕虫/mL S 完全培养基、50 μg/mL 羧苄青霉素、0.1 μg/mL 两性霉素和 6 mg/mL OP50，在第 1 部分中制备。 注：要制备的总体积取决于板的数量（每个 96 孔板 ~12 mL）。每个井中应有 6-12 条蠕虫。或者，可以使用大颗粒流式细胞术，例如 Union Biometrica 的 COPAS FP，将 10 条蠕虫精确分选到每个孔中（参见拥挤效应部分）。</li>
			<li>还包括 无蠕虫 液体混合物：第 1 部分制备的 50 μg/mL 羧青霉素、0.1 μg/mL 两性霉素和 6 mg/mL OP50。</li>
			<li>如前所述，将 120 μL 无蠕虫混合物放入 96 孔板的 H 行中，用于确定自解值。 每孔放入 120 μL 带有蠕虫的混合物，排在 A-G 行中。 注意：确保在移液过程中将蠕虫保持悬浮状态（参见 图 2A、B）。我们使用平底的透明 96 孔板。我们的板布局将板划分为产生 4 或 6 个条件，其中每对列将是不同的药物治疗或蠕虫菌株，底行将用作“无蠕虫”对照（图 2A、B）。</li>
			<li>为避免污染和蒸发，请使用胶带密封机密封板。将板在 20 °C 下孵育约 65 小时，直到动物变成 L4 蠕虫。</li>
		</ol></li>
	</ol></li>
	<li>第 0 天：星期四（第 2 周）中午前。<ol><li>通过添加氟脱氧尿嘧啶 （FUdR） 对蠕虫种群进行绝育。<ol><li>加入 30 μL 的 0.6 mM FUdR 储备溶液，对每个孔中的动物进行灭菌。使用胶带密封剂重新密封板，并在微量滴定板振荡器上以 800 rpm 的速度摇动板 20 分钟。将板放回 20 °C 培养箱中。 注：在此步骤中，最终 OP50 浓度从 6 mg/mL 降低到 5 mg/mL（1 × 109 个细菌/mL），每个孔的最终体积为 150 μL。在动物成年之前将 FUdR 添加到 L4 阶段至关重要。</li>
		</ol></li>
	</ol></li>
	<li>第 1 天：周五（第 2 周）<ol><li>向培养物中添加药物。<ol><li>到上午 9：00，确认大多数动物都怀孕了，并且每个孔都包含几个鸡蛋。如有必要，将任何药物添加至所需浓度（参见讨论）。</li>
			<li>添加药物后，用胶带密封剂密封板，并在板振荡器上以 800 rpm 的速度摇动板 20 分钟。 注意：摇动可确保在不接触密封剂的情况下溶解所有细菌团块。密封剂上的细菌团块或液体都会使 OD600 读数失真。如果密封剂上有液滴，请以 310 × g 的速度短暂旋转 10-20 秒。在摇床上再次摇晃 5 分钟并测量。</li>
			<li>在微量滴定板振荡器上摇动 20 分钟后，取下盖子和密封件，并在读板器中测量 OD600 ;这是 第 1 天的 OD600 测量值。密封板并将其放回 20 °C 培养箱中。 注意：由于细菌会迅速沉淀，因此读数应在摇动板后 10 分钟内出现。</li>
			<li>如果添加了药物，请使用倒置显微镜检查蠕虫种群是否有污染或毒性迹象。将板放回 20 °C 培养箱中。</li>
		</ol></li>
	</ol></li>
	<li>第 4 天：周一（第 3 周）<ol><li>读取第 4 天的 OD600 读数。<ol><li>从第 1 天开始重复读取程序（步骤 2.5.1.3）以获得 第 4 天的 OD600 值。在 OD600 测量之前，在微量滴定板振荡器上以 800 rpm 的速度摇动板 20 分钟。</li>
			<li>在倒置显微镜上，理想情况下使用 2 倍物镜，计算每个孔中的蠕虫数量并将其记录在电子表格中。计数后，将板放回 20 °C 培养箱中。 注意：请参阅补充材料中提供的评分表样本。</li>
			<li>第 4 天读数后，食物摄入量测量完成。如果需要，请保留这些板以进行寿命分析。 注意：该协议与 Solis 和 Petrascheck31 兼容。</li>
		</ol></li>
	</ol></li>
</ol>
3. 食物摄入量数据分析
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/66422/66422fig2_v1.jpg"> 图 2：设计和分析。（A、B） 4 和 6 种条件的可能板设计，H 行中“无蠕动”对照孔。这些孔对于确定自溶速率是必要的。在每个板中，始终包括 N 2 未处理的对照群体作为参考。（C） 在提供的电子表格中收集的样本数据，包括蠕虫数量（绿框）、第 1 天和第 4 天的两次 OD600 测量（橙色框）、自溶值（蓝框）以及使用公式 （2） 的评估（（OD600 -自解）/X0） 在红框中。此处显示的具体示例使用了 （B） 中所示的板设计。缩写：OD = 光密度;X0 = 每孔的蠕虫数。<a href="https://www.jove.com/files/ftp_upload/66422/66422fig2large_v1.jpg" style="font-size: 14px;" target="_blank">请单击此处查看此图的较大版本。</a>
注意：本节介绍如何分析本协议第 2 节的食物摄入量数据。要计算的第一个值是自解控制值。细菌会在液体中随着时间的推移而裂解，即使没有蠕虫也是如此。这个自解值也会受到许多化学物质的影响，需要控制，因为与蠕虫的食物摄入量相比，它相对较大。如步骤 2.3.1.4 中所述，如图 2A 中的板布局所示，B 行 H 包含相同的解决方案，不包括蠕虫。在图 2B 所示的板设置中，每个条件有 14 个孔，有两个相应的自解对照孔（H 行，“无蠕虫”）。
<ol><li>使用等式 （1） 计算每种条件下无蠕虫对照孔的自溶对照值。对于每个重复的孔组，确保至少有两个无蠕虫对照孔，如步骤 2.3.1.4 中所述。使用所有无蠕虫对照孔的平均值作为重复组的自溶值。 自拍 = 平均值（OD600 第 1 天 - OD600 第 4 天） （1） 注意：对于图 2B 所示的板设置，我们将通过计算 H 行中两个自解对照孔的平均值来计算六个自解值，六组中每组一个。</li>
	<li>使用方程式计算每条蠕虫的食物摄入量 <img alt="Equation 1" src="/files/ftp_upload/66422/66422equation2v2.jpg">     (2). 注意：电子表格中使用的公式（红框， 图 2C），其中 X0 是每孔的蠕虫数。</li>
	<li>在确定每个井的每条蠕虫的食物摄入量后，计算整个条件的平均值和标准差。 注意：对于 图 2B 所示的板设置，每个条件 14 个重复孔足以进行统计比较。</li>
	<li>将每种情况标准化为各自的 N2 未处理对照群体。将饲喂的变化表示为 N2 饲喂的倍数变化或百分比。 注：进行归一化是因为方程 （2） 中 OD 600/X0 的绝对值在不同日期进行的实验之间可能会有所不同。</li>
</ol>

