肯塔基大学生物系，本科课程和艺术与科学学院办公室支持。

1。简介最近期的小龙虾肠进行全面的概述，遵守在论文中，由芭芭拉博士E. Musolf"（2007年，美国佐治亚州亚特兰大，佐治亚州立大学，），主要集中在-羟色胺的调制和支配（Musolf等人，2009 ）。甲壳类hindguts首先研究了100年前Alexandrowicz，在比较解剖学的先驱（Alexandrowicz 1909年），描述了他们的神经支配的各个方面。继续研究多年来，确定肌肉层和结构类型，解决的整体功能，在整个动物渗透调节肠和各种化合物的研究肠收缩的调节控制（见审查，2007年由Musolf）。有趣的是，要注意肛门的后肠部分的行为不仅要排出粪便，但也采取了从水的渗透调节环境。这个地区的肠道可以进行向前或逆蠕动，根据动物的需求。 Alexandrowicz（1909年）确定的两个神经丛，支配的甲壳类动物的肠，一种内在的神经和外丛，后来被证明是用于识别和表征神经递质的丰富来源。在1950年的博士恩斯特弗洛里开始了一系列小龙虾肠药理研究表明收缩是由乙酰胆碱及其相关化合物，并通过肾上腺素和去甲肾上腺素（弗洛里，1954年）调制。弗洛里，被称为抑制性物质的发现者"因子- I"，研究这种物质上的各项准备工作，包括在小龙虾的胃肠道系统（弗洛里，1961年）的影响。因子- I后来被证明是GABA的。因此，小龙虾肠突触抑制的早期研究中发挥重要作用。 发现的GABA之后，其他公认的发射机也显示肠丛引起的生理反应。这种发射器，包括多巴胺（Elekes 等 1988年; Elofsson 等1968年），proctolin（RYLPG - OH;名士等 1997年）两个orcokinin肽（NFDEIDRSGFGFN和AFDEIDRSGFGFN; Bungart 等1994;。Dircksen等 2000） ，orcomyotropin（FDAFTTGFamide; Dircksen 等 2000）和一个myosuppressin肽（名士等1997;。最有可能的pQDLDHVFLRFamide，比照Stemmler 等 2007年）。这些物质中的每个调节自发性肠收缩，除GABA的似乎是兴奋。肠也响应谷氨酸和quisqualate（琼斯，1962;错误等，2003），但谷氨酸能神经支配的明确证据尚未见报道。细胞内信使，调解各种变送器的影响主要是未开发的，但有证据表明多巴胺的能力，以加强宫缩（Knotz名士，1995年）中cAMP参与。 虽然甲壳类动物的肠合同自发去神经后，这种收缩是典型的薄弱和混乱（威尔士，1982年; Winlow Laverack，1972a）。蠕动运动，需要协调的中枢神经系统的电机的输出，显然在过去的腹神经节（Winlow＆Laverack，1972a，B，C）始发。小龙虾，电机的输出进行肠通过的第 7 次腹部根，其中包含75轴突，其胞体在过去的腹神经节（Kondoh久田荣，1986年）的本地化。至少出现一些肽肠神经丛神经元在过去的腹神经节（Dircksen等人2000年，名士等1991b; Siwicki主教，1986年）的提供，但多巴胺是提供更多的神经元前神经节（名士等。1991a）。由于电机输出通过的第 7 次腹部根大，协调的收缩是必要的，它很可能部分或所有上面列出的假定发射机蠕动的方式在一些贡献。然而，他们的相对贡献尚不清楚。由圆形和纵肌（名士与李，2002），学习他们的影响，可能获得一些有识之士。 除了控制未消化食物的运动，与甲壳类动物肠相关的神经丛，似乎发挥了重要的内分泌功能与蜕皮有关。蟹肠，Carcinus maenas，包含内分泌细胞释放甲壳动物高血糖激素和其相关的前体肽在蜕皮（ 韦伯斯特等人，2000），建议在吸水作用和肿胀与蜕皮。因此，甲壳类动物的肠参与了几个重要的生理过程。 这份报告主要是为先进的高中和本科学生在生理学课程，在实验中参与的教学工具。的意义，以及背后的肠合同和功能可以由学生解决的潜在机制。将用于药理剂，神经递质和神经激素，剂量反应曲线，将兴建，这将从事学生如何获取和解释的生理和药理数据。一个一般的理解受体激动剂和拮抗剂方面的功能也可以实现。学生还将学习目前在图形化的形式进行统计分析的数据。 这是很容易的解剖和记录小龙虾肠收缩。