作者要感谢Andrea Thomson，Jon Hash，Hope Woods和Autumn Anthony为QST处理狗，Masataka Enomoto帮助筛选狗，以及Sam Chiu为建立热热QST协议所做的贡献。

所有程序均由北卡罗来纳州立大学机构动物护理和使用委员会批准。
1. 房间设置和学习科目适应
<ol><li>在专用空间中进行 QST，该空间有足够的空间供 QST 操作员、处理员和任何大小的狗舒适地移动。尽量减少潜在的听觉和视觉干扰，并使用白噪声机器阻挡环境声音。</li>
	<li>在地板上放置一个大的瑜伽垫或类似的衬垫，以确保狗在测试期间在侧卧时感到舒适。</li>
	<li>让狗至少 10 分钟自由探索和适应房间，并与 QST 操作员和处理者相处融洽。在客房内随意提供淡水，并偶尔提供食物奖励。</li>
	<li>通过抛硬币随机化测试站点（左侧或右侧）。在第三和第四跖骨背表面之间的空间周围夹一个大约 2 x 4 cm 的皮毛部分，中间位于跗跖关节和跖趾关节之间。在腕骨上方靠近臂前腕关节的外侧臂前夹一个大约 1 x 2 cm 的毛皮切片。</li>
</ol>2. 电子冯弗雷麻醉仪
<ol><li>仪器设置<ol><li>轻轻地将刚性 0.9 mm 冯弗雷尖端施加到称重传感器上，并确保称重传感器牢固地拧入手机中。通过M0通道将电源线从手机连接到录音设备（图1A，B）。</li>
		<li>打开记录设备并按下 MAX 按钮，以便设备将记录并显示狗对施加的刺激做出反应时达到的最大力（图1C）。</li>
		<li>按下 CLR 按钮将仪器归零。</li>
	</ol></li>
	<li>数据采集<ol><li>将狗放在侧面卧位以测量阈值。 注意：将狗放置在右侧卧位以测量左肢的阈值，或放置在左侧卧位以测量右肢的阈值。如果狗在给予口头提示时不愿意躺在侧卧中，QST 操作员和处理员可以手动将狗放在侧卧位。</li>
		<li>根据需要应用最小到中度的约束，以保持狗在侧卧和相对静止。 注意：处理程序执行此步骤。</li>
		<li>一旦狗平静和放松并且被测试的肢体至少伸展 70%，就应用刺激。为被测试的肢体提供温和的手动支撑，使肢体远离地板，并提供稳定的支撑以施加力，同时不阻止狗抽出肢体。 注意：QST 运算符执行此步骤。</li>
		<li>垂直于被测区域的皮肤应用冯弗雷尖端。如果狗表现出反射运动（例如，爪子抽搐或在用力前肢体退出），因为冯弗雷尖端在皮肤上的感觉，让狗在重新应用冯弗雷尖端之前再次放松肢体。当皮肤不引起反射运动时，通过应用冯弗雷尖端进行测量。</li>
		<li>用冯弗雷尖端（~20 g/sec）施加稳定增加的力，直到狗撤回肢体、发声、转身看刺激，或表现出其他表明对刺激的有意识感知的动作或行为反应。当狗抽出肢体或达到最大力时，移除刺激。 注意：不要超过 1，000 克的力。</li>
		<li>记录记录设备上显示的最大力。 注意：如果达到 1，000 g 力的安全截止值，则记录 1，000 g 作为感官阈值，并且注意在安全截止之前没有响应。</li>
		<li>重复测量总共五次试验，每次测量之间留出1分钟（试验间隔）。通过按 CLR 按钮在每个步骤之间将仪器归零。<ol><li>允许狗在试验间隔期间保持侧卧，如果它们保持相对平静和放松，没有或很少的约束。否则，让狗在 QST 房间内坐下、站立或移动以保持舒适度。在随后的测量之前，将狗放回侧面卧位。</li>
		</ol></li>
		<li>记录 0-5 的可行性分数，以指示收集数据的难易程度。 注意：可行性分数如下：0 = 没有问题，1 = 轻度难度，2 = 中等难度，3 = 显着难度，4 = 极端困难，5 = 不可能。用于分配可行性分数的量规如 表1所示。</li>
		<li>让狗休息 5 分钟，然后开始用钝探头的压力测藻力计进行测量。</li>
	</ol></li>
</ol>3. 钝探头压力测数计
<ol><li>仪器设置<ol><li>确保将小钝探头牢固地拧入设备（图2A）。</li>
		<li>打开录音设备，并在屏幕上出现提示时按 MAX 按钮继续。按 UNIT 按钮，直到设备在屏幕顶部显示为克 （g）（图 2B）。</li>
		<li>按 ZERO 按钮将仪器归零。</li>
	</ol></li>
	<li>数据采集<ol><li>将狗放在侧面卧位以测量阈值。 注意：将狗放置在右侧卧位以测量左肢的阈值，或放置在左侧卧位以测量右肢的阈值。如果狗在给予口头提示时不愿意躺在侧卧中，QST 操作员和处理员可以手动将狗放在侧卧位。</li>
		<li>根据需要应用最小到中度的约束，以保持狗在侧卧和相对静止。 注意：处理程序执行此步骤。</li>
		<li>一旦狗平静和放松并且被测试的肢体伸展约 70%，就应用刺激。为被测试的肢体提供温和的手动支撑，使肢体远离地板，并提供稳定的支撑以施加力，同时不阻止狗抽出肢体。 注意：QST 运算符执行此步骤。</li>
		<li>垂直于被测区域的皮肤应用钝探针（图2C）。如果狗在皮肤上感受到钝探针后表现出反射运动（例如，爪子抽搐或在用力前肢体撤回），请在重新应用钝探针之前让狗再次放松肢体。当将钝探头施加到皮肤上不引起反射运动时进行测量。</li>
