安东尼仁达支持由维多利亚大学研究生奖学金奖。这项研究是由自然科学和工程研究理事会，加拿大发现格兰特，NARSAD青年研究者奖（RN）的维多利亚基金会 - Myre和王佩瑜SIM基金，创新补助金，加拿大不列颠哥伦比亚省知识发展基金，基金会的支持一个自然的科学和工程研究理事会，加拿大研究工具和仪表格兰特。我们感谢优秀的鼠标畜牧业吉利安麦基，克里斯蒂娜巴恩斯，阿里尔·沙利文，珍妮弗·麦克唐纳和丹尼尔·莫尔加多。

1。泵植入术<ol><li>泵植入前，填写，并准备在Alzet微型渗透泵（Alzet，型号2002年，库珀蒂诺，美国），小心不要引入气泡。这种微型渗透泵的模型提供了0.5微升/小时的速度在14天的解决方案。保证无菌的条件。权衡空和填充泵。在实验（移植后10天）结束，剩余的液体在泵可去除注射器和针头，称重，计算容积泵。 </li><li>泵与控制解决方案包含生理盐水（0.9％W / V，Teknova，S5819，霍利斯特，美国）。准备尼古丁溶液，1 M的原液（ - ） - 尼古丁酒石酸氢盐（Sigma公司，猫＃N5260）生理盐水稀释（0.9％W / V），并通过0.22微米年底注射器过滤器，过滤消毒。之前我们已经在0.4和2毫克/千克/小时（免费的尼古丁基础计算）为10天的尼古丁管理。 </li><li>准备盐水浴三（三生理盐水填充10厘米的培养皿）。一旦该泵已填写并加盖，在每个连续的盐水浴的外壳，以消除任何毒品的痕迹彻底清洗泵。浸入，直到手术填充储存的盐溶液泵，保持控制和独立的盐水容器中的尼古丁泵。 </li><li>鼓励健康的恢复和减少手术后感染的风险，有干净的笼子里准备长期复苏。 </li><li>对短期恢复后立即手术，准备一个笼子里，包含一个加热垫和热灯。 </li><li>检查慢性尼古丁的影响，我们有5至6的合子α4YFP敲在各组小鼠（2-3月龄）（控制或尼古丁）。 α4YFP敲鼠标线，已为10代的C57BL/6J小鼠品系回交。我们要确保所有小鼠的年龄和性别是相同的，所有的手术都是在同一天进行，以尽量减少变异的研究。为了防止HYpothermia期间和手术后的小鼠，配备一个加热垫在无菌手术悬垂覆盖的手术台上。 </li><li>与3 L / min的氧和3％的异氟醚诱导的α4YFP敲鼠标，然后维持在2.5 L / min的氧气和1％异氟醚麻醉。我们宁愿异氟醚异氟醚麻醉，因为手术后迅速清除系统和老鼠〜2分钟内意识和移动。适用于眼睛立即下降（催泪凝胶，诺华），以避免角膜损伤。 </li><li>泵通过皮肤切口植入皮下颈后泵下推背背方面尾端。擦拭该地区95％的乙醇磨砂皮毛前肢之间的背面。捏皮肤0.8毫米格雷夫钳（精细的科学工具，11050-10）和一个1厘米的中央横向截用虹膜剪刀（精细的科学工具，14060-10）。 </li><li>要创建一个泵皮下空间，插入标准模式的剪刀（精细的科学工具，14101-14）切口，仔细推向尾端的动物。 </li><li>使用1.0毫米格雷夫钳（精细的科学工具，11650-10），删除从盐水泵。举行切口开放使用镊子和插入切口泵，面临动物的尾端与渗透泵帽的泵推到尾端。 </li><li>捏伤口关闭使用0.8毫米钳和应用适量的Vetbond（3M公司，猫＃1469SB）胶水。按住直到伤口被密封。 </li><li>从动物，异氟醚面罩注入镇痛，意识和移动到一个短期的回收笼，直到美洛昔康（0.1毫克/千克SC）和地点。然后将其放置在一个长期的回收笼的食物和水的随意 。 </li></ol> 2。 α4YFP敲鼠标固定腔内灌注<ol><li>使解决方案之一的前一天的程序，并在4°C，离开为了减少变异所有小鼠将灌注在同一天，同一批次的解决方案。 </li><li>在通风良好的地方进行灌注的录制品。冲洗DDH 2 O的线peristatic泵（轻松的Masterflex负载，7518-00;的Masterflex泵控制器，60648） </li><li> 〜0.0015克，抗凝血剂肝素（西格玛，猫＃H4784），加入20毫升的PBS pH值7.6（Invitrogen公司，猫＃70011）。 </li><li>麻醉α4YFP敲除小鼠肌肉注射，注射了氯胺酮的混合物（25毫克/公斤，惠氏动物健康）和medatomidine盐酸（0.25毫克/千克，辉瑞公司）的后腿肌肉，并立即将其家乡笼动物。 </li><li>针插入一个金属托盘到发泡胶盖的动物。 95％的乙醇擦拭胸部。捏皮肤与adson钳（精细的科学工具，91106-12）和剪断与虹膜剪刀打开胸腔。使用超细止血钳（精细的科学工具，13021-12）夹住胸腔，暴露心脏。 </li><li>开始抽4毫升/分钟，我PBSnsert到动物左心室1 23G的蝴蝶针（Becton Dickinson公司，367253）。立即剪断右心房，让血液和灌注液逃跑。 </li><li>灌注20毫升的PBS（pH值7.6），然后30毫升4％多聚甲醛（pH值7.6，用PBS稀释从16％PFA股票，电子显微学，猫＃15710），然后20毫升5％的蔗糖（pH值7.6） 。我们发现，超过固定增加自体荧光。 5％的蔗糖灌注冲洗大脑，从而降低自体荧光的残余煤灰。 </li><li>取出大脑在3天30％的蔗糖和商店。 </li><li>冻结的大脑冠状切片，在一个塑料嵌入模具切断刀片和地方的大脑与小脑（厂商VWR，猫＃18986-1）喙侧和淹没在华侨城安装复合（组织TEK，猫＃4583） 。冻结在-20℃的干冰和商店前切片。 </li><li>部分大脑（30微米厚）在低温恒温器，然后转移到涂层的幻灯片。储存在-20脑切片度;在幻灯片包含一个无水硫酸钙石盒ç。幻灯片中应存放于-20°C时，在拉链密封袋，以避免水分和后续冷冻烧伤</li></ol> 3。利用光谱共焦显微镜的成像荧光nAChRs <ol><li>确保幻灯片有任何光源，以尽量减少漂白的风险最小。 </li><li>盖玻片与安装介质，不抑制荧光蛋白荧光的大脑部分幻灯片。我们有良好的成功与：MOWIOL 4-88（pH值8.5，在Tris-HCl和甘油EMD Calbiochem公司，猫＃475904），几个小时后变硬。确保MOWIOL达到平衡至室温前滑倒，为了避免气泡的封面。不要使用指甲油时盖下滑，因为它会熄灭YFP的荧光。 </li><li>使用尼康C1si光谱共焦显微镜系统获得的图像。光谱共焦成像和线性分解方法的详细资料提供ð其他地方13-15。是固定的脑组织有固有的自体荧光光谱共焦显微镜使用的理由。光谱共焦成像的尼康C1si上使用的32个光电倍增管探测器阵列样品，指定范围内的荧光灯发出的光的波长，折射空间到其不同波长通过光栅色散元件，就像一个棱镜折射成彩虹的颜色白光16。什么是收集到的是一个图像的lambda栈 - 拉姆达栈的图像的每个像素，这样的发射光谱为收集不同波长光的收集到的图像。由于YFP的和组织自体荧光每个有特色的光谱特征，在lambda栈可以使用到单独的YFP的萤光信号（图2）线性代数算法混溶deconvolved。因此，可以非常精确的YFP的荧光定量测定Au的显着水平，即使在组织tofluorescence。 </li><li>各种设置可以进行调整，以优化图像质量和荧光收集效率。我们将报告我们通常使用的设置，但根据样品和共聚焦显微镜，可以调整这些设置。准确量化在α4YFP亚基表达的变化与慢性尼古丁我们确保灰度级别的所有像素的信号强度低于饱和值（&lt;4095为12位灰阶）。此外，我们容纳信号由于受体上调的潜在增长，调整聚焦的设置，使像素饱和值约三分之一（约1300-1400）或更少。一旦设置进行了优化，设置保持所有成像会议和样品相同。 </li><li>我们使用成像α4YFP以下设置，使用原讼法庭1 60X石油计划脂蛋白VC的目标（不适用1.40，0.13毫米的工作距离）：在488 nm的激光线15％，最大40毫瓦的氩激光传输，496.5 nm到656.5 nm的光谱范围从5 nm的分辨率成像光谱检测器的增益，在220，超过50微米×50微米，面积512×512像素，中等针孔大小（直径60微米），4.08像素的停留时间，平均两个扫描灰度和12位。 </li></ol> 4。光谱共焦图像和图像分析的线性分解<ol><li>执行线性光谱分离光谱共聚焦显微镜拍摄的样本图像，必须首先获得一个YFP的参考谱和组织自体荧光的参考谱。 </li><li>任何参考谱的一个重要条件是获得较高的信号噪声荧光光谱成像对样品的成像使用相同的激光线。虽然氩514 nm激光线具有较高的激发效率，激发YFP的，我们愿意与488 nm激光线图像YFP的，因为有较好的分离，从峰值发射了YFP和488纳米线是升少ikely扭曲的高峰，包括上升到峰值，可以得到整个YFP的发射光谱。我们的形象可溶性YFP的转染细胞系中得到这样一个强大的YFP的信号，以及由此产生的YFP的频谱被储存在我们的参考光谱库。染过表达细胞株α4YFP可能也可以使用，因为它也将提供一个高信号背景光谱。 </li><li>为了获得从野生型小鼠不同脑区的脑节的自体荧光，我们的形象自体荧光的参考谱，我们打算从α4YFP鼠标图像，并获得其相应的光谱。我们使用相同的激光线，488纳米，使用几乎相同的参数和图像，虽然我们可能会改变探测器的增益，激光强度或执行的平均成像信噪比最大化。 </li><li>从的α4YFP鼠标的大脑区域后获得的光谱共聚焦图像，图像，然后将其YFP的和autofluor deconvolved采用线性光谱分离算法，使用相同的大脑区域YFP的参考谱和野生型小鼠的自发荧光的参考谱escence信号。 </li><li>然后的纯色α4YFP图像可以被打开ImageJ的（如图像分析软件<a href="http://rsbweb.nih.gov/ij/" target="_blank">http://rsbweb.nih.gov/ij/</a> ），然后对感兴趣的区域平均像素强度计算。