在解剖的准备，肠道中很容易暴露于外源性物质。蠕动活动的改变，可以监视目视或用一个力传感器，还可以监视的收缩力。由于它的简单性和可靠性作为生物活性制剂，小龙虾肠仍然是调查有关调制电机输出和肌肉的收缩，蠕动，及受体功能的机制的许多研究问题非常有用。后肠也可用于测试杀虫剂，crustaceancides，并为具体的行动机制的污染物。 2。方法 2.1）材料<ul><li>小龙虾</li><li>小龙虾生理盐水</li><li>清扫仪器（粗精剪刀，粗与细的镊子） </li><li> Sylgard平底培养皿</li><li>钢解剖引脚</li><li>烧杯（持有化学解决方案） </li><li>封口膜（覆盖和混合解决方案） </li></ul> 2.2）夹层<ol><li>龙虾（ 克氏螯虾 ）测量体长6-10厘米，应在冰上放置5至10分钟，麻醉开始前清扫动物。 </li><li>从后面的爪子，用一只手握住麻醉的小龙虾。很快，削减从眼窝中间的头两侧，然后斩首小龙虾（注:从筹备的血液会被粘当它变干，所以洗的工具时完成）。 <img src="/files/ftp_upload/2324/2324fig1.jpg" alt="图1" /> 图1:小龙虾斩首</li><li>切断螯和腿走路。 </li><li>切断左，右的尾翼，只留下中间的尾鳍（uropod）。 </li><li>在背侧的小龙虾腹部切背角质层的左侧和右侧。 <img src="/files/ftp_upload/2324/2324fig2.jpg" alt="图2" /> 图2:背角质层去除</li><li>横向剪切背侧的角质层，确保切割浅，以防止损害胃肠，然后取出背腹角质层下段。 </li><li>解剖小龙虾放入一个Sylgard内衬的菜。 </li><li>垂尾尖端脚的小龙虾的菜。有人可能会使用更多的引脚上胃肠两侧必要的身体。 </li><li>小龙虾生理盐水填充盘覆盖胃肠确保不断扑灭胃肠使用吸管生理盐水。生理盐水的修改后的范Harreveld解决方案（1936年），这是用205毫米氯化钠，5.3毫米氯化钾，氯化钙2 · 2H 2 O 13.5毫米，2.45 毫米氯化镁2 · 6H 2 O 5毫米的HEPES和调整pH至7.4。解剖小龙虾应该会出现如图3所示。 <img src="/files/ftp_upload/2324/2324fig3.jpg" alt="图3" /> 图3:解剖与GI完整的小龙虾。 </li></ol> 2.3）实验在动物2.3.1）宫缩<ol><li>有解决方案的化合物进行测试准备和在相同的温度洗澡盐水。可能使用的个性化解决方案的5 - 羟色胺（100纳米，1microM），谷氨酸（1microM），和小龙虾生理盐水开始进行测试的物质多巴胺（1microM）。 </li><li>允许解剖坐了大约10分钟的小龙虾，以小龙虾的解决方案，使其能够调整夹层和盐水接触的冲击。从肠缓慢蠕动收缩，开始出现。 </li><li>一旦宫缩开始，记录收缩30秒（注意收缩，发生的类型:即蠕动类型，或痉挛，任何蠕动波方向）发生的数量。 表1:小龙虾盐水收缩 <table border="1"><tbody><tr><td> 收缩的数目</td><td> 宫缩类型 </td></tr><tr><td></td><td></td></tr></tbody></table></li><li>使用一个PIpette内拆除的小龙虾的腹部暴露腔，生理盐水，并应用生理盐水包含的实质内容进行审查直接到肠。 </li><li>后立即加入该解决方案中，创纪录的30秒后发生的收缩，并注意发生的收缩（即蠕动，或痉挛）的类型。 表2:与接触的化合物的收缩 <table border="1"><tbody><tr><td> 复合测试 </td><td> 集中 </td><td> 收缩的数目</td><td> 宫缩类型 </td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr><tr><td></td><td></td><td></td><td></td></tr></tbody></table></li><li>记录测试物质的反应后，立即与正常的小龙虾生理盐水冲洗胃肠几次。