测量 秀丽隐杆 线虫食物摄入量和寿命的高通量测定

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作者没有需要声明的利益冲突。

该方案将测量细菌清除率作为 96 孔微量滴定板中秀丽隐杆线虫食物摄入量的读数。代表性结果部分中的剂量反应曲线显示，该测定定量测量食物摄入量，这一概念使用同位素标记的细菌11 得到独立证实。使用该测定法，我们成功测试了 FDA 批准的药物，例如具有已知人类代谢副作用的抗精神病药，并表明这些药物增加了秀丽隐杆线虫的摄食 10,26。该测定可用于研究喂养的遗传学或药理学，并为秀丽隐杆线虫研究新陈代谢增加了新工具。该测定提供了一种低成本的方法，以可扩展和时间友好的方式研究摄食，这在大多数其他生物体中是不可能的。我们对 FDA 批准的药物的研究表明，在秀丽隐杆线虫中也观察到在人类患者身上看到的许多副作用，揭示了进化保守性 24,26,32。
该测定受可在 OD600 测量的线性范围内测量的细菌浓度的限制。因此，可以检测的蠕虫数量和细菌浓度范围是有限的。过多的蠕虫会因过快地消耗细菌而影响 OD600 ，从而“吃掉”线性 OD600 中的细菌浓度。根据我们的经验，细菌制备的可变性是导致结果因实验而异的关键参数。我们曾尝试冷冻大批量细菌或使用冻干细菌，但未成功。在这些情况下，要么 自溶 率变得太高，要么细菌的质量减慢 了秀丽隐杆线 虫的发育。但是，我们的测试并不详尽，这个问题可能是可以解决的。
一般来说，细菌的 自溶 率是最难控制的，它会导致相当大的变异性。我们发现一些药物可以将 自溶率提高到测定法无法可靠地确定食物摄入量的水平。由于测定中的 自溶 率随着时间的推移而增加，因此我们尚未测量成年后第 5 天后的食物摄入量。到那个年龄时，细菌已经培养了 ~10 天。要测量成年后第 5 天后的食物摄入量，必须洗掉旧细菌并用新鲜细菌代替。
所提出的测定是我们基于 96 孔微量滴定板的寿命测定31 的扩展。这种组合可以测量同一种群的寿命和食物摄入量。虽然这里介绍的细菌清除测定法由于细菌自溶而无法确定秀丽隐杆线虫整个生命周期的食物摄入量，但它为成年的前四天提供了可靠的数据，即食物摄入量最高的时间。特别是对于小分子药物的发现，控制食物摄入量是必不可少的。总之，我们预计所提出的检测将对秀丽隐杆线虫社区有用，并扩展工具集以研究新陈代谢或在衰老和寿命研究的背景下控制它。
规划： 
在计划测量食物摄入量之前，必须考虑一些因素。
杀死细菌：
我们通过在 50 mL 锥形管中用 X 射线照射来杀死细菌（900 格雷，设置为 160.0 kV 和 25.0 mA，持续 4 小时）。我们使用 Rad Source 的 RS2000 进行照射。根据设备的不同，具体时间和辐射剂量可能因设施而异。需要通过在照射后对细菌进行电镀来确定细菌的完全杀灭，以通过生长检测幸存者。在检测过程中杀死所有细菌以防止生长是必不可少的，因为这会导致低估摄食量甚至负摄食值。重要的是，必须杀死细菌，以便秀丽隐杆线虫仍然会吃掉它们。根据我们的经验，有三种方法可以可靠地杀死大量细菌：（i） X 射线照射，（ii） γ射线照射，以及 （iii） 添加甲醛29,30。这三种方法之间的自解速率是可比的，图 3B 所示的实验自解速率之间的变异系数为 8%。在我们手中不起作用的方法是紫外线照射、抗生素混合物、热灭活和叠氮化钠。这些方法要么不能完全杀死细菌（紫外线和抗生素），要么产生秀丽隐杆线虫不吃的细菌（加热），要么改变食物摄入量（叠氮化钠）。
重复数： 
根据我们的经验，与观察到的变化相比，喂养的褶皱变化通常很小。因此，测量食物摄入量的变化需要几个重复井。图 2A、B 显示了专为 4 或 6 种不同条件设计的 96 孔板，其中有 21 或 14 个重复孔和 2 个无蠕虫的自解对照孔。鉴于较大的可变性和预期的相对较小的变化，0.5-2.5 倍的饲喂变化往往是下限和上限。我们建议每种情况有 14 到 21 个重复孔，并且至少有两个自溶对照孔，如下所述。这些重复足以为改变喂养的药物生成定量剂量-反应曲线11,26。
自我溶解对照： 
OD600 可测量溶液中的颗粒数量，主要与颗粒大小无关。然而，死细菌会裂解，失去其颗粒性质。我们称之为 自溶，它在蠕虫存在的情况下独立发生，并在蠕虫不吃的情况下降低 OD600 测量值。 自溶 发生在检测过程中，需要在每种条件下至少两个孔中独立测量。这些井在相同条件下接受与其他井相同的处理，只是它们不包含任何蠕虫。我们发现许多药物也可以改变 自溶率。因此，每种情况都需要具有各自浓度的独立 自溶 对照孔。 图 2 显示了用于 4 或 6 种条件的板设置，所有 自解 控制孔都位于板底部的 H 行。
N2 参考人群： 
