由于戴安娜卡萨-Culaki，克里斯塔尔桑普森和神学Karagiorgis为他们在这个项目上努力工作。

这些实验方案已获得批准的机构动物护理和使用委员会证明所有的主体和程序均符合健康指南护理全国学院和实验动物使用。 1.主题<ol><li>购买和/或品种雄性SD大鼠（260 - 300克）。 </li><li>房子只单独在铁丝网笼子里。保持他们与大鼠饲料和水可随意 12:12小时的光/暗周期。 </li><li>分配适当的样本大小（例如 ，N≈6 - 8）随机分组。 </li></ol> 2.测试仪器和收容程序<ol><li>使用校准的离心管用橡胶塞和一个45°角金属吸管，以提供精确的测量（±0.1毫升）所提出的解决方案。通过拉紧金属弹簧它们固定到饲养笼，以允许校准的能见度。 </li><li>限制粮食配给（〜大鼠的15 /克/天），以减轻重量，以它们的原始体重的85％，以增加动力消耗的解决方案。注:减重应该采取3之间 - 天5。 </li><li>在1小时的会话四天提供0.2％的糖精的训练前溶液（10毫升）以最大化大鼠将与短（少于1分钟）延迟采样随后的测试解决方案的可能性。 </li><li>由溢出几滴确认通过离心管流动。 </li><li>在每次会议结束后称取管获得的摄入量的测量。 </li><li>执行在接收六溶液1子组在第五天的进气试验（10毫升，1小时）:a）水，二）新颖味（0.05％樱桃香料）0.2％糖精，C）8％果糖，D）8 ％葡萄糖，E）为3.5％的玉米油悬浮于0.3％黄原胶，和f）0.3％的黄原胶。 </li><li>确保营养解决方案等热量;因此，3.5％的玉米油浓度等热量至8％的糖溶液。 </li><li>确保日在短的等待时间（小于1分钟）的大鼠的样品溶液。如果这个要求不能满足，则丢弃从研究的课题。 </li></ol> 3.组织准备<ol><li>由最初暴露于每个测试溶液后的戊巴比妥90分钟腹膜内注射麻醉各动物。确认动物被适当地证明该动物不再响应这样的反射，撤退到脚捏，闪烁下的角膜直接压力或摇头来深耳廓刺激麻醉。 </li><li>如前面所述69灌注transcardially每个动物。 <ol><li>麻醉大鼠的戊巴比妥钠（65毫克/千克）的过量，取出肋骨和揭露胸部免费进入心脏69。 </li><li>放置针在左心脏瓣膜的先端，并切断腔静脉。施用接着是磷酸盐缓冲的固定剂共磷酸盐缓冲液（PBS，〜180ml）中ntaining 4％多聚甲醛（〜180毫升）中。 </li><li>确保动物实际被正确地通过检查液体是否正在离开其他腔，如鼻，口，和生殖器区域灌注。注:多聚甲醛适当固定将大肌肉运动陪同。如果这种情况不会发生，直到发生这种反应重新调整针。 </li></ol></li><li>快速通过削减皮毛和皮肤从头骨上卸下从头骨的大脑。用咬骨钳破解，并动起了脑筋，从后到前去除骨头。最初的工作在下面和小脑后面的区域，确保咬骨钳是骨和脑膜软脑膜之间。一旦头骨的顶部和侧面被去除，用小刮刀从底座提起大脑，并剪断颅神经的小剪刀。小心不要损伤大脑在试图消除骨。 </li><li>固定在4％多聚甲醛溶液的大脑在4℃下过夜。放置的大脑中有30％的蔗糖/ 70％PBS中在室温下溶液中，直至它们沉降在容器的底部。 </li><li>阻止脑<ol><li>取出大脑切割横向尾椎嗅球的延髓一部分。 </li><li>取出大脑在小脑和脑桥水平横向切割的尾的部分。 </li></ol></li><li>冠装入大脑与固定在滑动切片机的阶段尾椎部位，切冠状切片（40微米）通过内侧前额叶皮质（2.86 - 2.20毫米喙前囟门），南汽芯和外壳和背侧纹状体（+ 1.76 - 1.60毫米喙前囟门），艾米（-2.12 - -2.92毫米尾鳍前囟）和VTA（-5.20 - -5.60毫米尾鳍前囟）。使用大鼠脑图谱70指导。 </li><li>收集自由浮动切片到充满PBS中，以便最终免疫组织化学分析71 24孔板的各个孔中。用封口膜密封24，我们LL板以确保PBS不会在容器蒸发和干涸大脑。储存在4℃的脑组织。 </li></ol> 4，c-fos程序（从71改编） <ol><li>对待每个部分用5毫升5％正常山羊血清和0.2％的Triton X-100的PBS中1小时。 </li><li>孵育初级抗体处理过的部分（兔抗c-fos和1:5000）4℃36小时在含有1毫升的PBS的孔中。 </li><li>冲洗切片3倍，用PBS（5毫升），每次10分钟。 </li><li>在RT 2小时在含有1毫升的PBS的孔中:;具有第二抗体孵育（200 1生物素化的山羊抗兔）。 </li><li>漂洗，每次10分钟的PBS每个部分3×（5毫升）中。 </li><li>孵育在附带在一个5毫升的PBS Avadin DH（100微升）和生物素化辣根过氧化物酶的H（100微升）构成试剂盒的市售抗生物素蛋白 - 辣根过氧化物酶混合物2小时的漂洗部分。 </li><li>再冲洗部3X的PBS（5ml）中，每次10分钟。 </li><li>反应在0.0015％H 2 O 2的存在下进行5用0.05％二氨基联苯胺（DAB）的章节- 10分钟，这取决于组织在含有5毫升DAB溶液的孔中的反应性。 </li><li>双击标签VTA部分。在PBS（5毫升）在4℃过夜:用酪氨酸羟化酶（TH）抗体（2,000兔抗大鼠TH，1）孵育它们。 </li><li>冲洗切片3倍于PBS（5ml）中，每次10分钟。 </li><li>在室温下在PBS中（5毫升）处理2小时:;具有第二抗体孵育（200 1生物素化的山羊抗兔）。 </li><li>冲洗切片3倍于PBS（5ml）中，每次10分钟。 </li><li>通过使用二次抗体 - 过氧化物酶复合物可视化的抗体。用0.05％DAB的组合，0.3％的硫酸镍溶液进行反应，5 - 10分钟，这取决于组织在含有5毫升的DAB /的NiCl溶液的孔中的反应。 </li><li>确保DAB溶液的颜色是乳白色浅绿色从中用0.3％的硫酸镍的反应。如果溶液是太绿，然后将反应将太暗。 </li><li>安装所有章节到明胶涂层的幻灯片。让他们干通宵，然后盖玻片用甲苯基础的解决方案（TBS）几滴。 </li><li>代码幻灯片，使实验条件是未知的观察员。 </li></ol> 5.测定c-fos的免疫反应计数<ol><li>对分配公正的观察员来算感兴趣这些区域（ROI）的Fos阳性神经元:前度内侧前额叶皮质，infralimbic内侧前额叶皮质，NAC核心，NAC壳，基底核AMY，中央皮质内侧AMY，背侧纹状体和VTA。划定的c-Fos的免疫反应是否存在于TH +和TH-细胞中的VTA。 图1提供了从显微镜NAC的屏幕捕获的图像。 </li></ol><img alt="图1" src="/files/ftp_upload/53897/53897fig1.jpg" /> <stroNG&gt;按网格由c-fos计数制成轮廓利益图1伏隔核的代表科（NAC）显示区域。NAC的外壳内侧和腹部发现了NAC的核心。在NAC核心环绕前连合（前通信）。侧脑室（横向通风）的腹侧程度是可见的。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <ol start="2"><li>在所有的测试条件分析每个通用于所有动物的网站至少有三个有代表性的切片。 </li><li>使用软件并在光学显微镜通过跟踪轮廓（ 图1）以分析每个ROI的整个区域。 <ol><li>对于给定的网站，打开应用程序，并单击收购下拉菜单中，点击"实时图像"。带来的投资回报率成为关注的焦点，并点击屏幕来建立一个参考点。然后TR王牌使用网格作为指导所选择的脑区域。一旦跟踪完成后，细胞计数（步骤5.3.1.1 - 5.3.1.3）。 <ol><li>双击软件图标。转到菜单栏，点击"获取"，然后选择"实时图像"。带来的投资回报率成为关注的焦点，并点击屏幕来建立一个参考点。 </li><li>转到网格工具栏，然后单击"显示网格"和"使用网格标签"。外形与预定的一丝投资回报率。 </li><li>计算所有细胞的每个ROI区域中，在左侧栏选择一个"+"，以保持c-fos的细胞计数。点击每个单元单独注册计数。当一个定义暗红色圆圈观察（ 图1）考虑为阳性的c-fos基因的细胞。 </li></ol></li><li>重复此过程为每个站点。 </li><li>记录在实验室笔记本和电脑供日后分析的计算。转到菜单栏中，单击"文件"，"保存数据文件"保存跟踪和计数。 </li></OL&gt; </li><li>保证两个不知情的评估者的评判间的可靠性（使用计数的相关性），使各ROI每一节总是超过0.8。 </li></ol> 6.统计<ol><li>使用重复测量方差分析的1路分析比较，3天1 2和4 69的糖精摄入量在评估前四天基线糖精摄入量。 </li><li>比较糖精摄入量（第4天），使用一个随机块双因子ANOVA 69的六组试验摄入量（第5天）。 </li><li>使用杜克比较（P &lt;0.05），以确定个人显著影响69。 </li><li>确定评判间的可靠性，然后用普通观察者的计数。 </li><li>平均C-FOS计数为每个站点69的三个有代表性的切片。 <ol><li>执行由六种溶液（3.5％的玉米油，8％的葡萄糖，8％果糖，0.2％调味糖精，XA摄入诱导的c-fos的激活的单因素方差分析对于perilimbic内侧前额叶皮质69 nthan胶控制和水）。 </li><li>六组的infralimbic内侧前额叶皮质，NAC核心，NAC外壳，基底AMY，中央皮质内侧AMY，VTA和背侧纹状体的重复平行分析。使用杜克比较（P &lt;0.05），以显示个别显著影响69。 </li></ol></li><li>比较玉米油的摄入量与饮水量都和进它的悬挂剂，黄原胶。比较取水和非营养性甜味剂，糖精的摄取都果糖和葡萄糖摄入。 </li><li>建立使用邦费罗尼ř相关（P &lt;0.05）是否观察每个站点的溶液摄入和c-fos激活之间显著关系。 <ol><li>比较系统地在perilimbic和infralimbic前额叶皮层的3.5％，玉米油组中的每个动物的c-fos的计数。 </li><li>重复每对六个站点的系统并行分析（VTA，背侧纹状体，infralimBIC内侧前额叶皮质，perilimbic内侧前额叶皮质，NAC核心，NAC外壳，基底AMY，中央皮质内侧AMY）为3.5％，玉米油。 </li><li>重复上述六个站点的系统并行分析其他五个实验条件的摄入量（8％葡萄糖，8％的果糖，0.2％调味糖精，黄原胶控制和水）。 </li></ol></li><li>取的一个事实，即一个溶液条件中的相同动物通过跨解决方案，并使用邦费罗尼每种溶液中确定的c-fos的激活之间显著关系在所有站点评价优点ř相关（P &lt;0.05）。 </li></ol>