一个ImageJ的插件称为“loci_tools.jar”（ <a href="http://www.loci.wisc.edu/bio-formats/imagej" target="_blank">http://www.loci.wisc.edu/bio-formats/imagej</a> ）可以用来导入尼康ICS / IDS聚焦文件。 </li><li>我们重复相同的线性光谱分离和分析野生型小鼠在相同的大脑区域，以获得背景的剩余价值。 </li><li>然后减去残留背景值（4.6），平均裸α4YFP值（4.5）获得的校正的平均α4YFP强度。 </li></ol> 5。代表结果我们展示了一个图像一个纯α4YFP鼠标的光谱共聚焦显微镜（图3A）内侧缰的lambda栈的代表真彩色投影。我们还表明发射光谱，从一个地区包含来自同一波长堆叠图像（图3B）α4YFP阳性神经元的利益。在〜527 nm的一个独特的发射峰是显而易见的，这是YFP的荧光发射峰。该地区邻近的内侧缰显示发射光谱在527 nm处缺乏一个谱峰，表明没有α4YFP胆碱受体亚基。以下是可能使用与参考光谱和YFP的小鼠大脑autofluoresence的排放量显着重叠（图4），YFP的分离和萤光信号的线性光谱解混高产1α4YFP纯色的图像，萤光的纯色图像和其余通道。明确定位α4YFP流感orescence可以识别体躯紧凑内侧缰核（图4）。 在海马α4YFP是本地化主要内侧perforant路径，的tempero ammonic路径和alveus 12。这些都是海马谷氨酸能神经支配。我们研究α4YFP表达的的海马perforant路径（图5）慢性尼古丁的影响。导致慢性尼古丁管理（2毫克/千克/小时为10天）在一个重要的增长（69±14％）从控制生理盐水治疗的老鼠在荧光α4YFP慢性尼古丁处理小鼠（P = 0.001，Wilcoxon秩和检验）（图5）。 <img alt="图1" src="/files/ftp_upload/3516/3516fig1.jpg" /> 图1。流程图α4YFP慢性尼古丁胆碱受体亚基的形象变化的过程。微型渗透泵都充满了盐水或尼古丁和植入皮下及ALP公顷; 4YFP纯合子小鼠。经过10天的小鼠灌注和固定用4％多聚甲醛和老鼠大脑切片（厚30微米）的幻灯片上。大脑部分的成像光谱共焦显微镜（尼康C1si）和光谱未混合成YFP的和自体荧光图像。然后α4YFP图像分析进一步ImageJ的软件。 <img alt="图2" src="/files/ftp_upload/3516/3516fig2.jpg" /> 图2。成像光谱共焦显微镜和线性到其光谱成分未混合的一个lambda栈示意图。 （A）图像的lambda堆栈收集。 （B）的lambda堆栈由不同波长的光发射光谱收集，使整个堆栈中的每个像素收集的图像。 （三）由于YFP的和组织自体荧光各有特征光谱特征，在lambda栈可以deconvolved使用线性分解成一个代数算法eparate YFP的萤光信号。因此，甚至可以决定在组织高层次的自体荧光YFP的荧光非常准确的量化。 <img alt="图3" src="/files/ftp_upload/3516/3516fig3.jpg" /> 图3。谱脑表达α4YFPnAChRs地区的共聚焦图像。 （A）的lambda栈的内侧缰从尼康C1si光谱共焦显微镜α4YFP鼠标的图像真彩色投影。 （二）地块的光谱，从一个地区的利益，其中包括α4YFP神经元（绿色），内侧缰核（红色）以外的地区利益。 <img alt="图4" src="/files/ftp_upload/3516/3516fig4.jpg" /> 图4。内侧缰核线性混合光谱。 （一）图片的纯色α4YFP和（乙）自体荧光线性光谱分离。 （三）参考光谱YFP的（绿色三用于光谱分离的角度）和荧光（黄色圆圈）。 <img alt="图5" src="/files/ftp_upload/3516/3516fig5.jpg" /> 图5。α4nAChRs上调海马α4YFP敲除小鼠在暴露于慢性尼古丁。 （一）平铺的α4YFP荧光蒙太奇海马。两个虚线选择区域的海马分析每个鼠标执行的perforant路径下肢体上的大致位置。 （二）α4YFP荧光显着高于在小鼠慢性生理盐水（P = 0.001，Wilcoxon秩和检验），慢性尼古丁暴露比perforant路径。结果代表平均值±SEM从n = 20生理盐水和慢性尼古丁处理小鼠（每个治疗组的5只）测量。 <img alt="图6" src="/files/ftp_upload/3516/3516fig6.jpg" /> 图6。更好ðα4YFPepth表达相比，抗体标记。作为一个次要的标签（二）Cy5的α4YFP荧光（一）和VGLUT2抗体的新征正交意见。 （三）情节相比α4YFP（空心圆），显示抗体染色深度（黑色正方形）更大的荧光信号强度退化。