我们还注意到，消耗的细菌绝对量在实验之间可能会波动，很可能与细菌的质量有关。尽管我们尽最大努力每次都以完全相同的方式准备它们，但还是存在波动。例如，在对数生长期，细菌可以在分裂状态下收获，因此，其大小比在固定相中收获的细菌大得多。因此，细菌大小的这些简单差异会导致消耗的细菌数量发生变化和 OD600 值不同，因为 OD600 测量值不区分大小。出于这个原因，我们始终包括一组未经处理的 N2 对照动物作为参考值，设置为 1 或 100%，以确定实验组是否比 N2 对照吃得更多或更少。这种做法允许在实验之间进行比较，即使 OD600 值不同。
时间间隔： 
使用 OD600 值来测量食物摄入量需要细菌浓度显着下降。由于培养物体积比蠕虫大得多，因此它们必须吃掉大量的细菌才能产生可检测的差异。OD600 值保持在线性范围内需要细菌浓度保持在 ~1 mg/mL 和 10 mg/mL 之间，因此必须消失大量细菌才能检测到它。由于产卵，蠕虫从 L4 晚期到成年后第 4 天吃得最多。因此，此间隔是理想的。较短的间隔（如 24 小时）可以测量11 个，但这要困难得多，并且每个条件需要 ~40 个井。测量幼虫食物摄入量的另一种选择是将每孔的动物数量增加一倍。然而，这种方法的问题在于，当它们长大后，太多的蠕虫会开始影响 OD600 读数。
待测药物的储备液： 
如果测试了 5-羟色胺等药物调节喂养的能力，请在制备储备溶液时考虑以下几点。我们通常为水溶性药物制备 50 倍储备液，并向每个孔中加入 3 μL （1：50），但其他储备液浓度（100 倍、300 倍）也有效。但是，如果药物在水以外的溶剂（例如 DMSO）中稀释，则最终浓度不应超过 0.5%。溶剂很快就会对 秀丽隐杆线虫有害。因此，溶解在 DMSO 等有机溶剂中的药物储备液必须为 300 倍或更高。
对活蠕虫和死蠕虫进行评分： 
确定活动物和死动物是基于蠕虫的运动。使用强光，尤其是蓝光，因为它会导致动物移动。此外，对每孔的动物数量进行评分将揭示是否有超额死亡。有毒物质或多种溶剂（包括 DMSO）的高浓度 （>0.5%） 可能导致超额死亡。一般来说，动物的死亡应该少于 1：100 （1%）。如果食物已经沉淀并且种群变得难以计数，则可以在微量滴定板振荡器上以 800 rpm 的速度摇动板 30 秒。忽略有雄性或超过 16 条蠕虫的孔（见问题）。
潜在问题：
摇晃不充分： 
在液体介质中，细菌很容易聚集并沉淀在孔底部。根据我们的经验，摇晃少于 20 分钟可能无法打碎细菌团块并重新悬浮，从而导致 OD600 测量不准确。在存在细菌团块的情况下进行 OD600 测量会低估细菌浓度。摇床设置过多可能会导致液体粘在密封器上。一旦根据 OD600 读数移除封口剂，粘在封口剂上的液体将导致 OD600 读数降低。如果封口机上有液体，请以低速 （500-1,000 rpm） 快速旋转板并再次摇晃 5 分钟。此外，确保按照 第 1 天 和 第 4 天的 读数所述，在摇动板后 10 分钟内读取，以避免细菌沉淀。
实验之间的绝对食物摄入量值不同： 
尽管我们尽最大努力使细菌制剂标准化，但它们在影响蠕虫食物摄入的方式上往往有所不同。例如，由于细菌处于分裂状态，细菌的大小取决于它是在对数生长期还是固定期收获的，对数细菌更大。由于 OD600 测量不区分大小，由于细菌收获阶段引起的细菌大小差异可能导致在细菌制剂之间观察到的基础食物摄入量存在明显差异。比较实验之间的最佳方法是始终包括未处理的 N2 对照群体，并将结果归一化为该 N2 群体。
拥挤效果： 
该方案中的细菌浓度足以将 OD600 保持在每孔 2-16 条蠕虫的线性范围内 ~72 小时。根据感兴趣的药物，含有超过 16 条蠕虫的孔可能会在第二次测量之前耗尽细菌浓度。我们还发现，只有一只动物的井往往不可靠。裂解因子超过了一条蠕虫吃的东西。因此，确定裂解因子的微小错误会不成比例地影响蠕虫较少的孔。
如果满足以下条件之一，我们建议从统计分析中排除孔。井里有雄性（我们没有比较雄性和雌雄同体食物摄入量的数据）;井中蠕虫少于 2 条或井中蠕虫超过 16 条;蠕虫在第二次阅读时死亡;或者如果由于 FUdR 失败而在井中有后代。FUdR 对热敏感，即使在低至 37 °C 的温度下也会失活。 始终在室温水中解冻 FUdR。
负食物摄入量：
食物摄入量值偶尔为负值，表明第 4 天的食物比第 1 天多。负的食物摄入量值可能表明以下因素之一：高 自溶 率，可能是由添加作用于细菌的药物引起的，或负的食物摄入量值，这可能表明井被污染并且正在生长除蠕虫以外的其他东西。随着细菌年龄的增长， 自溶 率也会增加;因此，我们建议使用不超过一周的细菌。添加了一种随时间沉淀的药物，影响 OD600 值。第 1 天的摇晃不足可能会因细菌团块而导致 OD600 读数降低。蠕虫经历了低氧应激。当表面与空气的比值不足以进行氧气交换时，就会发生这种情况。在材料中使用特定的 96 孔板，每孔 不超过 150 μL（120 μL + 30 μL FUdR）。井表面与蠕虫生活的底部之间的距离决定了氧气浓度。
局限性：
该测定仅限于液体培养。在固体板中观察到的摄食行为，例如进出食物草坪，不能用测定法评估。