让我们站在5分钟的准备，而约每30秒小龙虾生理盐水漂洗。 </li><li>使用移液器，将一些包含在未来的化合物进行检查，或直接到肠测试的最后物质的不同浓度的盐水。最好是先用较低的浓度和工作之一的浓度较高。 </li><li>后立即加入每一个新的物质或每一个新的浓度，记录的30秒后发生的收缩，并注意宫缩的类型。 </li></ol> 2.3.2）记录在切除的准备收缩力量<img src="/files/ftp_upload/2324/2324fig4.jpg" alt="图4" /> 图4:安装程序<ol><li>将传感器的桥梁POD。 </li><li> PowerLab系统26T连接的桥梁POD。 </li><li> PowerLab系统26T连接到计算机上的U​​SB端口。 </li><li> LabChart7打开，通过点击labchart7桌面上的图标。 <ul><li> LabChart欢迎中心中会弹出打开。关闭它。 </li><li>点击安装程序</li><li>点击通道设置。渠道的数量更改为1（盒左下角），按下OK。 </li><li>在图表的左上角设置每秒约2K的周期。设置伏特（Y轴），约500或200毫伏。 </li><li>通道1上按一下右边的图表。点击输入放大器。确保设置:单端AC耦合，并反转（反转的信号，如果需要的话），抗混叠，检查。 </li><li>要开始录制按开始。 </li></ul></li><li>拿起准备和减少胃肠道之间的胸部和腹部的时刻。然后小心地切断周围的尾部的尾节的一部分。删除从消化道解剖小龙虾放入Sylgard菜。有一个血管，沿背消化道方面运行。小心地拉出远离消化道。倒到准备新鲜的小龙虾生理盐水。 </li><li>放置在附近的解剖小龙虾钳力传感器。 </li><li>钩在小龙虾胃肠道的力传感器，如图5所示。 <img src="/files/ftp_upload/2324/2324fig5.jpg" alt="图5" /> 图5:钩力传感器连接到孤立的肠生理盐水中的小龙虾</li><li>等待，直到胃肠开始收缩，并推开始的LabChart7。 </li><li>让程序运行约20秒。按下"停止"，并添加注释标记，"吊只"。填写表4。 表四:化合物收缩 <table border="1"><tbody><tr><td> 收缩的数目</td><td> 收缩幅度（MV） </td></tr><tr><td></td><td></td></tr></tbody></table></li><li>添加感兴趣的测试化合物，并立即按下"开始"上LabChart7"。 </li><li>让程序运行约20秒。按下"停止"，并添加评论，直接添加了什么物质，其浓度的图表文件。请填写下面的表格。 表5:_________复合收缩 <table border="1"><tbody><tr><td> 收缩的数目</td><td> 收缩幅度（MV） </td></tr><tr><td></td><td></td></tr></tbody></table></li><li>使用吸管的小龙虾生理盐水冲洗掉的准备。 </li><li>添加其他物质或不同浓度，以类似的方式。立即按下"开始"上LabChart7。 </li><li>让程序运行约20秒。按下"停止"，并直接在图表上文件中添加一条注释和标签，说明在Compound使用和浓度。 </li></ol> 3。数据分析<ol><li>从2.3.1的实验，图中的每个溶液（生理盐水，血清素，谷氨酸）随时间的收缩。说明任何趋势。 </li><li>从2.3.2实验，图中的每个解决方案的收缩的数量（生理盐水，血清素，谷氨酸） - 在mV的收缩幅度。说明任何趋势。 </li></ol> 3.1）测量的收缩速度和力量索引上的收缩率可以衡量的时间，从开始一个偏转未来的开始或数超过一两分钟的总挠度。的值，然后可以在每分钟或每秒取决于他们正在迅速发生收缩。 索引收缩力的一个相对值可以用来比较不同的条件。所保存的文件，可以使用的"M"标志和移动基线。然后使用光标移动到峰值的偏转，并注意屏幕右上角列为一个delta值（从M的差异，以峰）值。请注意光标需要保持固定为衡量的变化。然后移动的M下一波形式的利益和重复测量。