		<li>用探针施加稳定增加的力（~20 g / s），直到狗撤回肢体，发声，转身看刺激，或表现出其他表明对刺激的有意识感知的运动或行为反应。当狗抽出肢体或达到最大力时，移除刺激。 注意：不要超过 2，500 克的力。</li>
		<li>记录记录设备上显示的最大力。 注意：如果达到 2，500 g 力的安全截止值，则记录 2，500 g 作为感官阈值，并注意在安全截止之前没有响应。</li>
		<li>重复测量总共五次试验，每次测量之间留出1分钟（试验间隔）。按 ZERO 按钮，在每一步之间将仪器归零。<ol><li>允许狗在试验间隔期间保持横向卧位，如果它们保持相对平静和放松，没有或很少的约束。否则，让狗在 QST 房间内坐下、站立或移动以保持舒适度。在随后的测量之前，将狗放回侧面卧位。</li>
		</ol></li>
		<li>记录 0-5 的可行性分数，以指示收集数据的难易程度。 注意：可行性分数如下：0 = 没有问题，1 = 轻度难度，2 = 中等难度，3 = 显着难度，4 = 极端困难，5 = 不可能。</li>
		<li>让狗休息 5 分钟，然后开始使用热探头进行测量。</li>
	</ol></li>
</ol>4. 热探头
<ol><li>仪器设置<ol><li>通过USB电缆 将 热感分析仪连接到计算机，并确保16 x 16 mm温度计连接到分析仪。打开分析器。</li>
		<li>打开计算机上的热感分析仪软件，然后从启动菜单中选择TSA II分析仪。在分析器自检的弹出警告上单击 确定 。确保在自检期间，恒温器未连接到研究对象。</li>
		<li>在“ 测试 ”选项卡（右上角）的“选择患者”提示（屏幕左侧）下，双击列表中的姓名来 选择相应的患者 。<ol><li>要创建新患者，请单击“测试”选项卡右侧的“患者”选项卡。单击左下角的新患者图标并填写患者详细信息（部门、患者姓名、ID、性别和出生日期）。</li>
		</ol></li>
		<li>在“测试”选项卡的“选择程序”提示下，双击列表中的程序来选择相应的程序。<ol><li>要创建新程序，请单击“患者”选项卡右侧的“程序”选项卡。单击左下角的“新建程序”图标并填写程序详细信息。 注意：对于此协议，程序详细信息在表 2 中给出。不需要在“测试”选项卡的“选择正文站点”提示下选择正文站点。</li>
		</ol></li>
		<li>选择适当的患者和程序后，单击“测试”选项卡下的“转到测试”提示。单击左下角的“预测试”按钮，将分析仪校准到指定的程序。</li>
		<li>预测试完成后，“预测试”按钮将替换为“开始”按钮，并显示测试窗口：按“开始”按钮开始测试（图 3A）。</li>
		<li>松开热度仪电缆，确保热度仪易于取用。</li>
	</ol></li>
	<li>数据采集<ol><li>将狗放在侧面卧位以测量热潜伏期。 注意：将狗放置在右侧卧位以测量左肢的阈值，或放置在左侧卧位以测量右肢的阈值。如果狗在给予口头提示时不愿意躺在侧卧中，QST 操作员和处理员可以手动将狗放在侧卧位。</li>
		<li>根据需要应用最小到中度的约束，以保持狗在侧卧和相对静止。 注意：处理程序执行此步骤。</li>
		<li>一旦狗平静和放松并且被测试的肢体伸展约 70%，就应用刺激。为被测试的肢体提供温和的手动支撑，使肢体远离地板，同时不阻止狗抽出肢体。此外，用手支撑肢体握住并操作秒表。 注意：QST 运算符执行此步骤。</li>
		<li>将热化物涂在被测区域的皮肤上（图3B）。如果狗表现出反射运动（例如，在加热之前爪子抽搐或肢体退出），因为皮肤上的热德感觉，在重新涂抹热化物之前让狗再次放松肢体。当将热敷物应用于皮肤不引起反射运动时进行测量。</li>
		<li>单击“测试”选项卡左下角的“开始”按钮以开始测试。 注意：QST 操作员向处理程序发出信号以开始测试（例如，通过点头），QST 操作员同时启动秒表。</li>
		<li>当狗抽出肢体、发声、转身看刺激或表现出其他表明对刺激的有意识感知的运动或行为反应时，或者在停止秒表的同时达到最大潜伏期时，取下热物。 注意：QST 运算符执行此步骤。不要超过20秒的热化物应用或49°C的最高温度。</li>
		<li>记录提款延迟。如果达到20 s的热化物应用安全截止，记录20 s作为感官潜伏期，并注意在安全截止之前没有响应。</li>
		<li>重复测量总共五次试验，每次测量之间留出1分钟（试验间隔）。单击“ 结束 测试”按钮，然后单击每次测量之间的 “预测试 ”按钮，停止加热温度计并重新校准温度计，为下一次应用做准备。 注意：处理程序执行此步骤。<ol><li>允许狗在试验间隔期间保持横向卧位，如果它们保持相对平静和放松，没有或很少的约束。否则，让狗在 QST 房间内坐下、站立或移动以保持舒适度。在随后的测量之前，将狗放回侧面卧位。</li>
		</ol></li>
		<li>记录 0-5 的可行性分数，以指示收集数据的难易程度。 注意：可行性分数如下：0 = 没有问题，1 = 轻度难度，2 = 中等难度，3 = 显着难度，4 = 极端困难，5 = 不可能。</li>
	</ol></li>
</ol>