秀丽隐杆线虫已广泛用于衰老研究。 它是研究饮食限制、线粒体活性降低或胰岛素信号传导减少诱导的新陈代谢介导长寿的遗传学的有力模型。 1,2,3,4,5,6,7。然而，由于动物的体型小，在长寿的背景下测量秀丽隐杆线虫的摄食已被证明是困难的 3,8,9,10,11,12,13,14,15。
摄食是生存所需的基本生物行为，其严格的调节是维持代谢健康、繁殖和衰老的核心。摄食行为受神经系统和外周的多种信号通路控制，因此只能在体内研究 16,17,18,19。摄食代表了三大支柱中的第一支柱：（i） 能量摄入，（ii） 能量储存，以及 （iii） 控制生物体能量稳态的能量消耗。秀丽隐杆线虫的摄食主要通过测量咽部泵送来研究，咽部泵送由咽部收缩速率定义。这种方法为秀丽隐杆线虫的摄食行为提供了重要的见解 3,4,8,12,13,20,21;然而，咽部泵血，尤其是在短时间的几分钟内测量，不一定与食物摄入量相关。
食物摄入量不仅取决于咽部泵送速率，还取决于喂食的持续时间和频率以及食物细菌的密度等参数。动物的咽部泵送量可能非常高，但其进食时间可能更短，从而抵消了增加的速度。另一个混杂因素是泵送可能会也可能不会摄入和研磨细菌的效率。将食物摄入与咽部泵送脱钩的一个极端例子是添加血清素，而没有任何食物（细菌）存在。在外源性血清素存在的情况下，动物以高速率泵送，但不存在细菌，高泵送速率不会导致食物摄入 2,6,13,22,23。
这里介绍的细菌清除方法测量单个孔中蠕虫种群的食物摄入量，以及细菌浓度随时间的下降（图 1）。这种方法类似于其他物种使用的方法，其中食物的消失代表了食物摄入量的量度 10,11,24,25,26,27,28。在最初的出版物中，我们验证了这种通过用 15N 同位素标记的细菌喂养秀丽隐杆线虫来测量食物摄入量的方法11。随后的质谱测量表明，测量细菌的消失与同位素标记的发生密切相关，揭示了细菌在动物中的实际吸收11。因此，我们有信心细菌清除测定在大多数情况下代表食物摄入量。该检测的目的不是取代咽泵检测，而是添加到检测工具箱中，以研究秀丽隐杆线虫的摄食和代谢以及它与衰老和长寿的关系。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Amphotericin B</td>
			<td>RPI</td>
			<td>A40030-0.1</td>
			<td>solvent: EtOH</td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Fisher Scientific</td>
			<td>BP176025</td>
			<td>solvent: water</td>
		</tr>
		<tr>
			<td>Bacto Peptone</td>
			<td>BD Biosciences</td>
			<td>211677</td>
			<td>use to make NGM plates</td>
		</tr>
		<tr>
			<td>Carbenicillin</td>
			<td>Fisher Scientific</td>
			<td>46-100-RG</td>
			<td>solvent: water</td>
		</tr>
		<tr>
			<td>Cell strainer</td>
			<td>Fisher Scientific</td>
			<td>22363547</td>
			<td>40 &#181;m to remove adults</td>
		</tr>
		<tr>
			<td>Cholesterol&#160;</td>
			<td>MP Biomedicals</td>
			<td>02101380-CF</td>
			<td>5 mg/mL stock</td>
		</tr>
		<tr>
			<td>Difco, Agar, Bacteriological</td>
			<td>BD Biosciences</td>
			<td>214510</td>
			<td>use to make NGM plates</td>
		</tr>
		<tr>
			<td>Fluorodeoxyuridine</td>
			<td>Sigma Aldrich</td>
			<td>F0503</td>
			<td>to sterilize worms on L4</td>
		</tr>
		<tr>
			<td>Luria Broth</td>
			<td>RPI</td>
			<td>L24045-1000.0</td>
			<td>open capsule, mix with 1 L of water, autoclave</td>
		</tr>
		<tr>
			<td>M9 Buffer&#160;</td>
			<td>Laboratory Prepared</td>
			<td></td>
			<td>Store in sterile conditions at room temperature 
			To prepare 1 L: 
			&#160;15 g Na2HPO4*12H2O (FW: 358) 
			3 g KH2PO4 (FW: 136) 
			&#160;5 g NaCl (FW: 58), 0.25 g MgSO4*7H2O (FW: 246) 
			&#160;autoclave</td>
		</tr>
		<tr>
			<td>Microplate Sealer</td>
			<td>Fisher Scientific</td>
			<td>236707</td>
			<td></td>
		</tr>
		<tr>
			<td>OP50</td>