作者有没有竞争经济利益。

这项研究的目的是利用细胞的c-fos的技术来确定源（VTA）和前脑投射目标DA奖励相关的神经元（NAC，AMY，内侧前额叶皮质）的脂肪和糖的新型摄入后同时激活大鼠。本研究是此前69发表的一项研究协议的详细描述。据推测，在VTA，其主要投影区域的前度和infralimbic mPFC的，在NAC的核和壳和基底外侧和中央皮质内侧AMY，以及背侧纹状体将充当分布式脑网络24 -27，并显示协调下同时FLI小说摄入葡萄糖（8％），果糖（8％），或相对于糖精（0.2％），水和其他控制解决方案，解决方案的玉米油（3.5％）的。玉米油，葡萄糖和果糖，但不糖精的VTA的前度和infralimbic mP在摄入产生显著和差分FLI活化FC，核心和NAC，基底外侧和中央皮质内侧AMY的外壳，和背侧纹状体。除了到c-fos的技术中，采用糖，脂肪和人造甜味剂摄取行为的措施。 其中一个关键的步骤包括摄入及时采样，这样他们会比较平等的，从而确保在跨站点的c-fos的激活任何差异是由于所消耗的解决方案，而摄入的任何图案或幅度。基线糖精摄入四天确保了食品限制动物快速采样的解决方案，并由此最小化非特异性的效果。第二个关键步骤是该程序造成最小的压力，或新奇的动物在情绪价独立摄入类型的变化也可能产生的c-fos的激活。因此，发现这种方法和协议相关的有效性提供了令人信服的"概念证明"识别急性暴露于脂肪（如玉米油），糖（葡萄糖和果糖）和非营养性甜味剂（糖精）解决方案是否同时启动DA介导的投资回报率在提示方式一个协调的分布式脑神经系统24-27。 因为最佳的c-Fos蛋白的活化需要时间敏感的反应之前牺牲51,52，以前验证程序42,44最大化溶液取样与在1小时的测试短潜伏期。因此，食品限制大鼠用0.2％糖精溶液受训（10毫升，1小时），进行4天，并给予在第五天试液。基线糖精摄入显著并逐渐增加，并且在第五天果糖和葡萄糖，但是不玉米油或糖精摄入量高于第四天糖精摄取显著更高。因此，增加FLI显著上升（葡萄糖，果糖）相关的解决方案或没有影响（玉米油）再摄取lative以前糖精训练，似乎通过一个奖励相关的行为激励机制介导的。仔细考虑需要采取以确保取样和行为的平等。其他研究人员可以有效地使用此过程来研究其他类型的新的解决方案或引入变化的范式来理解与适应和学习机制。 本协议的优点是比较充分研究的糖（果糖，葡萄糖）和脂肪的效果（玉米油），并比较它们的c-fos的激活作用比的重要对照（非营养性甜味剂，糖精的能力，控制乳化剂，黄原胶和水），然后检查在六个相关的大脑部位这些影响。虽然这种方法有在允许跨不同适口物质的脑部位同时检查明显的好处，它具有产生一个潜在的天文数据集的缺点显示的神经元细胞中的表达。为了使这更便于管理，我们把所有的测试条件分析每共同所有的动物现场三个有代表性的冠状切片的方法。当然，这是伴随着在这三个部分选择每个ROI的适当水平的警告。鉴于AMY，NAC，内侧前额叶皮质，背侧纹状体和腹侧被盖区的广泛rostro - 尾程度上，这应该告诫不可掉以轻心。此外，这是再经调查有责任在所有站点准确选择在所有动物每三个代表部分的一致。在这种选择小错误可以导致"假阳性"和"假阴性"。计算效率也是一个相关的变量。我们对这种潜在的变乱解决方案是分配两个不知情的评价者在每个ROI的每个部分，然后确保评定者间的可靠性（使用计数的相关性）总是超过0.8。这种方法虽然DUPLicative，给了我们关于精度远远更大的保证作为评判间可靠性轻松突破这个最低标准。南汽的分区域（核心与壳），AMY（BASO侧向与中央皮质，内侧）和内侧前额叶皮质（perilimbic与infralimbic）进行了分析。这些区域可以被进一步划分，特别是个体AMY核，背侧纹状体的贴片和矩阵隔间，并在NAC壳（顶点，拱，锥形，中间区）。因为在NAC壳未能始终显示以下玉米油，葡萄糖或果糖在FLI的变化，不进行该结构的进一步子的分析。背侧纹状体的贴片和矩阵区的明确的检查所需的未在本研究采用进一步免疫组化技术，但将是一个重要的后续研究。每个子区域内的各个AMY核分析也将额外的进一步研究。 以往的研究表明，SUC玫瑰摄取在中央AMY核增加FLI，所述VTA以及外壳，但不是核心，在NAC的，但口服或IG糖精输注在很大程度上是无效的55-57，60-62。葡萄糖和果糖摄入后FLI引起特定糖效果与两个有效的中央皮质内侧AMY和背侧纹状体，前者有效在NAC芯和基底外侧AMY，后者有效地将infralimbic mPFC的。糖精摄入量没有引起现场相对于水任何FLI的任何变化。脂肪的摄入量也增加FLI在accumbal和内侧前额叶皮质的网站在以往的研究65-67，而在VTA，infralimbic和前度内侧前额叶皮质，背侧纹状体，南汽生产的核心同时显著激活和基底外侧和中央皮质内侧AMY。 尽管以前的研究表明，糖和脂肪的摄入量诱导脑细观corticolimbic和黑质纹状体DA系统FLI，本系统的研究同时评估激活FLI在VTA，基底和中央皮质内侧AMY，背侧纹状体，前度和infralimbic内侧前额叶皮质，伏隔核和壳急性摄入玉米油，果糖，葡萄糖或糖精。显著FLI上升的高度相互关联的跨站点前脑，支持分布式网络的脑激活调解糖和脂肪的摄入量的想法。识别多发性脑位点同时改变这样的协议可以慢性和暴食的条件下，以及根据条件和喜好加以利用。这些研究表明，一个很强的解剖相关成分（C-FOS）可以在多个脑部位有效地用于同时识别用于介导适口摄取和偏好中的动物，可能提供深入了解有关肥胖，糖尿病人的医疗条件和其它饮食紊乱的候选。

脑内多巴胺（DA）已通过牵连到可口的糖的摄入量响应中央提出的享乐1,2，努力有关的3和习惯为基础的行动4,5机制。在这些效应牵连主DA通路起源于腹侧被盖区（VTA），和项目到伏隔核（NAC）芯和壳，基底外侧和中央皮质内侧杏仁核（AMY），以及前度和infralimbic内侧前额叶皮层（内侧前额叶皮质）（查看评论6,7）。该VTA有牵连的蔗糖摄入量8,9，和DA释放后观察糖的摄入量在10-15 NAC，AMY 16,17和内侧前额叶皮质18-20。脂肪的摄入量也刺激多巴胺释放的NAC 21，另有丰富DA投影带的背侧纹状体（尾状核）已与DA介导的喂养22,23也被相关。凯利24-27提出，这些多项目该DA介导系统的离子区形成通过广泛而亲密互连28-34集成和交互的分布式网络的大脑。 除了​​DA D1和D2受体拮抗剂来减少糖35-37和脂肪38-40的摄取的能力，DA信令也已牵涉于介导糖和脂肪以产生调节味道偏好（CFP）的能力41- 46。一个DA D1受体拮抗剂进入NAC，AMY或内侧前额叶皮质47-49显微注射消除CFP的收购胃内引起的葡萄糖。而无论是DA D1或D2受体拮抗剂显微注射到内侧前额叶皮质消除收购果糖CFP 50，收购和果糖CFP表达的差异由NAC和AMY 51,52 DA受体拮抗剂阻断。 在c-fos技术53,54已被用来研究神经活化法制由可口的摄入和神经激活ñ诱导。术语"C-fos的激活"，将整个手稿中使用，并且通过增加的c-Fos蛋白的转录神经元去极化期间可操作限定。蔗糖摄取在中央AMY核增加fos的样免疫反应（FLI），所述VTA以及外壳，但不是核心，在NAC 55-57的。而糖水摄取量假喂养大鼠的AMY和NAC显著上升FLI，但不是VTA 58，胃内蔗糖或葡萄糖输注显著的NAC和AMY 59,60中央和基底外侧核增加FLI。重复加入蔗糖至预定食物访问在mPFC的增加FLI以及在NAC壳和芯61。蔗糖浓度降档模式显示，最大的FLI增加发生在基底AMY和NAC，但不是VTA 62。继空调，糖有关的自然雷瓦灭绝RD行为的基底AMY和NAC 63增加FLI。此外，配对糖可用性达到了口气导致口气随后在基底64 AMY增加FLI水平。高脂肪的摄入量也NAC和内侧前额叶皮质部位增加65-67 FLI。 大多数先前引用的研究中单个位点，不提供关于奖赏相关分布式大脑网络24-27的识别信息检测上的c-fos的激活糖和脂肪的效果。此外，许多研究也没有描绘在NAC（核和壳），AMY（基底和中央皮质内侧）和内侧前额叶皮质的子区域的相对贡献（前度和infralimbic）可能潜在的被检查优势出色的空间，在C-FOS映射68个单细胞的决议。我们的实验室69最近使用的c-fos的激活和同时测量改建的VTA DA通路及其亲jection区（NAC，AMY和内侧前额叶皮质）大鼠脂肪和糖的摄入小说之后。本研究描述的程序和方法步骤，同时分析是否急性暴露六个不同的解决方案（玉米油，葡萄糖，果糖，糖精，水和脂肪乳剂控制）在NAC，AMY的子区域将差异激活FLI，内侧前额叶皮质以及背侧纹状体。这种差异同时检测系统允许的每个站点和决心FLI显著效果的确认，是否与相关网站的变化密切相关，从而为分布式网络的脑支持24-27日在一个特定的站点变化。这些经过测试的程序是否VTA中，前度和infralimbic内侧前额叶皮质的NAC的核心和壳，以及基底和中央皮质，内侧AMY）以及背侧纹状体会显示协调，口服后，无条件的摄入同时FLI激活葡萄糖（8％），果糖（8％），玉米油（3.5％）和糖精（0.2％）的解决方案。