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Nikon+C1si+combines+high+spectral+resolution,+high+sensitivity,+and+high+acquisition+speed.">The Nikon C1si combines high spectral resolution, high sensitivity, and high acquisition speed.</a> Cytometry A. 69, 825-8234 (2006).</li><li>Melvin, N. R., Sutherland, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+caveats+of+standard+immunohistochemical+procedures:+implications+for+optical+disector-based+designs.">Quantitative caveats of standard immunohistochemical procedures: implications for optical disector-based designs.</a> J. Histochem. Cytochem. 58, 577-5784 (2010).</li><li>Jones, I. W., Wonnacott, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+doesn't+nicotinic+ACh+receptor+immunoreactivity+knock+out.">Why doesn't nicotinic ACh receptor immunoreactivity knock out.</a> Trends Neurosci. 28, 343-345 (2005).</li><li>Moser, N., Mechawar, N., Jones, I., Gochberg-Sarver, A., Orr-Urtreger, A., Plomann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evaluating+the+suitability+of+nicotinic+acetylcholine+receptor+antibodies+for+standard+immunodetection+procedures.">Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures.</a> J. Neurochem. , (2007).</li><li>Whiteaker, P., Cooper, J. F., Salminen, O., Marks, M. J., McClure-Begley, T. D., Brown, R. W., Collins, A. C., Lindstrom, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunolabeling+demonstrates+the+interdependence+of+mouse+brain+a4+and+b2+nicotinic+acetylcholine+receptor+subunit+expression.">Immunolabeling demonstrates the interdependence of mouse brain a4 and b2 nicotinic acetylcholine receptor subunit expression.</a> The Journal of Comparative Neurology. 499, 1016-1038 (2006).</li><li>Marks, M. J., McClure-Begley, T. D., Whiteaker, P., Salminen, O., Brown, R. W. B., Cooper, J., Collins, A. C., Lindstrom, J. 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R., Zoli, M., Rimondini, R., Lena, C., Marubio, L. M., Pich, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+containing+the+Î²2+subunit+are+involved+in+the+reinforcing+properties+of+nicotine.">Acetylcholine receptors containing the Î²2 subunit are involved in the reinforcing properties of nicotine.</a> Nature. 391, 173-177 (1998).</li><li>Fowler, C. D., Lu, Q., Johnson, P. M., Marks, M. J., Kenny, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habenular+&#945;5+nicotinic+receptor+subunit+signalling+controls+nicotine+intake.">Habenular &#945;5 nicotinic receptor subunit signalling controls nicotine intake.</a> Nature. 471, 597-601 (2011).</li><li>Maskos, U., Molles, B. E., Pons, S., Besson, M., Guiard, B. P., Guilloux, J. 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我们什么都没有透露。