			<td colspan="2">Caenorhabditis Genetics Center</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate 96-well&#160;</td>
			<td>Falcon</td>
			<td>351172</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>Tecan</td>
			<td>30016056</td>
			<td>use 600 nm filter lens</td>
		</tr>
		<tr>
			<td>Potassium Citrate, 1 M , pH 6</td>
			<td>Laboratory Prepared</td>
			<td></td>
			<td>Store in sterile conditions at room temperature 
			To prepare 1 L: 
			268.8 g Potassium citrate tribasic monohydrate (FW: 324) 
			26.3 g citric acid monohydrate (FW: 210) 
			900 mL of dH2O 
			pH to 6 using 5 M KOH 
			autoclave&#160;</td>
		</tr>
		<tr>
			<td>Potassium Phosphate, 1 M , pH 6&#160;</td>
			<td>Laboratory Prepared</td>
			<td></td>
			<td>Store in sterile conditions at room temperature 
			To prepare 1 L: 
			136 g KH2PO4 (FW: 136) 
			900 mL dH2O 
			&#160;pH to 6 using 5 M KOH 
			autoclave</td>
		</tr>
		<tr>
			<td>S-Basal</td>
			<td>Laboratory Prepared</td>
			<td></td>
			<td>Store in sterile conditions at room temperature 
			To prepare 1 L: 
			5.9 g NaCl (FW: 58) 
			&#160;50 mL 1 M potassium phosphate (pH 6) 
			add 900 mL dH2O 
			autoclave, cool to 55 &#176;C</td>
		</tr>
		<tr>
			<td>S-Complete</td>
			<td>Laboratory Prepared</td>
			<td></td>
			<td>Store in sterile conditions at room temperature 
			To prepare 1 L: 
			Add to 1 L of S-basal (cooled to 55 &#176;C or RT) 
			&#160;10 mL of 1 M potassium citrate 
			&#160;pH 6, 10 mL of trace metal solutions 
			&#160;3 mL of 1 M CaCl2 
			3 mL of 1 M MgSO4</td>
		</tr>
		<tr>
			<td>Serotonin hydrochloride</td>
			<td>Thermo Scientific</td>
			<td>AAB2126309</td>
			<td>used at 5 mM</td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S7653-5KG</td>
			<td>to make buffers and NGM plates</td>
		</tr>
		<tr>
			<td>Terrific Broth</td>
			<td>Thermo Scientific</td>
			<td>J75856-A1</td>
			<td>12.5 g in 250 mL of water, autoclave</td>
		</tr>
		<tr>
			<td>Titer plate Shaker</td>
			<td>Thermo Scientific</td>
			<td>88880023</td>
			<td>shaken at 800 rpm, depends on shaker</td>
		</tr>
		<tr>
			<td>Trace Metals Solution&#160;</td>
			<td>Laboratory Prepared</td>
			<td></td>
			<td>Store in sterile conditions at room temperature 
			To prepare 1 L: 
			1.86 g Na2EDTA (FW: 372.24) 
			0.69 g FeSO4*7H2O (FW: 278) 
			&#160;0.20 g MnCl2*4H2O (FW: 198) 
			0.29 g ZnSO4*7H2O (FW: 287) 
			&#160;0.016 g CuSO4 (FW: 158) 
			autoclave 
			wrap in aluminum foil to keep in the dark</td>
		</tr>
	</tbody>
</table>