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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Feasibility+and+repeatability+of+cold+and+mechanical+quantitative+sensory+testing+in+normal+dogs.">Feasibility and repeatability of cold and mechanical quantitative sensory testing in normal dogs.</a> The Veterinary Journal. 199 (2), 246-250 (2014).</li><li>Knazovicky, 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=Replicate+effects+and+test-retest+reliability+of+quantitative+sensory+threshold+testing+in+dogs+with+and+without+chronic+pain.">Replicate effects and test-retest reliability of quantitative sensory threshold testing in dogs with and without chronic pain.</a> Veterinary Anesthesia and Analgesia. 44 (3), 615-624 (2017).</li><li>Sanchis-Mora, 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=Development+and+initial+validation+of+a+sensory+threshold+examination+protocol+(STEP)+for+phenotyping+canine+pain+syndromes.">Development and initial validation of a sensory threshold examination protocol (STEP) for phenotyping canine pain syndromes.</a> Veterinary Anesthesia and Analgesia. 44 (3), 600-614 (2017).</li><li>Backonja, 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=Value+of+quantitative+sensory+testing+in+neurological+and+pain+disorders:+NeuPSIG+consensus.">Value of quantitative sensory testing in neurological and pain disorders: NeuPSIG consensus.</a> Pain. 154 (9), 1807-1819 (2013).</li><li>Wylde, V., Palmer, S., Learmonth, I. D., Dieppe, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somatosensory+abnormalities+in+knee+OA.">Somatosensory abnormalities in knee OA.</a> Rheumatology. 51 (3), 535-543 (2012).</li></ol>