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J., Orchard, I., TeBrugge, V., Skerrett, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+two+FMRFamide-related+peptides+from+crayfish+pericardial+organs.">Isolation of two FMRFamide-related peptides from crayfish pericardial organs.</a> Peptides. 14, 137-143 (1993).</li><li>Musolf, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonergic+modulation+of+the+crayfish+hindgut:+Effects+on+hindgut+contractility+and+regulation+of+serotonin+on+hindgut.">Serotonergic modulation of the crayfish hindgut: Effects on hindgut contractility and regulation of serotonin on hindgut.</a> Biol Bull. , (2007).</li><li>Musolf, B. E., Spitzer, N., Antonsen, B. L., Edwards, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonergic+modulation+of+crayfish+hindgut.+Biol+Bull.+217(1),+50-64+(2009).">Serotonergic modulation of crayfish hindgut. Biol Bull. 217(1), 50-64 (2009).</a> Biol Bull. 217, 50-64 (2009).</li><li>Shuranova, Z. P., Burmistrov, Y. M., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+hundred+years+ago+and+now:+A+short+essay+on+the+study+of+the+crustacean+hindgut.+(Vor+hundert+Jahren+und+nun:+Eine+kurze+Geschichte+von+die+Forschung+des+Hinterdarmes+der+Crustaceen.">A hundred years ago and now: A short essay on the study of the crustacean hindgut. (Vor hundert Jahren und nun: Eine kurze Geschichte von die Forschung des Hinterdarmes der Crustaceen.</a> Crustaceana. 76, 755-670 (2003).</li><li>Shuranova, Z. P., Burmistrov, Y. M., Strawn, J. R., Cooper, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+an+Autonomic+Nervous+System+in+Decapod+Crustaceans.">Evidence for an Autonomic Nervous System in Decapod Crustaceans.</a> International Journal of Zoological Research. 2 (3), 242-283 (2006).</li><li>Siwicki, K. K., Bishop, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+proctolinlike+immunoreactivity+in+the+nervsou+systems+of+lobster+and.">Mapping of proctolinlike immunoreactivity in the nervsou systems of lobster and.</a> 243, 435-435 (1986).</li><li>Stemmler, E. A., Cashman, C. R., Messinger, D. I., Gardner, N. P., Dickinson, P. S., Christie, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-mass-resolution+direct-tissue+MALDI-FTMS+reveals+broad+conservation+of+three+neuropeptides+(APSGFLGMRamide,+GYRKPPFNGSIFamide+and+pQDLDHVFLRFamide)+across+members+of+seven+decapod+crustaean+infraorders.">High-mass-resolution direct-tissue MALDI-FTMS reveals broad conservation of three neuropeptides (APSGFLGMRamide, GYRKPPFNGSIFamide and pQDLDHVFLRFamide) across members of seven decapod crustaean infraorders.</a> Peptides. 28, 2104-2115 (2007).</li><li>Wales, W., Sandeman, D. C., Atwood, H. L. <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+mouthparts+and+gut.">Control of mouthparts and gut.</a> The Biology of Crustacea. 4, 165-191 (1982).</li><li>Webster, S. G., Dircksen, H., Chung, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocrine+cells+in+the+gut+of+the+shore+crab+Carcinus+maenas+immunoreactive+to+crustacean+hyperglycaemic+hormone+and+its+precursor-related+peptide.">Endocrine cells in the gut of the shore crab Carcinus maenas immunoreactive to crustacean hyperglycaemic hormone and its precursor-related peptide.</a> J. Exp. Biol. 300, 193-205 (2000).</li><li>Winlow, W., Laverack, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+hindgut+motility+in+the+lobster+Homarus+gammarus+(L.).+1.+Analysis+of+hindgut+movements+and+receptor+activity.">The control of hindgut motility in the lobster Homarus gammarus (L.). 1. Analysis of hindgut movements and receptor activity.</a> Mar. Behav. Physiol. 1, 1-28 (1972).</li><li>Winlow, W., Laverack, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+hindgut+motility+in+the+lobster+Homarus+gammarus+(L.).+2.+Motor+output.">The control of hindgut motility in the lobster Homarus gammarus (L.). 2. Motor output.</a> Mar. Behav. Physiol. 1, 29-47 (1972).</li><li>Winlow, W., Laverack, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+control+of+hindgut+motility+in+the+lobster+Homarus+gammarus+(L.).+3.+Structure+of+the+sixth+abdominal+ganglion+(6+A.G.)+and+associated+ablation+and+microelectrode+studies.">The control of hindgut motility in the lobster Homarus gammarus (L.). 3. Structure of the sixth abdominal ganglion (6 A.G.) and associated ablation and microelectrode studies.</a> Mar. Behav. Physiol. 1, 93-121 (1972).</li><li>Wrong, A. D., Sammahin, M., Richardson, R., Mercier, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+properties+of+glutamate+receptors+associated+with+the+crayfish+hindgut.">Pharmacological properties of glutamate receptors associated with the crayfish hindgut.</a> J. Comp. Physiol. 189, 371-378 (2003).</li></ol>