quantifying food intake in caenorhabditis elegans by measuring bacterial clearance

摄食是生物体生长、繁殖和生存的重要生物过程。该测定旨在测量 秀丽隐杆线虫 （C. elegans） 的食物摄入量，这是研究衰老或新陈代谢遗传学的重要参数。在大多数物种中，摄食是通过测量提供的食物量与给定时间间隔后剩余的量之间的差值来确定的。这里介绍的方法使用相同的策略来确定 秀丽隐杆线虫的摄食。它测量在 72 小时内清除的细菌量， 即秀丽隐杆线虫的食物来源。该方法使用 96 孔微量滴定板，并允许筛选数百种药物，以确定它们以其他动物模型无法实现的速度和深度调节食物摄入的能力。该测定法的优势在于，它可以同时测量摄食和寿命，并直接测量食物的消失情况，因此，它基于用于其他生物体的相同原理，便于物种间比较。

<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/66422/66422fig3_v1.jpg"> 图 3：代表性数据。 （A） 该图显示了食物摄入量倍数变化的剂量反应曲线与血清素浓度的函数关系。所有数据均显示未刺激的基础食物摄入量标准化为 N2 基础喂养。请注意，当没有添加血清素时， daf-16（mu86） 动物比野生型 N2 吃得更多，但它们控制进食的能力范围变慢。（B） 图表显示了用 50 μM 抗精神病药物洛沙平处理但喂食被 X 射线、γ射线或多聚甲醛杀死的细菌的蠕虫食物摄入量倍数变化的小提琴图 （C） 图表显示了不同突变体食物摄入量倍数变化的小提琴图，其基因型在 x 轴中表示。这些数据表明， exc-4 和 cgr-1 突变体吃得更少，而 srp-6 突变体吃得更多。星号表示 ** p < 0.001，****p < 0.0001，由方差分析和 Brown-Forsythe-Welch 确定，事后校正以说明多个假设检验。误差线显示 ±S.E.M <a href="https://www.jove.com/files/ftp_upload/66422/66422fig3large_v1.jpg" target="_blank">. 请点击此处查看此图的较大版本。</a>
图 1 突出显示了该协议的整体工作流程。它说明了从制造细菌到在第 1 天和第 4 天获得 OD600 读数的整个过程。此外，该信息图还引用了所用方法的具体步骤。 图 2A、B 显示了两种可能的实验板布局，包括 H 行中的“无蠕虫”对照孔，这是计算讨论部分中描述的 自解 速率所必需的。根据我们实验室的经验，有必要将未经处理的条件之一 N2 对照 解释在实验之间观察到的蠕虫细菌摄入的可变性（参见讨论：规划 [N2 参考人群]）。
图 2C 是用于分析该协议中生成的数据的电子表格的示例。它以绿色突出显示蠕虫数量，以蓝色突出显示自溶 ，以橙色突出显示第 1 天 OD600 和第 4 天 OD600，以红色突出显示每条蠕虫的食物摄入量。此外，还跟踪每个孔的菌株、药物、浓度、实验日期和添加的其他处理。有关所用具体方程的更多信息，请参见协议第 3 节。总体而言，这些电子表格（作为补充材料提供的模板）提供了该协议的高效数据收集和分析。
图 3A 显示了血清素如何使用该方案调节 N2 和 daf-16 突变体进食的剂量反应曲线的代表性结果。在这个实验中，计算了五次增加剂量的血清素从基础食物摄入量（曲线中的第一个点）的倍数变化。正如这些结果所强调的那样，N2 菌株能够以剂量依赖性方式暴饮暴食。然而，对于 daf-16 （mu86） 突变体，尽管基础喂养高于 N2，但突变体不能以与 N2 菌株相同的剂量依赖性方式对血清素做出反应。该协议使我们能够确定两种菌株之间的摄食行为，其中血清素的浓度是生成剂量 - 反应曲线的可变因素。 图 3B 显示了用抗精神病药洛沙平治疗但以被 X 射线、γ射线或多聚甲醛杀死的细菌为食的蠕虫的食物摄入量。请注意，这些实验不是平行进行的，并且差异并不代表杀死细菌的不同方法，而是由于实验间的可变性。 图 3C 显示了一系列遗传菌株的食物摄入量，其中两个菌株显示相对于 N 2 的摄食行为减少，一个菌株显示摄食行为增加。该方案的这种应用能够在不同的遗传背景中测试一种浓度的药物。它适用于鉴定在给予相同药物时显示与野生型 N2 对照不同的食物摄入反应的遗传背景。它可以与我们的寿命方案配对，以在同一板设置中快速筛选食物摄入量和寿命31。
补充材料：图 2C 所示的电子表格模板。 <a href="https://www.jove.com/files/ftp_upload/66422/66422.zip" target="_blank">请点击此处下载这些资料。</a>

Read this Scientific Article Protocol about Author Spotlight: Exploring the Relationship Between Lipotoxicity and HFpEF at JoVE.com

作者聚焦：探索脂毒性与 HFpEF 之间的关系 

该方案描述了一种基于测量液体培养物中细菌清除率来量化 秀丽隐杆线 虫摄食率的测定法。

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

Feeding is an essential biological process for an organism&#39;s growth, reproduction, and survival. This assay aims to measure the food intake of ...

我的研究重点是了解药物诱导的摄食行为变化背后的转录和代谢途径。我正在努力寻找降低食物摄入量和延长寿命的药物，表明激活健康的禁食途径。使用我们的细菌清除测定，我们确定了调节血清素诱导的食物摄入和抗精神病药诱导的食物摄入的两种不同途径。我现在正在研究这些途径与长寿突变体的摄食有何关系。该协议允许研究人员研究喂养与长寿之间的关系。喂养速率通常以咽部泵送为特征。然而，秀丽隐杆线虫的泵送速率不一定与吃的食物量成正比。当添加到以前的分析中时，该方案给出了每个蠕虫种群的可量化食物摄入量。我们的分析使我们能够使用小分子定量调节摄食，并确定诱导的变化如何同时改变寿命。

quantifying-food-intake-caenorhabditis-elegans-measuring-bacterial

Research

JoVE Journal

Behavior

Quantifying Food Intake in Caenorhabditis elegans by Measuring Bacterial Clearance

2.7K 次观看.  The Scripps Research Institute. 我的研究重点是了解药物诱导的摄食行为变化背后的转录和代谢途径。我正在努力寻找降低食物摄入量和延长寿命的药物，表明激活健康的禁食途径。使用我们的细菌清除测定，我们确定了调节血清素诱导的食物摄入和抗精神病药诱导的食物摄入的两种不同途径。我现在正在研究这些途径与长寿突变体的摄食有何关系。该协议允许研究人员研究喂养与长寿之间的关系。?...

视频: 作者聚焦：探索脂毒性与 HFpEF 之间的关系 

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

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

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

一个过程来研究延长食品的限制对海洛因寻求戒断大鼠的影响

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

科研

行为学

Studying Food Reward and Motivation in Humans

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as hedonics, motivation, and goal value in more objective ways. This is particularly relevant for the understanding of overeating and obesity as well as their potential treatments. In this paper are described a set of measures of food-related motivation using handgrip force as a motivational measure. These methods can be used to examine changes in food related motivation with metabolic (satiety) and pharmacological manipulations and can be used to evaluate interventions targeted at overeating and obesity. However to understand food-related decision making in the complex food environment it is essential to be able to ascertain the reward goal values that guide the decisions and behavioral choices that people make. These values are hidden but it is possible to ascertain them more objectively using metrics such as the willingness to pay and a method for this is described. Both these sets of methods provide quantitative measures of motivation and goal value that can be compared within and between individuals.

This article describes a set of methods for the measurement of food related motivation and food related goal values in humans.

研究食品奖赏和动机在人类

A key challenge in studying reward processing in humans is to go beyond subjective self-report measures and quantify different aspects of reward such as ...