作者没有利益冲突需要披露。

对于获取反映狗的感官阈值的准确数据至关重要，狗在每次测量中都尽可能平静、放松和适当的位置。之前的一项研究指出，测试环境内外因素的约束或分心的躁动会影响狗对 QST 刺激的反应16。如果狗因卧位或约束而变得激动或分心，则应在进行测量之前给狗时间安定下来;没有很快安顿下来的狗应该从测试过程中短暂休息一下。过度焦虑或因测试程序而感到压力的狗可能会表?...

定量感官测试（QST）评估外部应用刺激引起的反应;它用于评估人类和动物的体感系统的功能1。点状压力或深层压力形式的机械刺激作为斜坡刺激应用。感觉阈值被确定为唤起心理物理反应的力量1.热或冷热刺激可用作斜坡刺激或固定强度刺激。感觉阈值确定为对刺激有反应的温度或反应的潜伏期。使用电子冯弗雷麻醉仪或冯弗雷毛丝测量点状压力感觉阈值，使用手持式压力测高仪测量深压力，使用各种接触式热计确定热感觉阈值。
QST提供有关外周和中枢感觉通路功能的信息，可用于评估各种疾病过程中这些感觉通路的改变（可塑性），特别是那些引起慢性疼痛的疾病过程1。迈斯纳微粒检测点状压力，当刺激强度为有害强度时，感觉由非有害水平的 Aβ 传入纤维和 Aδ 传入纤维传递。深压力由帕西尼亚小体检测并由C传入纤维传递，有害热量由鲁菲尼小体检测并由Aδ和C传入纤维传递，有害寒冷由Krause小体检测并由C传入纤维1，2传递。QST可用于检测这些受体和途径的抑制（敏感性降低，感觉减退）和促进（敏感性增加，感觉过敏）。在狗中，QST 已被用于评估继发于急性脊髓损伤3，4，5、小脑扁桃体扁桃体下疝样畸形和脊髓空洞症6、颅交叉韧带断裂5，7 和骨关节炎 （OA） 的感觉阈值改变8，9，10。此外，一些研究使用QST来评估某些镇痛药6，11，12，13和外科手术提供的疼痛缓解14。这些研究为狗的疼痛感机制提供了重要的见解，例如手术后外周和中枢致敏的证据以及导致慢性疼痛状态的疾病，如颅十字韧带断裂和OA。这些信息可以帮助改善狗疼痛的检测和治疗。
狗的机械和热热 QST 验证研究表明，随着时间的推移，正常狗和患有 OA8、9、15、16 慢性疼痛的狗的 QST 结果具有良好的可行性、可重复性和可靠性。然而，一些研究发现冷热和偶尔的冯弗雷QST1，15，17的重复性和可靠性较差。这些研究使用了不同的设备和方法，但提供了证据表明机械和热热QST是测量狗感觉阈值的准确，半定量方法。然而，注意精确的细节，包括测量的设置，对于优化狗的QST至关重要，因此需要标准化的QST协议。Sanchis-Mora等人详细介绍了机械和冷热QST的感觉阈值检查协议（STEP），但狗对冷热QST或研究中使用的最高克力冯弗雷灯丝没有反应遇到了困难17。以下协议提供了狗的机械和热热QST的标准方法;该协议可以评估正常狗或具有影响体感系统的各种疾病过程的狗的感觉阈值。标准化方案的开发可以允许比较研究的结果和数据荟萃分析，以提高QST在兽医学中的效用。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Electronic von Frey anesthesiometer</td>
			<td>IITC Life Science Inc.</td>
			<td>Item # 23931</td>
			<td>Custom made with a 1000g max force load cell</td>
		</tr>
		<tr>
			<td>Medoc Main Station software</td>
			<td>Medoc</td>
			<td>(supplied with TSA-II)</td>
			<td></td>
		</tr>
		<tr>
			<td>SMALGO: SMall Animal ALGOmeter</td>
			<td>Bioseb</td>
			<td>Model VETALGO</td>
			<td></td>
		</tr>
		<tr>
			<td>TSA-II NeuroSensory Analyzer</td>
			<td>Medoc</td>
			<td>DC 00072 TSA-II</td>
			<td>No longer manufactured - new model is TSA-2 with same probes and same function</td>
		</tr>
	</tbody>
</table>