在相关的电影和文字所提供的细节描 ​​述所需的关键步骤，在小龙虾的肠活动记录在原位，以及在体外。我们报告的目标之一是增加的意识，在学生跑教生理学和药理学的基本概念的调查实验室，该制剂的潜力。 这些准备工作，可用于研究实验的问题，这将导致更好地了解了肠生理功能。蠕动波及其逆转的调控的内在机制仍然不知道。较高的控制整个消化道的机制还没有完全理解。较高的中心是如何整合他们的活动与自主输出，直接控制胃肠道系统的问题仍然是一个开放的调查面积（Shuranova等，2006） 。此外，渗透调节能力的甲壳动物的肠和蜕皮和环境压力的过程中渗透调节功能没有得到充分的阐述。还有许多问题等待在这准备的答案。

<ol><li>小龙虾（ 克氏螯虾 ）。阿查法拉亚生物供应公司，Raceland，洛杉矶，美国。 </li><li>小龙虾生理盐水（标准）:范Harreveld的解决方案（1936年）修改。 （205毫米）氯化钠氯化钾5.3; 13.5氯化钙2 · 2H 2 O; 2.45氯化镁2 · 6H 2 O 5 HEPES和调整pH至7.4。羟色胺，谷氨酸和多巴胺是在小龙虾生理盐水。所有化学品都是从Sigma公司（圣路易斯，密苏里州）。 </li><li>解剖工具:精细＃5镊子，精剪，刀刀座，昆虫引脚＃26002-20（所有获得精细科学的工具（美国）公司，373 - G的复古公园径，福斯特市，CA 94404-1139） </li><li> Sylgard平底培养皿</li><li>烧杯（持有化学溶液） </li><li>电信号记录在一个PowerLab系统26T接口计算机（ADInstruments，CO，美国科罗拉多州的斯普林斯，）线。我们使用标准软件名为图表或范围ADInstruments。 <li>桥的力传感器（力传感器的不同类型需要不同的桥豆荚）吊舱。 </li><li> MLT0402（2克）力传感器的研究级的视频显示。 MLT 0210 / D转换（10毫克25克）和MLT 500 /（0 - 500G）的教育等级的换能器（ADInstruments）。 </li></ol>

physiological experimentation with the crayfish hindgut: a student laboratory exercise

该报告的目的是描述准备的小龙虾肠的解剖技术，并演示如何使一个力传感器的生理录音监控收缩的力量。此外，我们演示了如何在视觉监控蠕动的活性，可作为各种肽的生物活性，生物胺神经递质。这种准备是适合学生在生理和药理概念展示给学生实验室。这个准备工作已经在使用了100多年，它仍然提供了作为一个负责调查的蠕动节奏的产生和调节，并描述的内在机制，其调制的模型。药理实验和受体的分型，50年前开始对肠仍然有助于今天的研究。这个强大的准备非常适合于训练学生在生理学和药理学。

Watch this Scientific Journal Video about Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise at JoVE.com

Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

在这份报告中，我们展示了可以用于研究的小龙虾肠的生物学技术。我们展示了如何剖析一个小龙虾腹部和研究相关的解剖学，生理学和调制的活动。蠕动活动和收缩力测力传​​感器使用。

生理实验与小龙虾肠:学生实验室的工作

The purpose of the report is to describe dissection techniques for preparing the crayfish hindgut and to demonstrate how to make physiological recordings ...

physiological-experimentation-with-crayfish-hindgut-student

Research

JoVE Journal

Neuroscience

15.7K 次观看.  University of Kentucky. 在这份报告中，我们展示了可以用于研究的小龙虾肠的生物学技术。我们展示了如何剖析一个小龙虾腹部和研究相关的解剖学，生理学和调制的活动。蠕动活动和收缩力测力传​​感器使用。

视频: Physiological Experimentation with the Crayfish Hindgut: A Student Laboratory Exercise

Recording Behavioral Responses to Reflection in Crayfish

Social behavior depends on sensory input from the visual, mechanical and olfactory systems. One important issue concerns the relative roles of each sensory modality in guiding behavior. The role of visual inputs has been examined by isolating visual stimuli from mechanical and chemosensory stimuli. In some studies (Bruski &amp; Dunham, 1987: Delgado-Morales et al., 2004) visual inputs have been removed with blindfolds or low light intensity, and effects of remaining sensory modalities have been elucidated. An alternative approach is to study the effects of visual inputs in the absence of any appropriate mechanical and chemosensory cues. This approach aims to identify the exclusive role of visual inputs. 
We have used two methods to provide visual stimuli to crayfish without providing chemical and mechanical cues. In one method, crayfish are videotaped in an aquarium where half of the walls are covered in mirrors to provide a reflective environment, and the other half are covered in a non-reflective (matte finish) plastic. This gives the crayfish a choice between reflective and non-reflective environments. The reflective environment provides visual cues in the form of reflected images of the crayfish as it moves throughout half of the tank; these visual cues are missing from the non-reflective half of the tank. An alternative method is to videotape the behavior of crayfish in an aquarium separated by a smaller chamber at each end, with a crayfish in one small chamber providing visual cues and an inert object in the opposite small chamber providing visual input from a non-moving, non-crayfish source. 
Our published results indicate that responses of crayfish to the reflective environment depend on socialization and dominance rank. Socialized crayfish spent more time in the reflective environment and exhibited certain behaviors more frequently there than in the non-reflective environment; isolated crayfish showed no such differences. Crayfish that were housed in same-sex pairs developed a social rank of either dominant or subordinate. Responses to reflection differed between dominant and subordinate crayfish (May &amp; Mercier, 2006; May &amp; Mercier, 2007). Dominant crayfish spent more time on the reflective side, entered reflective corners more frequently and spent more time in reflective corners compared to the non-reflective side. Subordinate crayfish walked in reverse more often on the reflective side than on the non-reflective side. Preliminary data suggest similar effects from visual cues provided by a crayfish in a small adjoining chamber (May et al., 2008).