<p class="jove_content"&gt;在敲在小鼠模型，以确定一个特定的离子通道的数量和本地化的荧光受体的使用提供了许多优势。相反，如肌动蛋白，它是无处不在所有细胞中表达的蛋白质，离子通道是目前人数要少得多，并作出准确的分析，通过免疫组化技术挑战传统的神经元亚型之间的表达变化。在α4YFP基因产物表达野生型水平相同的启动子，增强和贩卖机制的控制下，<sup&gt; 12</sup&gt;。荧光受体也显示了相同的WT离子通...

<table border="1"><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>微型渗透泵</td><td> alzet </td><td>模型2002年</td><td></td></tr><tr><td>盐水</td><td> teknova </td><td> S5819 </td><td></td></tr><tr><td> （ - ） - 尼古丁酒石酸氢盐</td><td>西格玛</td><td> N5260 </td><td></td></tr><tr><td>眼药水</td><td>诺华公司</td><td>撕裂凝胶</td><td></td></tr><tr><td> vetbond胶水</td><td> 3M </td><td> 1469SB </td><td></td></tr><tr><td>肝素钠盐</td><td>西格玛</td><td> H4784 </td><td></td></tr><tr><td> 10X PBS </td><td> Invitrogen公司</td><td> 70011 </td><td></td></tr><tr><td>氯胺酮</td><td> wyetĤ动物健康</td><td> 0856-4403-01 </td><td></td></tr><tr><td> medatomidine盐酸</td><td>辉瑞公司</td><td> 1950673 </td><td></td></tr><tr><td> 23G蝴蝶针</td><td>碧迪</td><td> 367253 </td><td></td></tr><tr><td>多聚甲醛</td><td>电子显微镜科学</td><td> 15710 </td><td></td></tr><tr><td>塑料包埋模具</td><td>厂商VWR </td><td> 18986-1 </td><td></td></tr><tr><td>华侨城安装复合</td><td>组织TEK </td><td> 4583 </td><td></td></tr><tr><td> MOWIOL 4-88 </td><td> EMD的Calbiochem公司</td><td> 475904 </td><td> pH值8.5 </td></tr></table>

spectral confocal imaging of fluorescently tagged nicotinic receptors in knock-in mice with chronic nicotine administration

配体门控离子通道，在中枢神经系统（CNS）有牵连的有严重的医疗和社会后果的许多条件。例如，通过吸烟尼古丁成瘾的首要原因是全球过早死亡（世界卫生组织）和可能造成的离子通道分布在大脑中的变更1。慢性尼古丁暴露于啮齿动物和人类的烟碱乙酰胆碱受体（nAChRs）的脑组织1-3人数增加的结果。同样，在谷氨酸GluN1或GluA1渠道，改变已牵连到其它能使人成瘾的药物，如可卡因，安非他明和鸦片4-6引发过敏。 因此，映射和量化特定的离子通道的分布和表达模式的能力是极为重要的理解成瘾的机制。研究大脑的特定区域的EF个别药物fects先进的技术，如放射性配体的问世。然而，低放射性配体结合的空间分辨率，防止量化在特定的神经元亚型配体门控离子通道的能力。 基因编码的荧光记者，如绿色荧光蛋白（GFP）和它的许多颜色的变种，7生物学领域的一个革命性的基因标记内源性蛋白，可以可视化的蛋白质在体内 7-10荧光记者。用探针荧光标记蛋白质的优势之一是消除抗体的使用，有目标蛋白的非特异性和无障碍的问题。我们已经用这种战略荧光标签nAChRs，这使福斯特共振能量转移（FRET）在转染细胞培养11受体装配研究。最近，我们已经使用了KNOCK-工程师与黄色荧光蛋白标签胆碱受体α4亚基（α4YFP）的，能够精确量化的亚微米决议通过光谱共焦显微镜12中枢神经系统的神经元的受体体外小鼠的方法。针对性的荧光敲在突变中的内源性的轨迹和其本地子的控制之下，成立生产受体的表达和调控的正常水平相比，在野生型小鼠无标记的受体时。这个连锁反应中的方法可以扩展到其他离子通道的荧光标记，并提供了一​​个可视化和量化受体在中枢神经系统的功能强大的方法。 在本文中，我们描述了一种方法来量化在特定的中枢神经系统神经元暴露后慢性尼古丁胆碱受体表达的变化。我们的方法包括微型渗透泵植入术，心内灌注固定，成像和荧光标记的烟碱REC分析eptor亚基从α4YFP敲入小鼠（图1）。我们已经优化了固定技术，从固定脑tissue.We的，以尽量减少自体荧光，在详细描述我们的成像方法，利用光谱共聚焦显微镜结合线性光谱分离算法减去autofluoresent为了准确地获取α4YFP荧光信号的信号。最后，我们表明慢性尼古丁诱导的海马内侧perforant的路径α4YFP受体上调的结果。