assessment of sensory thresholds in dogs using mechanical and hot thermal quantitative sensory testing

定量感觉测试 （QST） 用于通过评估对施加的机械和热刺激的反应来评估狗的体感系统功能。QST 用于确定正常狗的感觉阈值，并评估由各种疾病状态引起的外周和中枢感觉通路的改变，包括骨关节炎、脊髓损伤和颅交叉韧带断裂。机械感觉阈值由电子冯弗雷麻醉仪和压力测高仪测量。它们被确定为狗表现出指示有意识刺激感知的反应的力。热热感官阈值是对接触式热化物施加的固定或斜坡温度刺激做出响应的延迟。
遵循一致的方案进行 QST 并注意测试环境、程序和个体研究对象的细节对于获得准确的狗 QST 结果至关重要。尚未详细描述狗QST数据标准化收集的协议。QST 应在安静、无干扰的环境中进行，对狗、QST 操作员和训导员来说都是舒适的。确保狗在每次测量中保持冷静、放松和正确的位置有助于对刺激产生可靠、一致的反应，并使测试过程更易于管理。QST操作员和处理者应该熟悉并熟悉处理狗和解释狗对潜在疼痛刺激的行为反应，以确定测试的终点，减轻压力，并在测试过程中保持安全。

已经进行了机械和热QST以检测研究和客户拥有的狗在各种临床条件下的感觉阈值，包括正常，健康的狗，患有慢性疼痛状况（如OA）的狗，患有急性脊髓损伤的狗，并评估术后疼痛和镇痛药的有效性。尽管在狗身上的QST方面的工作越来越多，但尚未为任何测试方式建立正常的值范围。然而，一些研究已经评估了狗的机械和热QST的可行性和可重复性，显示QST数据是狗感觉阈值的准确测量值<sup class="xr...