<table border="0" cellpadding="0" cellspacing="0" width="613">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:273px;">Equipment</td>
			<td style="width:195px;"></td>
			<td style="width:127px;"></td>
			<td style="width:19px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sprague-Dawley rats</td>
			<td>Charles River Laboratories</td>
			<td>CD-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Wire Mesh Cages</td>
			<td>Lab Products, Seaford, DE</td>
			<td>30-Cage rack</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Chow</td>
			<td>PMI Nutrition International</td>
			<td>5001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Taut Metal Spring</td>
			<td>Lab Products, Seaford, DE</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rat Weighing Scale</td>
			<td>Fisher Scientific Company</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nalgene Centrifuge Tubes</td>
			<td>Lab Products, Seaford, DE</td>
			<td>10-0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Rubber Stopper</td>
			<td>Lab Products, Seaford, DE</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Metal Sippers</td>
			<td>Lab Products, Seaford, DE</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Saccharin</td>
			<td>Sigma Chemical Co</td>
			<td>82385-42-0</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kool-Aid, Cherry</td>
			<td>Kool-Aid</td>
			<td>Commerical</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Kool-Aid, Grape</td>
			<td>Kool-Aid</td>
			<td>Commercial</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fructose</td>
			<td>Sigma Chemical Co</td>
			<td>F0127</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glucose</td>
			<td>Sigma Chemical Co</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Corn Oil</td>
			<td>Mazzola</td>
			<td>Commerical</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Xanthan Gum</td>
			<td>Sigma Chemical Co</td>
			<td>11138-66-2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sliding Microtome</td>
			<td>Microm International</td>
			<td>n/a</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Neurolucida Camera</td>
			<td>MBF Bioscience</td>
			<td colspan="2">Software application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin-coated Slides</td>
			<td>Fisher Scientific Company</td>
			<td>12-550-343</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cover glass</td>
			<td>Fisher Scientific Company</td>
			<td>12-545-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Golden Nylon Brushes</td>
			<td>Loew-Cornell&#160;</td>
			<td>2037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Natural Hair Sable&#160;</td>
			<td>Loew-Cornell&#160;</td>
			<td>2022</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 Well Plates</td>
			<td>Fisher Scientific</td>
			<td>3527</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 Well Plates</td>
			<td>Fisher Scientific</td>
			<td>3506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1L Pyrex bottles</td>
			<td>Fisher Scientific</td>
			<td>1395-1L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue insert (tissue strainer)</td>
			<td>Fisher Scientific</td>
			<td>7200214</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Eagle pipettes&#160;</td>
			<td>World Precision Instruments</td>
			<td>E10 for 1-10ul&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eagle pipettes&#160;</td>
			<td>World Precision Instruments</td>
			<td>E100 for 20-100ul</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eagle pipettes&#160;</td>
			<td>World Precision Instruments</td>
			<td>E200 for 50-200ul</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eagle pipettes&#160;</td>
			<td>World Precision Instruments</td>
			<td colspan="2">E1000 for 100-1000ul</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Eagle pipettes&#160;</td>
			<td>World Precision Instruments</td>
			<td colspan="2">E5000 for 1000-5000ul&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Universal Tips .1-10ul</td>
			<td>World Precision Instruments</td>
			<td>500192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Universal Tips 5-200ul</td>
			<td>World Precision Instruments</td>
			<td>500194</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Universal Tips 500-5000ul</td>
			<td>World Precision Instruments</td>
			<td>500198</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blade Vibroslice 100</td>
			<td>World Precision Instruments</td>
			<td>BLADE</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DPX Mounting Medium&#160;</td>
			<td>Electron Microscopy&#160;</td>
			<td>13510</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL centrifuge tubes</td>
			<td>Biologix Research Co.</td>
			<td>10-0501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Slide Boxes</td>
			<td>Biologix Research Co.</td>
			<td>41-6100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Orbital Shaker&#160;</td>
			<td>Madell Corporation&#160;&#160;</td>
			<td>ZD-9556</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">weigh boats&#160;</td>
			<td>Fisher Scientific</td>
			<td>02-202-100</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">5mL disposable pipettes</td>
			<td>Fisher Scientific</td>
			<td>13-711-5AM</td>
			<td></td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Stereo Investigator Software</td>
			<td>Micro Bright Field</td>
			<td colspan="2">Software application</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde Granular</td>
			<td>Fisher Scientific</td>
			<td>19210</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaCl</td>
			<td>Fisher Scientific</td>
			<td>S271-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phophate Monobasic</td>
			<td>Fisher Scientific</td>
			<td>S468-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Phosphate Diphasic</td>
			<td>Fisher Scientific</td>
			<td>BP332-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hydrogen Peroxide&#160;</td>
			<td>Fisher Scientific</td>
			<td>H324-500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">SafeClear II&#160;</td>
			<td>Fisher Scientific</td>
			<td>23-044-192</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Methanol&#160;</td>
			<td>Fisher Scientific</td>
			<td>A412-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Normal Goat Serum</td>
			<td>Vector</td>
			<td>S-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Biotinylated Anti-Rabbit IgG (H+L)</td>
			<td>Vector</td>
			<td>BA-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ABC Kit Peroxidase Standard</td>
			<td>Vector</td>
			<td>PK-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Anti-cFos (Ab-5) Rabbit</td>
			<td>EMD chem/Cal Biochem</td>
			<td>PC38</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X 100</td>
			<td>SigmaAldrich</td>
			<td>X-100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3,3&#39; diaminobenzidine tetra hydrochloride&#160;</td>
			<td>SigmaAldrich</td>
			<td>D5905</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Hydroxide</td>
			<td>SigmaAldrich</td>
			<td>5881</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Primary TH anti body</td>
			<td>EMD Millipore</td>
			<td>AB152</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Euthosol</td>
			<td>Virbac AH</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

simultaneous detection of c-fos activation from mesolimbic and mesocortical dopamine reward sites following naive sugar and fat ingestion in rats