The Modified Hole Board - Measuring Behavior, Cognition and Social Interaction in Mice and Rats

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure multiple dimensions of unconditioned behavior in small laboratory mammals (e.g., mice, rats, tree shrews and small primates). This paradigm is a valuable alternative for the use of a behavioral test battery, since a broad behavioral spectrum of an animal&#8217;s behavioral profile can be investigated in one single test.
The apparatus consists of a box, representing the &#8216;protected&#8217; area, separated from a group compartment. A board, on which small cylinders are staggered in three lines, is placed in the center of the box, representing the &#8216;unprotected&#8217; area of the set-up. The cognitive abilities of the animals can be measured by baiting some cylinders on the board and measuring the working and reference memory. Other unconditioned behavior, such as activity-related-, anxiety-related- and social behavior, can be observed using this paradigm. Behavioral flexibility and the ability to habituate to a novel environment can additionally be observed by subjecting the animals to multiple trials in the mHB, revealing insight into the animals&#8217; adaptive capacities.
Due to testing order effects in a behavioral test battery, na&#239;ve animals should be used for each individual experiment. By testing multiple behavioral dimensions in a single paradigm and thereby circumventing this issue, the number of experimental animals used is reduced. Furthermore, by avoiding social isolation during testing and without the need to food deprive the animals, the mHB represents a behavioral test system, inducing if any, very low amount of stress.

This protocol describes the modified hole board, which is a behavioral test set-up that comprises the characteristics of an open field and a traditional hole board. This set-up enables the differential analysis of unconditioned behavior of small laboratory mammals as well as the analysis of cognitive abilities.

修改后的孔板 - 测量行为，认知和社会互动小鼠和大鼠

This protocol describes the modified hole board (mHB), which combines features from a traditional hole board and open field and is designed to measure ...

Quantifying Social Motivation in Mice Using Operant Conditioning

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered shuttle boxes were equipped with two operant levers (left and right) and a food receptacle in one chamber, which was then divided from the second chamber by an automated guillotine door covered by a wire grid. Different stimulus mice, rotated across testing days, served as a social stimulus behind the wire grid, and were only visible following the opening of the guillotine door. Test mice were trained to lever press in order to open the door and gain access to the stimulus partner for 15 sec. The number of lever presses required to obtain the social reward progressively increased on a fixed schedule of 3. Testing sessions ended after test mice stopped lever pressing for 5 consecutive minutes. The last reinforced ratio or breakpoint can be used as a quantitative measure of social motivation. For the second paradigm, test mice were trained to discriminate between left and right lever presses in order to obtain either a food reward or the social reward. Mice were rewarded for every 3 presses of each respective lever. The number of food and social rewards can be compared as a measurement of the value placed upon each reward. The ratio of each reward type can also be compared between mouse strains and the change in this ratio can be monitored within testing sessions to measure satiation with a given reward type. Both of these operant conditioning paradigms are highly useful for the quantification of social motivation in mouse models of autism and other disorders of social behavior.

This article describes a new protocol for the quantitative measurement of social motivation in mice using operant conditioning for a social reward. The protocol is useful for the measurement of social motivation in mouse models of autism and other disorders of social behavior.

量化的社会动机小鼠使用操作条件反射

In this protocol, social motivation is measured in mice through a pair of operant conditioning paradigms. To conduct the experiments, two-chambered ...

Feeding Experimentation Device (FED): Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in mice: the Feeding Experimentation Device (FED). FED is an open-source system that was designed to facilitate flexibility in food intake studies. Due to its compact and battery powered design, FED can be placed within standard home cages or other experimental equipment. Food intake measurements can also be synchronized with other equipment in real-time via FED's transistor-transistor logic (TTL) digital output, or in post-acquisition processing as FED timestamps every event with a real-time clock. When in use, a food pellet sits within FED's food well where it is monitored via an infrared beam. When the pellet is removed by the mouse, FED logs the timestamp onto its internal secure digital (SD) card and dispenses another pellet. FED can run for up to 5 days before it is necessary to charge the battery and refill the pellet hopper, minimizing human interference in data collection. Assembly of FED requires minimal engineering background, and off-the-shelf materials and electronics were prioritized in its construction. We also provide scripts for analysis of food intake and meal patterns. Finally, FED is open-source and all design and construction files are online, to facilitate modifications and improvements by other researchers.

Feeding Experimentation Device FED: Construction and Validation of an Open-source Device for Measuring Food Intake in Rodents

Feeding Experimentation Device (FED) is an open-source device for measuring food intake in mice. FED can also synchronize food intake measurements with other techniques via a real-time digital output. Here, we provide a step-by-step tutorial for the construction, validation, and usage of FED.

饲养实验装置（FED）:建筑和验​​证的开源设备的在鼠害测量摄食

Food intake measurements are essential for many research studies. Here, we provide a detailed description of a novel solution for measuring food intake in ...

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

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

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

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

在果蝇中结合定量食物摄入量测定和强迫激活神经元研究食欲

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

Continuous Noninvasive Measuring of Crayfish Cardiac and Behavioral Activities

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as a useful biological indicator of water quality. Even though crayfish cannot directly specify the substances that cause water quality deterioration, they can immediately (within a few seconds) warn humans of water quality deterioration via acute changes in their cardiac and behavioral activities.
In this study, we present a noninvasive method that is simple enough to be implemented under various conditions due to a combination of simplicity and reliability in one model.
This approach, in which the biological organisms are implemented into environmental evaluation processes, provides a reliable and timely alarm for warning of and preventing acute water deterioration in an ambient environment. Therefore, this noninvasive system based on crayfish physiological and ethological parameter recordings was investigated for the detection of changes in an aquatic environment. This system is now applied at a local brewery for controlling&#160;quality of the water used for beverage production, but it can be used at any water treatment and supply facility for continuous, real-time water quality evaluation and for regular laboratory investigations of crayfish cardiac physiology and behavior.