Watch this Scientific Journal Video about Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing at JoVE.com

Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing

这项工作描述了机械和热定量感官测试的标准协议，以评估狗的体感系统。使用电子冯弗雷麻醉仪、压力测藻计和热接触热敷物测量感觉阈值。

使用机械和热热定量感官测试评估狗的感官阈值

Quantitative sensory testing (QST) is used to evaluate the function of the somatosensory system in dogs by assessing the response to applied mechanical ...

assessment-sensory-thresholds-dogs-using-mechanical-hot-thermal

Research

JoVE Journal

Neuroscience

2.1K 次观看.  North Carolina State University. 这项工作描述了机械和热定量感官测试的标准协议，以评估狗的体感系统。使用电子冯弗雷麻醉仪、压力测藻计和热接触热敷物测量感觉阈值。

视频: Assessment of Sensory Thresholds in Dogs Using Mechanical and Hot Thermal Quantitative Sensory Testing

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (&alpha;, &beta;I, &beta;II and &gamma;), novel type I PKCs (&#949; and &eta;), novel type II PKCs (&delta; and &theta;), and atypical PKCs (&zeta; and &iota;). The classical PKCs are activated by Ca2+ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca2+-independent. The atypical PKCs are activated by neither Ca2+ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation 1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail 3. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article 4, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.

In this video, we demonstrate visualization of PKC translocation in living cells using fluorescently tagged PKCs.

在Sf9细胞，并在海兔感觉神经元PKC移位实时成像

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

科研

神经科学

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

在完整的感觉器官前体细胞的活细胞成像果蝇蛹

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the olfactory bulb (OB). The molecular mechanisms of OSN axon growth and targeting are not well understood. Manipulating gene expression and subsequent 
visualizing of single OSN axons and their terminal arbor morphology have thus far been challenging. To study gene function at the single cell level within a specified 
time frame, we developed a lentiviral based technique to manipulate gene expression in OSNs in vivo. Lentiviral particles are delivered to OSNs by microinjection 
into the olfactory epithelium (OE). Expression cassettes are then permanently integrated into the genome of transduced OSNs. Green fluorescent protein expression 
identifies infected OSNs and outlines their entire morphology, including the axon terminal arbor. Due to the short turnaround time between microinjection and 
reporter detection, gene function studies can be focused within a very narrow period of development. With this method, we have detected GFP expression within as 
few as three days and as long as three months following injection. We have achieved both over-expression and shRNA mediated knock-down by lentiviral microinjection. 
This method provides detailed morphologies of OSN cell bodies and axons at the single cell level in vivo, and thus allows characterization of candidate gene function 
during olfactory development.

Lentivirus-mediated Genetic Manipulation and Visualization of Olfactory Sensory Neurons in vivo

We present a lentiviral technique for genetic manipulation and visualization of single olfactory sensory neuron axon and its terminal arborization in vivo.

嗅觉感官神经元的慢病毒介导的遗传操作和可视化在体内</em

Development of a precise olfactory circuit relies on accurate projection of olfactory sensory neuron (OSN) axons to their synaptic 
targets in the ...

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic development and axonal wiring. Recently the dendritic arborization (DA) sensory neurons of the Drosophila larval peripheral nervous system (PNS) have become powerful genetic models in which to elucidate both general and class-specific mechanisms of neuron differentiation.
There are four main DA neuron classes (I-IV)1. They are named in order of increasing dendrite arbor complexity, and have class-specific differences in the genetic control of their differentiation2-10. The DA sensory system is a practical model to investigate the molecular mechanisms behind the control of dendritic morphology11-13 because: 1) it can take advantage of the powerful genetic tools available in the fruit fly, 2) the DA neuron dendrite arbor spreads out in only 2 dimensions beneath an optically clear larval cuticle making it easy to visualize with high resolution in vivo, 3) the class-specific diversity in dendritic morphology facilitates a comparative analysis to find key elements controlling the formation of simple vs. highly branched dendritic trees, and 4) dendritic arbor stereotypical shapes of different DA neurons facilitate morphometric statistical analyses.
DA neuron activity modifies the output of a larval locomotion central pattern generator14-16. The different DA neuron classes have distinct sensory modalities, and their activation elicits different behavioral responses14,16-20. Furthermore different classes send axonal projections stereotypically into the Drosophila larval central nervous system in the ventral nerve cord (VNC)21. These projections terminate with topographic representations of both DA neuron sensory modality and the position in the body wall of the dendritic field7,22,23. Hence examination of DA axonal projections can be used to elucidate mechanisms underlying topographic mapping7,22,23, as well as the wiring of a simple circuit modulating larval locomotion14-17.
We present here a practical guide to generate and analyze genetic mosaics24 marking DA neurons via MARCM (Mosaic Analysis with a Repressible Cell Marker)1,10,25 and Flp-out22,26,27 techniques (summarized in Fig. 1).