本研究采用细胞的c-fos的激活来评估脂肪和糖对大鼠脑内多巴胺（DA）的新途径摄入的影响。糖和脂肪摄入量是由他们天生的景点，以及了解到的喜好调节。脑多巴胺，特别是内消旋 - 边缘和从腹侧被盖区（VTA）内消旋 - 皮质突起，已经牵涉在这两个未学习并了解到响应。分布式脑网络，其中的几个位点和发射机/肽系统交互的概念，已提出​​调解适口食物摄入量，但有证据有限经验证明这种行动。因此，糖的摄入引起从个体VTA多巴胺投影区，包括伏隔核（NAC），杏仁核（AMY）DA释放和增加的c-fos的样免疫反应（FLI）和内侧前额叶皮质（mPFC的）以及背侧纹状体。此外，选择性多巴胺受体拮抗剂的集中管理到这些网站Ş差异减少糖类和脂肪引起的空调味的喜好采集和表达。一种方法，借以确定这些网站是否响应糖或脂肪摄入互动作为分布式脑网络将同时评估是否对VTA及其主要mesotelencephalic DA投影区域（前度和伏隔核的infralimbic内侧前额叶皮质，核和壳，基底外侧和中央皮质，内侧AMY）以及背侧纹状体会显示协调，口服后，无条件的摄入玉米油（3.5％），葡萄糖（8％），果糖（8％）和糖精的同时FLI激活（0.2 ％）的解决方案。这种方法在鉴别同时使用到相关的大脑部位细胞的c-fos的激活，研究中啮齿类动物的可口食物摄取奖励有关学习的可行性了成功的第一步。