Watch this Scientific Journal Video about Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration at JoVE.com

Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

我们已经开发出一种量化烟碱型乙酰胆碱受体亚地区的中枢神经系统神经元的具体亚型内的变化，更好地了解使用爆震的方法和光谱用荧光蛋白标记的受体相结合的办法，包括对尼古丁成瘾的机制的新技术共聚焦成像。

光谱共焦成像的荧光标记的尼古丁受体敲在慢性尼古丁政府小鼠

Ligand-gated ion channels in the central nervous system (CNS) are implicated in numerous conditions with serious medical and social consequences.  For ...

spectral-confocal-imaging-fluorescently-tagged-nicotinic-receptors

Research

JoVE Journal

Neuroscience

13.4K 次观看.  University of Victoria. 我们已经开发出一种量化烟碱型乙酰胆碱受体亚地区的中枢神经系统神经元的具体亚型内的变化，更好地了解使用爆震的方法和光谱用荧光蛋白标记的受体相结合的办法，包括对尼古丁成瘾的机制的新技术共聚焦成像。

视频: Spectral Confocal Imaging of Fluorescently tagged Nicotinic Receptors in Knock-in Mice with Chronic Nicotine Administration

Subcutaneous Administration of Muscarinic Antagonists and Triple-Immunostaining of the Levator Auris Longus Muscle in Mice

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo pharmacological studies of the signals essential for the formation and maintenance of mammalian NMJs. However, drug penetration into these muscles after subcutaneous or intramuscular administration is often incomplete or uneven and many NMJs can remain unaffected. Although systemic administration with devices such as mini-pumps can improve the spatiotemporal effects, the invasive nature of this approach can cause confounding inflammatory responses and/or direct muscle damage. Moreover, complete analysis of the NMJs in a hind limb muscle is challenging because it requires time-consuming serial sectioning and extensive immunostaining. 
The mouse LAL is a thin, flat sheet of muscle located superficially on the dorsum of the neck. It is a fast-twitch muscle that functions to move the pinna. It contains rostral and caudal portions that originate from the midline of the cranium and extend laterally to the cartilaginous portion of each pinna. The muscle is supplied by a branch of the facial nerve that projects caudally as it exits the stylomastoid foramen. We and others have found LAL to be a convenient preparation that offers advantages for the investigation of both short and long-term in vivo effects of drugs on NMJs and muscles. First, its superficial location facilitates multiple local applications of drugs under light anesthesia. Second, its thinness (2-3 layers of muscle fibers) permits visualization and analysis of almost all the NMJs within the muscle. Third, the ease of dissecting it with its nerve intact together with the pattern of its innervation permits supplementary electrophysiological analysis in vitro9,5. Last, and perhaps most importantly, a small applied volume (&tilde;50&mu;l) easily covers the entire muscle surface, provides a uniform and prolonged exposure of all its NMJs to the drug and eliminates the need for a systemic approach1,8.

We describe procedures for repeated administration of inhibitors of muscarinic signaling to the levator auris longus (LAL) muscle of young adult mice and for subsequent immunostaining of its neuromuscular junctions (NMJs) in wholemounts. The LAL muscle has unique advantages for revealing in vivo pharmacological effects on NMJs.

皮下给药的毒蕈碱受体拮抗剂和上睑提的Auris在小鼠体内长肌的三重免疫染色

Hind limb muscles of rodents, such as gastrocnemius and tibialis anterior, are frequently used for in vivo  pharmacological studies of the signals ...