Morphological Analysis of Drosophila Larval Peripheral Sensory Neuron Dendrites and Axons Using Genetic Mosaics

The dendritic arborization sensory neurons of the Drosophila larval peripheral nervous system are useful models to elucidate both general and neuron class-specific mechanisms of neuron differentiation. We present a practical guide to generate and analyze dendritic arborization neuron genetic mosaics.

形态分析果蝇幼虫外周感觉神经元树突和轴突使用遗传马赛克

Nervous system development requires the correct specification of neuron position and identity, followed by accurate neuron class-specific dendritic ...

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this information to the brain. As such, determining how olfactory information is transferred to the brain, modifying both perception and behavior, merits investigation. Interestingly, there is emerging evidence that cellular transduction and transcriptional factors are involved in the diversification of olfactory receptor neuron. Here we provide a robust whole mount immunological labeling method to assay in vivo olfactory receptor neuron organization. Using this method, we identified all olfactory receptor neurons with anti-ELAV antibody, a known pan-neural marker and Or49a-mCD8::GFP, an olfactory receptor neuron specifically expressed in Nba neuron using anti-GFP antibody.

Whole Mount Immunolabeling of Olfactory Receptor Neurons in the Drosophila Antenna

Herein we describe the process of whole mount immunostaining of Drosophila antennae, which enables us to better understand the molecular mechanisms involved in the diversification of olfactory receptor neurons (ORN)s.

嗅觉受体神经元在全山免疫标记<em&gt;果蝇</em&gt;天线

Odorant molecules bind to their target receptors in a precise and coordinated manner. Each receptor recognizes a specific signal and relays this ...

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

吗啡诱发的痛觉过敏和镇痛耐受小鼠中利用热和机械伤害性的评估方式

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Production and Isolation of Axons from Sensory Neurons for Biochemical Analysis Using Porous Filters

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is required to shape functional neuronal circuits. The protocol described here explains how to use cell culture inserts bearing a porous membrane (filter) to obtain large amounts of pure axonal preparations suitable for examination by conventional biochemical or immunocytochemical techniques. The functionality of these filter inserts will be demonstrated with models of developmental pruning and Wallerian degeneration, using explants of embryonic dorsal root ganglion. Axonal integrity and function is compromised in a wide variety of neurodegenerative pathologies. Indeed, it is now clear that axonal dysfunction appears much earlier in the course of the disease than neuronal soma loss in several neurodegenerative diseases, indicating that axonal-specific processes are primarily targeted in these disorders. By obtaining pure axonal samples for analysis by molecular and biochemical techniques, this technique has the potential to shed new light into mechanisms regulating the physiology and pathophysiology of axons. This in turn will have an impact in our understanding of the processes that drive degenerative diseases of the nervous system.

This article describes a neuronal culture system that can be used to obtain large quantities of pure axonal samples for biochemical and immunocytochemical analyses. This technique will allow an improved understanding of normal axonal physiology and signaling pathways leading to neurodegeneration.

生产和轴突隔离从感觉神经元的生化分析使用多孔过滤器

Neuronal axons use specific mechanisms to mediate extension, maintain integrity, and induce degeneration. An appropriate balance of these events is ...