下面描述的所有代表性结果先前已发表​​69，并在此处重新提出支持"概念验证"中指示的技术的有效性。 解决方案摄入量 在基线糖精摄入显著差异观察到在第一四天所有动物（F（3108）= 57.27，P &lt;0.001）与摄入量（第1天:1.3（±0.2）毫升;第2天:3.9（±0.4）毫升;第3天:5.9（±0.6）毫升;第4天:7.1（±0.6）毫升）显著（p &lt;0.05，图基HSD检验）和逐渐增加。在第5天显著果糖和葡萄糖摄取，但不玉米油或糖精摄入量（P &lt;0.05，图基HSD检验）相对于第4天糖精摄取升高（p &lt;0.05，图基HSD检验）与果糖（9.6（±0.4）毫升）和葡萄糖（9.4（±0.6）毫升）比SACC显著更高harin摄入量。此外，玉米油摄取（7.4（±0.6）毫升）溶液显著（P &lt;0.05，图基HSD检验）高于黄原胶摄取更高。 这些结果提出，溶液摄取本身可能占任何站点的任何观察到的c-fos的激活的可能性。为了检验这一点，进行邦弗朗尼 - [R相关，其中五个解决方案的摄入量有关每个六个站点的c-fos激活。显著相关未能在芯溶液摄取和c-fos激活之间进行观察（R（29）= 0.186），壳（R（29）= 0.029）或总（R（29）= 0.10）伏隔，所述前度（ R（29）= 0.23），infralimbic（R（29）= 0.30）或总（R（29）= 0.14）内侧前额叶皮质的腹侧被盖区（R（29）= 0.10），背侧纹状体（R（29）= 0.14 ）或基底（R（29）= 0.47），炫酷，皮质中层（R（29）= 0.48）或总（R（29）= 0.409），AMY。由于摄入量和AMY的c-fos的激活，进一步correla之间的相关性较高系统蒸发散分别为每个单独的溶液来进行。显著（P &lt;0.05，图基HSD检验）关系失败摄入和AMY FLI之间被观察为果糖（基底（R = 0.15），中央 - 皮质内侧相关（r = 0.13），总相关（r = 0.13）），葡萄糖（基底（R = 0.17），中央 - 皮质内侧（R = 0.17），总R = 0.13）），糖精（基底（R = 0.42），中央 - 皮质内侧（R = 0.42），总相关（r = 0.42））或玉米油（基底（R = 0.54），中央 - 皮质内侧（R = 0.59），总相关（r = 0.64））。一个显著（P &lt;0.05，杜克HSD检验）呈负相关黄原胶的摄入量和总AMY FLI相关（r = 0.94）之间的观察。 内侧前额叶皮质c-fos激活 玉米油显著（P &lt;0.05，图基HSD检验）增加的总（ 图2A），infralimbic（ 图2B）和前度（ 图2C）mPFC的的c-Fos的计数相对于水（*）或黄原胶控制（＃）。显著果糖（P &lt;0.05，图基HSD检验）infralimbic mPFC的相对增加的c-Fos的计数到水（*）或糖精（+）（ 图2B），但不完全或前度mPFC的计数。相反，葡萄糖或糖精未能改变总，perilimbic或infralimbic mPFC的的c-Fos的计数。 图动物示出玉米油诱导FLI相对于水增加的3显示代表mPFC的部分。 <img alt="图2" src="/files/ftp_upload/53897/53897fig2.jpg" /> 图2. 脂肪或糖的摄入量增加差异的c-fos的激活在内侧前额叶皮质（mPFC的）变化的c-fos的激活（平均值±SEM）在整个内侧前额叶皮质（A组 ），该infralimbic内侧前额叶皮质区域中指出（ 面板B）和该前度mPFC的区域的水（ 图C）以下消费（1小时），糖精（0.2％），黄原胶（玉米油控制），葡萄糖（8％），果糖（8％），或玉米油（3.5％）。 （此前公布的69）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53897/53897fig3.jpg" /> 图3. 实际mPFC的的c-fos的激活之后的脂肪和糖。在暴露于是显著更大玉米油（ 图A（4倍放大倍率）和C（10倍放大倍率））的摄取动物中观察到的C-fos的激活比水（ 图B（4倍放大倍率）和D（10倍放大倍率））的摄入。的前度（PL）和infralimbic（IL）内侧前额叶皮质的划定子区域的代表性，可图示于图A（玉米油）和C（水），因为是T的一部分放大到在相应的板10倍的倍率（B和D）软管面板（A和C）。在面板C和D箭头指示代表c-fos阳性细胞。所有比例尺为100微米。 （此前公布的69）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> AMY c-fos激活 玉米油显著（P &lt;0.05，杜克HSD检验）增加总AMY（ 图4A），基底（ 图4B）和中央皮质内侧（ 图4C）子区域AMY的c-Fos的计算相对于水（*）或黄原胶控制（＃）。葡萄糖也显著（P &lt;0.05，杜克HSD检验）共增加（ 图 4A），基底（ 图4B）和中央皮质，内侧（ <s仲&gt;图4C）子区域AMY的c-Fos的计数相对于水（*）或糖精（+）葡萄糖和显著果糖（P &lt;0.05，图基HSD检验）增加AMY FLI相对糖精或水。显著果糖（P &lt;0.05，图基HSD检验）增加的总（ 图4A）和中央皮质内侧子区的AMY的（ 图4C）的c-Fos的计数相对于水（*）或糖精（+），但未在基底外侧AMY子区域。糖精没有改变总的，基底或中央皮质内侧AMY的c-Fos的计算相对于水。的AMY内的单个核的详细分析表明，在AMY的基底区域注意到显著变化在个体基底外侧和横向AMY细胞核也明显。在AMY的中央皮质区内侧注意到显著的变化在个人中心，皮层和内侧核AMY还注意到， 图5显示了代表AMY节的动物示出增加的玉米油，葡萄糖和果糖诱导的相对于水FLI。 （此前公布的69）。 <img alt="图4" src="/files/ftp_upload/53897/53897fig4.jpg" /> 图4. 脂肪或糖的摄入量差异增加的c-fos的激活在杏仁核（AMY）变化的c-fos的激活（平均值±SEM）在整个AMY（A组 ）都指出，基底外侧AMY区（B组 ）和以下的水，糖精，黄原胶，葡萄糖，果糖或玉米油消耗量（1小时）的中央皮质内侧AMY区域（ 图C）。在AMY的基底区域注意到显著变化在个人基底外侧和横向AMY细胞核也明显。在AMY的中央皮质膜面积注意到显著的变化在个人中心，皮层和内侧AMY细胞核也明显。 （此前公布的69）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/53897/53897fig5.jpg" /> 图5.实际AMY的c-fos的激活之后的脂肪和糖。在暴露于玉米油（ 图A（4倍放大倍率），D（10倍放大倍率）和G的摄入动物中观察到的C-fos的激活（60-折叠倍率）），葡萄糖（ 图B（4倍放大倍率）和E（10倍放大率））和果糖（ 图C（4倍放大倍率）和F（10倍）），该均高于显著大于的水（ 面板的H（4倍放大倍率）和I（10倍放大倍率））的摄入。的表示中央-皮质内侧（CMC）和基底（BLA）AMY的划定子区域中板A（玉米油），B（葡萄糖）的可图示，C（果糖）和H（水），因为是部分那些在相应的面板（D，E，F和I）放大到10倍的放大倍率面板（A，B，C和H）。划定子区域的D组（玉米油，10倍的放大倍率）被放大到60倍的放大倍率面板D.箭头在图D，E，F，G和我指示代表的c-Fos阳性细胞。所有的比例尺是100微米，除了面板G（50微米）。 （此前公布的69）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> NAC c-fos激活 玉米油显著（P &lt;0.05，杜克HSD检验）增加总的（ 图6A）和核心（ <s仲&gt;图6B）伏核的c-Fos的计数相对于水（*），但不是伏隔壳（ 图6C）。