We have developed two methods for studying effects of visual cues on behavior in the absence of tactile and chemical cues. One method involves videotaping responses of crayfish to reflective walls in an aquarium; the other examines effects of visual inputs provided by a live crayfish behind a transparent partition.

小龙虾的反思记录行为反应

Social behavior depends on sensory input from the visual, mechanical and olfactory systems.  One important issue concerns the relative roles of each ...

科研

神经科学

A Procedure for Studying the Footshock-Induced Reinstatement of Cocaine Seeking in Laboratory Rats

The most insidious aspect of drug addiction is the high propensity for relapse. Animal models of relapse, known as reinstatement procedures, have been used extensively to study the neurobiology and phenomenology of relapse to drug use. Although procedural variations have emerged over the past several decades, the most conventional reinstatement procedures are based on the drug self-administration (SA) model. In this model, an animal is trained to perform an operant response to obtain drug. Subsequently, the behavior is extinguished by withholding response-contingent reinforcement. Reinstatement of drug seeking is then triggered by a discrete event, such as an injection of the training drug, re-exposure to drug-associated cues, or exposure to a stressor 1.
Reinstatement procedures were originally developed to study the ability of acute non-contingent exposure to the training drug to reinstate drug seeking in rats and monkeys 1, 2. Reinstatement procedures have since been modified to study the role of environmental stimuli, including drug-associated cues and exposure to various forms of stress, in relapse to drug seeking 1, 3, 4.
Over the past 15 years, a major focus of the reinstatement literature has been on the role of stress in drug relapse. One of the most commonly used forms of stress for studying this relationship is acute exposures to mild, intermittent, electric footshocks. The ability of footshock stress to induce reinstatement of drug seeking was originally demonstrated by Shaham and colleagues (1995) in rats with a history of intravenous heroin SA5. Subsequently, the effect was generalized to rats with histories of intravenous cocaine, methamphetamine, and nicotine SA, as well as oral ethanol SA 3, 6.
Although footshock-induced reinstatement of drug seeking can be achieved reliably and robustly, it is an effect that tends to be sensitive to certain parametrical variables. These include the arrangement of extinction and reinstatement test sessions, the intensity and duration of footshock stress, and the presence of drug-associated cues during extinction and testing for reinstatement. Here we present a protocol for footshock-induced reinstatement of cocaine seeking that we have used with consistent success to study the relationship between stress and cocaine seeking.

Animal models of relapse, known as reinstatement procedures, have been used extensively to study the role of stress in relapse to drug seeking. Here, we report on a method for inducing the reinstatement of cocaine seeking in laboratory rats via acute exposures to mild, intermittent electric footshock.

在实验室老鼠研究求可卡因电击诱导恢复原状的程序

The most insidious aspect of drug addiction is the high propensity for relapse.  Animal models of relapse, known as reinstatement procedures, have been ...

Physiological Recordings of High and Low Output NMJs on the Crayfish Leg Extensor Muscle

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor neurons innervating the extensor muscle in the walking leg of a crayfish. Distinct differences are present in the physiology and morphology of the phasic and tonic nerve terminals. The tonic axon contains many more mitochondria, enabling it to take a vital stain more intensely than the phasic axon. The tonic terminals have varicosities, and the phasic terminal is filiform. The tonic terminals are low in synaptic efficacy but show dramatic facilitated responses. In contrast, the phasic terminals are high in quantal efficacy but show synaptic depression with high frequency stimulation. The quantal output is measured with a focal macropatch electrode placed directly over the visualized nerve terminals. Both phasic and tonic terminals innervate the same muscle fibers, which suggests that inherent differences in the neurons, rather than differential retrograde feedback from the muscle, account for the morphological and physiological differentiation.

This article demonstrates how to conduct electrophysiological recordings of synaptic responses on the extensor muscle in the walking leg of a crayfish and how the nerve terminals are visualized to show the gross morphological differences of high- and low-output nerve terminals.

小龙虾腿伸肌高和低输出NMJs生理录音

We explain in detail how to expose and conduct electrophysiological recordings of synaptic responses for high (phasic) and low (tonic) output motor ...