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an excellent model system for such investigations due to its complex behaviors, powerful genetic techniques, and compact nervous system. Laboratory behavioral assays have long been used with Drosophila to simulate properties of the natural environment and study the neural mechanisms underlying the corresponding behaviors (e.g. phototaxis, chemotaxis, sensory learning and memory)1-3. With the recent availability of large collections of transgenic Drosophila lines that label specific neural subsets, behavioral assays have taken on a prominent role to link neurons with behaviors4-11. Versatile and reproducible paradigms, together with the underlying computational routines for data analysis, are indispensable for rapid tests of candidate fly lines with various genotypes. Particularly useful are setups that are flexible in the number of animals tested, duration of experiments and nature of presented stimuli. The assay of choice should also generate reproducible data that is easy to acquire and analyze. Here, we present a detailed description of a system and protocol for assaying behavioral responses of Drosophila flies in a large four-field arena. The setup is used here to assay responses of flies to a single olfactory stimulus; however, the same setup may be modified to test multiple olfactory, visual or optogenetic stimuli, or a combination of these. The olfactometer setup records the activity of fly populations responding to odors, and computational analytical methods are applied to quantify fly behaviors. The collected data are analyzed to get a quick read-out of an experimental run, which is essential for efficient data collection and the optimization of experimental conditions.

Olfactory Behaviors Assayed by Computer Tracking Of Drosophila in a Four-quadrant Olfactometer

We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.

通过计算机跟踪检测大鼠嗅觉行为<em&gt;果蝇</em&gt;在四象限嗅

A key challenge in neurobiology is to understand how neural circuits function to guide appropriate animal behaviors. Drosophila melanogaster is an ...

Dynamic Quantitative Sensory Testing to Characterize Central Pain Processing

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain processing is present in many chronic pain conditions and can influence responses to specific therapies. Thus, the ability to precisely describe the state of central pain processing has profound clinical significance in both prognosis and prediction. Because it is not practical to record neuronal firings directly in the human spinal cord, surrogate behavior tests become an important tool to assess the state of central pain processing. Dynamic QST is one such test, and can probe both the ascending facilitation and descending modulation of incoming nociceptive signals via TS and CPM, respectively. Due to the large between-individual variability in the sensitivity to noxious signals, standardized TS and CPM tests may not yield any meaningful data in up to 50% of the population due to floor or ceiling effects. We present methodologies to individualize TS and CPM so we can capture these measures in a broader range of individuals than previously possible. We have used these methods successfully in several studies at the lab, and data from one ongoing study will be presented to demonstrate feasibility and potential applications of the methods.

By assessing pain in response to repetitive or different types of standardized stimuli, dynamic quantitative sensory testing (QST) can reveal changes in the central processing of pain. We present methods to optimize and individualize two dynamic QST measures: temporal summation (TS) and conditioned pain modulation (CPM).

动态定量感觉测试表征中央疼痛处理

Central facilitation and modulation of incoming nociceptive signals play an important role in the perception of pain. Disruption in central pain ...

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog (Rana catesbeiana)

The study of hearing and balance rests upon insights drawn from biophysical studies of model systems. One such model, the sacculus of the American bullfrog, has become a mainstay of auditory and vestibular research. Studies of this organ have revealed how sensory cells hair can actively detect signals from the environment. Because of these studies, we now better understand the mechanical gating and localization of a hair cell&#39;s transduction channels, calcium&#39;s role in mechanical adaptation, and the identity of hair cell currents. This highly accessible organ continues to provide insight into the workings of hair cells. Here we describe the preparation of the bullfrog&#39;s sacculus for biophysical studies on its hair cells. We include the complete dissection procedure and provide specific protocols for the preparation of the sacculus in specific contexts. We additionally include representative results using this preparation, including the calculation of a hair bundle&#39;s instantaneous force-displacement relation and measurement of a bundle&#39;s spontaneous oscillation.

Physiological Preparation of Hair Cells from the Sacculus of the American Bullfrog Rana catesbeiana

The American bullfrog&#39;s (Rana catesbeiana) sacculus permits direct examination of hair-cell physiology. Here the dissection and preparation of the bullfrog&#39;s sacculus for biophysical studies is described. We show representative experiments from these hair cells, including the calculation of a bundle&#39;s force-displacement relation and measurement of its unforced motion.