显著葡萄糖（P &lt;0.05，图基HSD检验）在NAC芯（ 图6B）增加的c-Fos的计数，而不是在总伏隔或伏隔相对糖精（+）或水（*）的壳。与此相反，果糖和糖精未能从水在伏隔核和/或壳中引发的c-Fos蛋白的激活不同。 图7显示在动物中显示出增加的玉米油或葡萄糖诱导相对于水FLI的伏隔核代表节。 <img alt="图6" src="/files/ftp_upload/53897/53897fig6.jpg" /> 图6.脂肪或糖的摄入量差异增加的c-fos的激活在NAC中的c-fos的激活变化（平均值±SEM）在整个NAC（A组 ），在NAC核心（B组 ）和NAC壳（以下的水，糖精，黄原胶，葡萄糖，果糖或玉米油消耗量（1小时）图C）。 （此前公布的69）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图7" src="/files/ftp_upload/53897/53897fig7.jpg" /> 图7.实际NAC核心，但不是NAC壳的c-fos的激活之后的脂肪和糖。在暴露于玉米油（ 图A（4倍放大倍率），D（10倍放大倍率的摄入动物中观察到的C-fos的激活）和G（60倍放大率））和葡萄糖（ 图B（4倍放大倍率）和E（10倍放大率）），该均高于水摄入显著更大（ 图C（4倍放大倍率）和F（10倍放大倍率化））。在伏隔核和伏隔核壳的划定子区域的表示在图A（玉米油）的可图示，B（葡萄糖）和C（水）为那些面板（A，B，C和H）的一部分放大到10倍的倍率在相应的面板（D，E和F）。所描绘的子区中图D（玉米油，10倍放大率）被放大到在面板D，E在面板G.箭头60倍的倍率，F和G指示代表的c-fos的阳性细胞。所有比例尺为100微米。 （此前公布的69）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig7large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 背纹状体c-fos激活 玉米油显著（P &lt;0.05，图基HSD检验）相对的背侧纹状体中增加的c-Fos的计数到水（*）或黄原胶（＃）（FIGURË8A）。葡萄糖或显著果糖（P &lt;0.05，图基HSD检验）背侧纹状体FLI相对糖精（+）（ 图8A）增加。与此相反，糖精未能从水中引出背纹状体的c-Fos蛋白的激活不同。 图动物示出增加的玉米油，相对于水葡萄糖或果糖诱导FLI的9上显示代表背侧纹状体切片。 <img alt="图8" src="/files/ftp_upload/53897/53897fig8.jpg" /> 图8.脂肪或糖的摄入量差异增加的c-fos的激活背侧纹状体和腹侧被盖区。背atriatal（A组 ）和改建被注意到的c-fos的激活腹侧被盖区（B组 ）（平均值±SEM）以下的水，糖精，黄原胶，葡萄糖，果糖或玉米油消耗量（1小时）。 （此前公布的69）。 <aHREF ="htt​​ps://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig8large.jpg"目标="_空白"&gt;点击此处查看该图的放大版本。 <img alt="图9" src="/files/ftp_upload/53897/53897fig9.jpg" /> 在暴露于玉米油（ 图A（4倍放大倍率），D（10倍放大倍率）和G的摄入动物中观察到图9的实际背纹状体的c-fos的激活之后的脂肪和糖。c-fos活化（60倍倍率）），葡萄糖（ 图B（4倍放大倍率）和E（10倍放大率）），果糖 （ 图C（4倍放大倍率）和F（10倍放大率）），该均高于水摄入显著更大（ 面板的H（4倍放大倍率）和I（10倍放大率））。划定子一在面板A（玉米油）的背侧纹状体的REAS，B（葡萄糖），C（果糖）和H（水）表示，放大到10倍的放大倍率相应的面板（D，E，F和I）。所描绘的子区中图D（玉米油，10倍放大率）被放大到在面板D，E在面板G.箭头60倍的倍率，F和G指示代表的c-fos的阳性细胞。所有的比例尺是100微米，除了面板G（50微米）。 （此前公布的69）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig9large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> VTA c-fos激活 玉米油显著（P &lt;0.05，杜克HSD试验）TH + VTA细胞相对于黄原胶控制（＃）（ 图8B）增加c-fos计数。相反，葡萄糖，果糖或糖精未能改变VTA相对中的c-Fos的计数<html>水。 图10显示了代表TH +和TH-和动物显示出玉米油引起的FLI相对于水增加的c-fos激活VTA细胞。 <img alt="图10" src="/files/ftp_upload/53897/53897fig10.jpg" /> 图10.实际腹侧被盖区的c-fos的激活之后的脂肪和糖。VTA中暴露于玉米油（ 图A（4倍）和C（10倍））和水（ 图B的动物中观察到的c-fos的激活（4倍）和D（10倍））。黑色箭头指示代表双标TH / c-fos阳性细胞，而灰色箭头指示代表的c-fos的唯一细胞。所有比例尺为100微米。 （此前公布的69）。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53897/53897fig10large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> ove_content"FO:保together.within页="1"&gt; c-fos激活在网站和解决方案的关系 的c-Fos蛋白的计数在暴露于玉米油动物的图案显露显著（P &lt;0.05）正面的伏隔核和任一伏隔壳之间的相关性（r = 0.971）或整个mPFC的相关（r = 0.670）时，前度mPFC的和之间任infralimbic mPFC的相关（r = 0.940）或背侧纹状体性（r = 0.849）时，infralimbic mPFC的和背侧纹状体之间相关（r = 0.749），基底外侧和中央皮质内侧AMY之间相关（r = 0.999）之间，以及背侧纹状体和腹侧被盖区（R = 0.723）。与此相反，c-fos的计数在暴露于玉米油动物图案显露显著（P &lt;0.05）的负基底外侧AMY和任一伏隔核之间的相关性（r = -0.712）或壳（R = -0.708），和中央-皮质内侧AMY，要么在伏隔核相关（r = -0.712）或shell相关（r = -0.710），c-fos的.The模式动物ë计数xposed 葡萄糖揭示显著（P &lt;0.05）正面的前度和infralimbic mPFC的相关（r = 0.930）之间的相关性，背侧纹状体和任VTA之间相关（r = 0.821），基底（R = 0.910）或中央皮质内侧（R = 0.911），AMY和基底外侧和中央皮质内侧之间的相关（r = 0.999），AMY。的c-Fos蛋白的计数在暴露于果糖动物图案显露显著（P &lt;0.05）正面的伏隔核和任一伏隔壳之间的相关性（r = 0.969）或前度mPFC的相关（r = 0.740），伏隔壳之间的前度mPFC的相关（r = 0.733）时，前度和infralimbic mPFC的之间相关（r = 0.959），和基底外侧和中央皮质内侧AMY之间相关（r = 0.996）的c-Fos蛋白的计数在暴露的动物.The图案糖精揭示显著（p &lt;0.05），伏隔核和伏隔核的外壳呈正相关（r = 0.792），在伏隔核的外壳和背侧纹状体相关（r = 0.715），与前度mPFC的一间第二infralimbic内侧前额叶皮质（R = 0.999）。