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

膜电位，突触反应，神经回路，神经调节和肌肉组织学用小龙虾:学生实验室练习

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

Muscle Receptor Organs in the Crayfish Abdomen: A Student Laboratory Exercise in Proprioception

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive information for an animal. An additional objective of this experiment is to learn anatomy of the preparation by staining, dissection and viewing of neurons and sensory structures under a dissecting microscope. This is performed by using basic neurophysiological equipment to record the electrical activity from a joint receptor organ and staining techniques. The muscle receptor organ (MRO) system in the crayfish is analogous to the intrafusal muscle spindle in mammals, which aids in serving as a comparative model that is more readily accessible for electrophysiological recordings. In addition, these are identifiable sensory neurons among preparations. The preparation is viable in a minimal saline for hours which is amenable for student laboratory exercises. The MRO is also susceptible to neuromodulation which encourages intriguing questions in the sites of modulatory action and integration of dynamic signals of movements and static position along with a gain that can be changed in the system.

The primary purpose of this experiment is to understand how primary sensory neurons convey information of joint movements and positions as proprioceptive information for an animal. An additional objective of this report is present the anatomy of the preparation by dissection and viewing of neurons under a dissecting microscope.

小龙虾腹部肌肉受体机关:在本体的学生实验室练习

The primary purpose of this experiment is to demonstrate primary sensory neurons conveying information of joint movements and positions as proprioceptive ...

VisualEyes: A Modular Software System for Oculomotor Experimentation

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and dysfunctional brain.1 However, development of a platform to stimulate and store eye movements can require substantial programming, time and costs. Many systems do not offer the flexibility to program numerous stimuli for a variety of experimental needs. However, the VisualEyes System has a flexible architecture, allowing the operator to choose any background and foreground stimulus, program one or two screens for tandem or opposing eye movements and stimulate the left and right eye independently. This system can significantly reduce the programming development time needed to conduct an oculomotor study. The VisualEyes System will be discussed in three parts: 1) the oculomotor recording device to acquire eye movement responses, 2) the VisualEyes software written in LabView, to generate an array of stimuli and store responses as text files and 3) offline data analysis. Eye movements can be recorded by several types of instrumentation such as: a limbus tracking system, a sclera search coil, or a video image system. Typical eye movement stimuli such as saccadic steps, vergent ramps and vergent steps with the corresponding responses will be shown. In this video report, we demonstrate the flexibility of a system to create numerous visual stimuli and record eye movements that can be utilized by basic scientists and clinicians to study healthy as well as clinical populations.

Neural control and cognitive processes can be studied through eye movements. The VisualEyes software allows an operator to program stimuli on two computer screens independently using a simple, custom scripting language. The system can stimulate tandem eye movements (saccades and smooth pursuit) or opposing eye movements (vergence) or any combination.

VisualEyes:一个模块化的软件系统动眼神经实验

Eye movement studies have provided a strong foundation forming an understanding of how the brain acquires visual information in both the normal and ...

Odorant-induced Responses Recorded from Olfactory Receptor Neurons using the Suction Pipette Technique

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex behaviors such as food choice, predator, conspecific and mate recognition and other socially relevant cues. Olfactory receptor neurons (ORNs) are located in the dorsal part of the nasal cavity embedded in the olfactory epithelium. These bipolar neurons send an axon to the olfactory bulb (see Fig. 1, Reisert &amp; Zhao1, originally published in the Journal of General Physiology) and extend a single dendrite to the epithelial border from where cilia radiate into the mucus that covers the olfactory epithelium. The cilia contain the signal transduction machinery that ultimately leads to excitatory current influx through the ciliary transduction channels, a cyclic nucleotide-gated (CNG) channel and a Ca2+-activated Cl- channel (Fig. 1). The ensuing depolarization triggers action potential generation at the cell body2-4.
In this video we describe the use of the "suction pipette technique" to record odorant-induced responses from ORNs. This method was originally developed to record from rod photoreceptors5 and a variant of this method can be found at jove.com modified to record from mouse cone photoreceptors6. The suction pipette technique was later adapted to also record from ORNs7,8. Briefly, following dissociation of the olfactory epithelium and cell isolation, the entire cell body of an ORN is sucked into the tip of a recording pipette. The dendrite and the cilia remain exposed to the bath solution and thus accessible to solution changes to enable e.g. odorant or pharmacological blocker application. In this configuration, no access to the intracellular environment is gained (no whole-cell voltage clamp) and the intracellular voltage remains free to vary. This allows the simultaneous recording of the slow receptor current that originates at the cilia and fast action potentials fired by the cell body9. The difference in kinetics between these two signals allows them to be separated using different filter settings. This technique can be used on any wild type or knockout mouse or to record selectively from ORNs that also express GFP to label specific subsets of ORNs, e.g. expressing a given odorant receptor or ion channel.