科研

神经科学

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Live Imaging of Nicotine Induced Calcium Signaling and Neurotransmitter Release Along Ventral Hippocampal Axons

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors (nAChRs) is an important mechanism for neuromodulation by acetylcholine (ACh). The difficulty with access to probing the signaling mechanisms within intact axons and at nerve terminals both in vitro and in vivo has limited progress in the study of the pre-synaptic components of synaptic plasticity. Here we introduce a gene-chimeric preparation of ventral hippocampal (vHipp)&#8211;accumbens (nAcc) circuit in vitro that allows direct live imaging to analyze both the pre- and post-synaptic components of transmission while selectively varying the genetic profile of the pre- vs post-synaptic neurons. We demonstrate that projections from vHipp microslices, as pre-synaptic axonal input, form multiple, reliable glutamatergic synapses with post-synaptic targets, the dispersed neurons from nAcc. The pre-synaptic localization of various subtypes of nAChRs are detected and the pre-synaptic nicotinic signaling mediated synaptic transmission are monitored by concurrent electrophysiological recording and live cell imaging. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of glutamatergic synaptic plasticity in vitro.

We developed a gene-chimeric preparation of ventral hippocampal &#8211; accumbens circuit in vitro that allows direct live imaging to analyze presynaptic mechanisms of nicotinic acetylcholine receptors (nAChRs) mediated synaptic transmission. This preparation also provides an informative approach to study the pre- and post-synaptic mechanisms of synaptic plasticity.

尼古丁诱导的钙信号和神经递质的释放了沿着腹侧海马轴突实时成像

Sustained enhancement of axonal signaling and increased neurotransmitter release by the activation of pre-synaptic nicotinic acetylcholine receptors ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

高分辨率定量免疫膜受体在视网膜带状突触分析

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Quantifying Infra-slow Dynamics of Spectral Power and Heart Rate in Sleeping Mice

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior are identified in freely moving animals, this three-fold subdivision becomes too simplistic. During wakefulness, ensembles of global and local cortical activities, together with peripheral parameters such as pupillary diameter and sympathovagal balance, define various degrees of arousal. It remains unclear the extent to which sleep also forms a continuum of brain states-within which the degree of resilience to sensory stimuli and arousability, and perhaps other sleep functions, vary gradually-and how peripheral physiological states co-vary. Research advancing the methods to monitor multiple parameters during sleep, as well as attributing to constellations of these functional attributes, is central to refining our understanding of sleep as a multifunctional process during which many beneficial effects must be executed. Identifying novel parameters characterizing sleep states will open opportunities for novel diagnostic avenues in sleep disorders.
We present a procedure to describe dynamic variations of mouse non-REM sleep states via the combined monitoring and analysis of electroencephalogram (EEG)/electrocorticogram (ECoG), electromyogram (EMG), and electrocardiogram (ECG) signals using standard polysomnographic recording techniques. Using this approach, we found that mouse non-REM sleep is organized into cycles of coordinated neural and cardiac oscillations that generate successive 25-s intervals of high and low fragility to external stimuli. Therefore, central and autonomic nervous systems are coordinated to form behaviorally distinct sleep states during consolidated non-REM sleep. We present surgical manipulations for polysomnographic (i.e., EEG/EMG combined with ECG) monitoring to track these cycles in the freely sleeping mouse, the analysis to quantify their dynamics, and the acoustic stimulation protocols to assess their role in the likelihood of waking up. Our approach has already been extended to human sleep and promises to unravel common organizing principles of non-REM sleep states in mammals.

Here, we present experimental and analytical procedures to describe the temporal dynamics of the neural and cardiac variables of non-REM sleep in mice, which modulate sleep responsiveness to acoustic stimuli.

量化睡眠小鼠的光谱功率和心率的慢速动力学

Three vigilance states dominate mammalian life: wakefulness, non-rapid eye movement (non-REM) sleep, and REM sleep. As more neural correlates of behavior ...

Automated Gait Analysis in Mice with Chronic Constriction Injury

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some disadvantages such as subjective data and the requirement of a skilled, experienced experimenter. To date, a variety of modifications have improved the von Frey method, but it still has a few limitations. Recent reports have suggested that gait analysis produces more accurate and objective data from the neuropathic animals. This protocol demonstrates how to perform the automated gait analysis to determine the degree of neuropathic pain in mice. After several days of acclimation, the mice were allowed to walk freely on the glass floor to illuminate footprints. Then, quantification of the footprints and gait were performed through video clips with automatic analysis of various walking parameters, such as area of paw print, swing time, angle of paw, etc.
The main purpose of this study is to describe the methodology of automated gait analysis and briefly compare it with data from the classical sensory test using von Frey filament.

The precise assessment of pain response in a neuropathic animal model is critical to investigate the pathophysiology of pain diseases and develop new analgesics. We present a sensitive and objective method to determine the sensory function of the rodent hind paw by an automated gait analysis system.