Watch this Scientific Journal Video about Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats at JoVE.com

Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

这项研究的目的是通过使用蜂窝的c-fos的激活来测量大鼠的脂肪和糖新颖摄入后多巴胺途径和终端站点同步变化划定一个可靠免疫组织学技术，以确定奖励相关的分布式脑网络。

从脑边缘和Mesocortical多巴胺奖赏网站继朴素的糖和脂肪摄入大鼠c-fos激活的同时检测

This study uses cellular c-fos activation to assess effects of novel ingestion of fat and sugar on brain dopamine (DA) pathways in rats. Intakes of sugars ...

simultaneous-detection-c-fos-activation-from-mesolimbic-mesocortical

Nanyang Technological University

Research

JoVE Journal

Neuroscience

9.0K 次观看.  CUNY, New York, NY. 这项研究的目的是通过使用蜂窝的c-fos的激活来测量大鼠的脂肪和糖新颖摄入后多巴胺途径和终端站点同步变化划定一个可靠免疫组织学技术，以确定奖励相关的分布式脑网络。 

视频: Simultaneous Detection of c-Fos Activation from Mesolimbic and Mesocortical Dopamine Reward Sites Following Naive Sugar and Fat Ingestion in Rats

Simultaneous fMRI and Electrophysiology in the Rodent Brain

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in which functional MRI data and in vivo intracortical recording can be performed simultaneously. The combination of MRI and electrical recording is technically challenging because the electrodes used for recording distort the MRI images and the MRI acquisition induces noise in the electrical recording. To minimize the mutual interference of the two modalities, glass microelectrodes were used rather than metal and a noise removal algorithm was implemented for the electrophysiology data. In our studies, two microelectrodes were separately implanted in bilateral primary somatosensory cortices (SI) of the rat and fixed in place. One coronal slice covering the electrode tips was selected for functional MRI. Electrode shafts and fixation positions were not included in the image slice to avoid imaging artifacts. The removed scalp was replaced with toothpaste to reduce susceptibility mismatch and prevent Gibbs ringing artifacts in the images. The artifact structure induced in the electrical recordings by the rapidly-switching magnetic fields during image acquisition was characterized by averaging all cycles of scans for each run. The noise structure during imaging was then subtracted from original recordings. The denoised time courses were then used for further analysis in combination with the fMRI data. As an example, the simultaneous acquisition was used to determine the relationship between spontaneous fMRI BOLD signals and band-limited intracortical electrical activity. Simultaneous fMRI and electrophysiological recording in the rodent will provide a platform for many exciting applications in neuroscience in addition to elucidating the relationship between the fMRI BOLD signal and neuronal activity.