Olfactory receptor neurons (ORNs) convert odor signals first into a receptor current that in turn triggers action potentials that are conveyed to second order neurons in the olfactory bulb. Here we describe the suction pipette technique to record simultaneously the odorant-induced receptor current and action potentials from mouse ORNs.

从使用吸吸液管技术的嗅觉受体神经元的气味引起的反应记录

Animals sample the odorous environment around them through the chemosensory systems located in the nasal cavity. Chemosensory signals affect complex ...

Proprioception and Tension Receptors in Crab Limbs: Student Laboratory Exercises

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons responsible for proprioception as they are detecting joint position and movement, and muscle tension. Electrical activity from crustacean proprioceptors and tension receptors is recorded by basic neurophysiological instrumentation, and a transducer is used to simultaneously measure force that is generated by stimulating a motor nerve. In addition, we demonstrate how to stain the neurons for a quick assessment of their anatomical arrangement or for permanent fixation. Staining reveals anatomical organization that is representative of chordotonal organs in most crustaceans. Comparing the tension nerve responses to the proprioceptive responses is an effective teaching tool in determining how these sensory neurons are defined functionally and how the anatomy is correlated to the function. Three staining techniques are presented allowing researchers and instructors to choose a method that is ideal for their laboratory.

Physiological and anatomical techniques are demonstrated to address function and structure for joint proprioceptors and muscle tension receptors in crustacean walking limbs.

本体感觉和张力感受器在蟹四肢:学生实验室练习

The primary purpose of these procedures is to demonstrate for teaching and research purposes how to record the activity of living primary sensory neurons ...

Measuring Oral Fatty Acid Thresholds, Fat Perception, Fatty Food Liking, and Papillae Density in Humans

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty acid receptors housed on taste cells. Previous research has shown that an individual&#39;s oral sensitivity to fatty acid, specifically oleic acid (C18:1) is associated with body mass index (BMI), dietary fat consumption, and the ability to identify fat in foods. We have developed a reliable and reproducible method to assess oral chemoreception of fatty acids, using a milk and C18:1 emulsion, together with an ascending forced choice triangle procedure. In parallel, a food matrix has been developed to assess an individual&#39;s ability to perceive fat, in addition to a simple method to assess fatty food liking. As an added measure tongue photography is used to assess papillae density, with higher density often being associated with increased taste sensitivity.

Oral chemoreception of fatty acids and the association with diet and fatty food preferences may enable the identification of mechanisms involved with the development of obesity and why dietary changes may be difficult for many individuals.

测量口腔脂肪酸阈值，感知脂肪，脂肪食物喜欢的，和乳头密度的人类

Emerging evidence from a number of laboratories indicates that humans have the ability to identify fatty acids in the oral cavity, presumably via fatty ...

Lineage-reprogramming of Pericyte-derived Cells of the Adult Human Brain into Induced Neurons

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis and enable new strategies for in vitro modeling or repairing the diseased brain. Identifying brain-resident non-neuronal cell types amenable to direct conversion into iNs might allow for launching such an approach in situ, i.e. within the damaged brain tissue. Here we describe a protocol developed in the attempt of identifying cells derived from the adult human brain that fulfill this premise. This protocol involves: (1) the culturing of human cells from the cerebral cortex obtained from adult human brain biopsies; (2) the in vitro expansion (approximately requiring 2-4 weeks) and characterization of the culture by immunocytochemistry and flow cytometry; (3) the enrichment by fluorescence-activated cell sorting (FACS) using anti-PDGF receptor-&#946; and anti-CD146 antibodies; (4) the retrovirus-mediated transduction with the neurogenic transcription factors sox2 and ascl1; (5) and finally the characterization of the resultant pericyte-derived induced neurons (PdiNs) by immunocytochemistry (14 days to 8 weeks following retroviral transduction). At this stage, iNs can be probed for their electrical properties by patch-clamp recording. This protocol provides a highly reproducible procedure for the in vitro lineage conversion of brain-resident pericytes into functional human iNs.

Targeting brain-resident cells for direct lineage-reprogramming offers new perspectives for brain repair. Here we describe a protocol of how to prepare cultures enriched for brain-resident pericytes from the adult human cerebral cortex and convert these into induced neurons by retrovirus-mediated expression of the transcription factors Sox2 and Ascl1.

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

Direct lineage-reprogramming of non-neuronal cells into induced neurons (iNs) may provide insights into the molecular mechanisms underlying neurogenesis ...