慢性压迫性损伤小鼠步态的自动分析

The von Frey test is a classical method that has been widely used to examine the sensory function of neuropathic pain animals. However, it has some ...

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains brain homeostasis. The principal sites of the barrier are endothelial cells of the brain capillaries whose barrier function results from tight intercellular junctions and efflux transporters expressed on the plasma membrane. This function is regulated by pericytes and astrocytes that together form the neurovascular unit (NVU). Several neurological diseases such as stroke, Alzheimer&#39;s disease (AD), brain tumors are associated with an impaired BBB function. Assessment of the BBB permeability is therefore crucial in evaluating the severity of the neurological disease and the success of the treatment strategies employed.
We present here a simple yet robust permeability assay that have been successfully applied to several mouse models both, genetic and experimental. The method is highly quantitative and objective in comparison to the tracer fluorescence analysis by microscopy that is commonly applied. In this method, mice are injected intraperitoneally with a mix of aqueous inert fluorescent tracers followed by anesthetizing the mice. Cardiac perfusion of the animals is performed prior to harvesting brain, kidneys or other organs. Organs are homogenized and centrifuged followed by fluorescence measurement from the supernatant. Blood drawn from the cardiac puncture just before perfusion serves for normalization purpose to the vascular compartment. The tissue fluorescence is normalized to the wet weight and serum fluorescence to obtain a quantitative tracer permeability index. For additional confirmation, the contralateral hemi-brain preserved for immunohistochemistry can be utilized for tracer fluorescence visualization purposes.

An In Vivo Blood-brain Barrier Permeability Assay in Mice Using Fluorescently Labeled Tracers

Here we present a mouse brain vascular permeability assay using intraperitoneal injection of fluorescent tracers followed by perfusion that is applicable to animal models of blood-brain barrier dysfunction. One hemi-brain is used for assessing permeability quantitatively and the other for tracer visualization/immunostaining. The procedure takes 5 - 6 h for 10 mice.

用荧光标记示踪剂对小鼠血脑屏障通透性的体内检测

Blood-brain barrier (BBB) is a specialized barrier that protects the brain microenvironment from toxins and pathogens in the circulation and maintains ...

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its highly-organized subcellular architecture, we recently developed a new imaging modality, named spectral reflectometry (SpeRe), which enables unprecedented label-free nanoscale imaging of the live myelinated axons in situ. The underlying principle is to obtain nanostructural information by analyzing the reflectance spectrum of the multilayered subcellular structure. In this article, we describe a detailed step-by-step protocol for performing a basic SpeRe imaging of the nervous tissues using a commercial confocal microscopic system, equipped with a white-light laser and a tunable filter. We cover the procedures of sample preparation, acquisition of spectral data, and image processing for obtaining nanostructural information.

Spectral Reflectometric Microscopy on Myelinated Axons In Situ

Here, we present a step-by-step protocol for imaging myelinated axons in a fixed brain slice using a label-free nanoscale imaging technique based on spectral reflectometry.

髓轴突原位光谱 Reflectometric 显微术

In a mammalian nervous system, myelin provides an electrical insulation by enwrapping the axon fibers in a multilayered spiral. Inspired by its ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Probing Nicotinic Acetylcholine Receptor Function in Mouse Brain Slices via Laser Flash Photolysis of Photoactivatable Nicotine

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with subcellular location within cells where this function is carried out. To study the subcellular location of nicotinic ACh receptors (nAChRs) in native brain tissue, an optical method was developed for precise release of nicotine at discrete locations near neuronal membranes during electrophysiological recordings. Patch-clamped neurons in brain slices are filled with dye to visualize their morphology during 2-photon laser scanning microscopy, and nicotine uncaging is executed with a light flash by focusing a 405 nm laser beam near one or more cellular membranes. Cellular current deflections are measured, and a high-resolution three-dimensional (3D) image of the recorded neuron is made to allow reconciliation of nAChR responses with cellular morphology. This method allows for detailed analysis of nAChR functional distribution in complex tissue preparations, promising to enhance the understanding of cholinergic neurotransmission.

This article presents a method for studying nicotinic acetylcholine receptors (nAChRs) in mouse brain slices by nicotine uncaging. When coupled with simultaneous patch clamp recording and 2-photon laser scanning microscopy, nicotine uncaging connects nicotinic receptor function with cellular morphology, providing a deeper understanding of cholinergic neurobiology.

利用可活性可选位的激光光致发光体研究小鼠脑片中的烟碱乙酰胆碱受体功能

Acetylcholine (ACh) acts through receptors to modulate a variety of neuronal processes, but it has been challenging to link ACh receptor function with ...