This article presents a noninvasive biomonitoring system for the continuous recording and analyses of crayfish cardiac and locomotor activities. This system consists of a near-infrared optical sensor, a video-tracking module, and software for evaluating crayfish heartbeats that reflects its physiological condition and characterizes crayfish behavior during heartbeat fluctuations.

小龙虾心脏的连续无创测量与行为活动

A crayfish is a pivotal aquatic organism that serves both as a practical biological model for behavioral and physiological studies of invertebrates and as ...

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) controls several functions in C. elegans including learning and motor activity. Conditions that stimulate DA release (e.g., amphetamine (AMPH) treatments) or that prevent DA clearance (e.g., animals lacking the DA transporter (dat-1) which are incapable of reaccumulating DA into the neurons) generate an excess of extracellular DA ultimately resulting in inhibited locomotion. This behavior is particularly evident when animals swim in water. In fact, while wild-type animals continue to swim for an extended period, dat-1 null mutants and wild-type treated with AMPH or inhibitors of the DA transporter sink to the bottom of the well and do not move. This behavior is termed "Swimming Induced Paralysis" (SWIP). Although the SWIP assay is well established, a detailed description of the method is lacking. Here, we describe a step-by-step guide to perform SWIP. To perform the assay, late larval stage-4 animals are placed in a glass spot plate containing control sucrose solution with or without AMPH. Animals are scored for their swimming behavior either manually by visualization under a stereoscope or automatically by recording with a camera mounted on the stereoscope. Videos are then analyzed using a tracking software, which yields a visual representation of thrashing frequency and paralysis in the form of heat maps. Both the manual and automated systems guarantee an easily quantifiable readout of the animals' swimming ability and thus facilitate screening for animals bearing mutations within the dopaminergic system or for auxiliary genes. In addition, SWIP can be used to elucidate the mechanism of action of drugs of abuse such as AMPH.

Swimming Induced Paralysis to Assess Dopamine Signaling in Caenorhabditis elegans

Swimming induced paralysis (SWIP) is a well-established behavioral assay used to study the underlying mechanisms of dopamine signaling in Caenorhabditis elegans (C. elegans). However, a detailed method to perform the assay is lacking. Here, we describe a step-by-step protocol for SWIP.

游泳诱导麻痹对线虫多巴胺信号的评价

The swimming assay described in this protocol is a valid tool to identify proteins regulating the dopaminergic synapses. Similar to mammals, dopamine (DA) ...

Providing Meaningful Environmental Enrichment and Measuring Saliva Cortisol in Pigs Housed on Slatted Flooring

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a substantial pressure coming from customers, legislators and other stakeholders, to make management changes to improve the well-being of these animals. Several studies have demonstrated that providing environmental enrichment to pigs allows them to express their natural behavior, such as rooting and exploring, as well as nesting prior to farrowing, and is associated with reduced stress and improved production and welfare. However, many considerations should be taken into account when providing environmental enrichment, such as the type of floor, drainage and sewage systems, the pigs&#39; stage in life, the material, as well as its hanging method, height and location within the pen. The objectives of this paper are (1) to give methodologic information on how to provide a relatively simple and practical meaningful environmental enrichment for pigs which are housed on slatted floors during the different stages of their life, and (2) to demonstrate how to collect saliva samples for the measurement of cortisol concentrations, as a biomarker for acute stress. Protocols include information regarding the use of jute, cotton ropes, straw in racks, as well as chewable silicone sticks devices as environmental enrichment in pens of farrowing and lactation, weaners and finishers. In addition, the use of cotton rope for a non-invasive saliva samples collection for cortisol concentrations analysis is detailed. The protocols provided are relevant for professionals aiming to improve and monitor animal welfare, in both research and industrial swine farming.

This protocol demonstrates how to provide practical meaningful environmental enrichment for pigs which are housed on slatted flooring during the different stages of their lives, and how to collect saliva samples in a non-invasive manner for the measurement of cortisol concentrations, as a biomarker for acute stress.

提供有意义的环境富集和测量唾液皮质醇在猪安置在板状地板

As pork is the most consumed meat worldwide, the welfare of animals in the swine industry has increasingly become a major public concern, which imposes a ...

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. Animals cope with these stimuli by modifying their behavior. Understanding the mechanisms underlying such behavioral modification requires quantification of neural activity during the behavior of interest. Here, we report a method for calcium imaging in freely behaving Caenorhabditis elegans with nonlocalized vibration of specific frequency, displacement, and duration. This method allows the production of well-controlled, nonlocalized vibration using an acoustic transducer and quantification of evoked calcium responses at single-cell resolution. As a proof of principle, the calcium response of a single interneuron, AVA, during the escape response of C. elegans to vibration is demonstrated. This system will facilitate understanding of neural mechanisms underlying behavioral responses to mechanical stimuli.

Calcium Imaging in Freely Behaving Caenorhabditis elegans with Well-Controlled, Nonlocalized Vibration

Reported here is a system for calcium imaging in freely behaving Caenorhabditis elegans with well-controlled, nonlocalized vibration. This system allows researchers to evoke nonlocalized vibrations with well-controlled properties at nano-scale displacement and to quantify calcium currents during responses of C. elegans to the vibrations.

自由行为 秀丽隐杆线虫 的钙成像，具有良好控制，非局部化振动

Nonlocalized mechanical forces, such as vibrations and acoustic waves, influence a wide variety of biological processes from development to homeostasis. ...

通过测量细菌清除率来量化 秀丽隐杆线虫 的食物摄入量

通过测量细菌清除率来量化秀丽隐杆线虫的食物摄入量