We have developed a method for simultaneous functional magnetic resonance imaging and electrophysiological recording in the rodent brain, providing a platform for the investigation of the relationship between neural activity and the blood oxygenation level dependent (BOLD) MRI signal.

同时在啮齿动物脑功能磁共振成像和电

To examine the neural basis of the blood oxygenation level dependent (BOLD) magnetic resonance imaging (MRI) signal, we have developed a rodent model in ...

科研

神经科学

Chromatin Immunoprecipitation from Dorsal Root Ganglia Tissue following Axonal Injury

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after injury (Teng et al., 2006). It is recognized that transcriptional programs essential for neurite and axonal outgrowth are 
reactivated upon injury in the PNS (Makwana et al., 2005). However the tools available to analyze neuronal gene regulation in vivo are limited and 
often challenging.
The dorsal root ganglia (DRG) offer an excellent injury model system because both the CNS and PNS are innervated by a 
bifurcated axon originating from the same soma. The ganglia represent a discrete collection of cell bodies where all transcriptional events occur, 
and thus provide a clearly defined region of transcriptional activity that can be easily and reproducibly removed from the animal. Injury of nerve 
fibers in the PNS (e.g. sciatic nerve), where axonal regeneration does occur, should reveal a set of transcriptional programs that are distinct from 
those responding to a similar injury in the CNS, where regeneration does not take place (e.g. spinal cord). Sites for transcription factor binding, 
histone and DNA modification resulting from injury to either PNS or CNS can be characterized using chromatin immunoprecipitation (ChIP). 
Here, we describe a ChIP protocol using fixed mouse DRG tissue following axonal injury. This powerful combination provides a means for characterizing the pro-regeneration chromatin environment necessary for promoting axonal regeneration.

We present a method for chromatin immunoprecipitation from dorsal root ganglia tissue following axonal injury. The approach can be used to identify specific transcription factor binding sites and epigenetic modification of histone and DNA important for the regeneration of injured axons in both the peripheral and central nervous system.

染色质免疫沉淀从以下轴索损伤的背根神经节组织

Axons in the central nervous system (CNS) do not regenerate while those in the peripheral nervous system (PNS) do regenerate 
to a limited extent after ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated. Thus, simultaneous recordings of neuronal activity and behavior are essential to correlate brain activity to behavior. For such behavioral analyses, the fruit fly, Drosophila melanogaster, allows us to incorporate genetically encoded calcium indicators such as GCaMP1, to monitor neuronal activity, and to use sophisticated genetic manipulations for optogenetic or thermogenetic techniques to specifically activate identified neurons2-5. Use of a thermogenetic technique has led us to find critical neurons for feeding behavior (Flood et al., under revision). As a main part of feeding behavior, a Drosophila adult extends its proboscis for feeding6 (proboscis extension response; PER), responding to a sweet stimulus from sensory cells on its proboscis or tarsi. Combining the protocol for PER7 with a calcium imaging technique8 using GCaMP3.01, 9, I have established an experimental system, where we can monitor activity of neurons in the feeding center &#x2013; the suboesophageal ganglion (SOG), simultaneously with behavioral observation of the proboscis. I have designed an apparatus ("Fly brain Live Imaging and Electrophysiology Stage": "FLIES") to accommodate a Drosophila adult, allowing its proboscis to freely move while its brain is exposed to the bath for Ca2+ imaging through a water immersion lens. The FLIES is also appropriate for many types of live experiments on fly brains such as electrophysiological recording or time lapse imaging of synaptic morphology. Because the results from live imaging can be directly correlated with the simultaneous PER behavior, this methodology can provide an excellent experimental system to study information processing of neuronal networks, and how this cellular activity is coupled to plastic processes and memory.

Simultaneous Recording of Calcium Signals from Identified Neurons and Feeding Behavior of Drosophila melanogaster

The fruit fly, Drosophila melanogaster, extends its proboscis for feeding, responding to a sugar stimulus from its proboscis or tarsus. I have combined observations of the proboscis extension response (PER) with a calcium imaging technique, allowing us to monitor the activity of neurons in the brain, simultaneously with behavioral observation.

从查明的神经元和取食行为的钙信号的同时记录果蝇</em

To study neuronal networks in terms of their function in behavior, we must analyze how neurons operate when each behavioral pattern is generated.  Thus, ...

Progressive-ratio Responding for Palatable High-fat and High-sugar Food in Mice

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can produce a rewarding effect that strengthens action-outcome associations and reinforces future behavior directed at obtaining these foods. Increasing evidence that the rewarding effects of energy-dense foods play a profound role in overeating and the development of obesity has heightened interest in studying the genes, molecules and neural circuitry that modulate food reward1,2. The rewarding impact of different stimuli can be studied by measuring the willingness to work to obtain them, such as in operant conditioning tasks3. Operant models of food reward measure acquired and voluntary behavioral responses that are directed at obtaining food. A commonly used measure of reward strength is an operant procedure known as the progressive ratio (PR) schedule of reinforcement.4,5 In the PR task, the subject is required to make an increasing number of operant responses for each successive reward. The pioneering study of Hodos (1961) demonstrated that the number of responses made to obtain the last reward, termed the breakpoint, serves as an index of reward strength4. While operant procedures that measure changes in response rate alone cannot separate changes in reward strength from alterations in performance capacity, the breakpoint derived from the PR schedule is a well-validated measure of the rewarding effects of food. The PR task has been used extensively to assess the rewarding impact of drugs of abuse and food in rats (e.g.,6-8), but to a lesser extent in mice9. The increased use of genetically engineered mice and diet-induced obese mouse models has heightened demands for behavioral measures of food reward in mice. In the present article we detail the materials and procedures used to train mice to respond (lever-press) for a high-fat and high-sugar food pellets on a PR schedule of reinforcement. We show that breakpoint response thresholds increase following acute food deprivation and decrease with peripheral administration of the anorectic hormone leptin and thereby validate the use of this food-operant paradigm in mice.

The present report details the protocol employed to measure the rewarding effects of high-fat food in mice using a progressive ratio operant conditioning task.

累进比率响应小鼠可口的高脂肪和高糖食品

Foods that are rich in fat and sugar significantly contribute to over-eating and escalating rates of obesity. The consumption of palatable foods can ...

Single Cell Measurement of Dopamine Release with Simultaneous Voltage-clamp and Amperometry

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is terminated by diffusion2-3, extracellular enzymes4, and membrane transporters5. The dopamine transporter, located in the peri-synaptic cleft of dopamine neurons clears the released amines through an inward dopamine flux (uptake). The dopamine transporter can also work in reverse direction to release amines from inside to outside in a process called outward transport or efflux of dopamine5. More than 20 years ago Sulzer et al. reported the dopamine transporter can operate in two modes of activity: forward (uptake) and reverse (efflux)5. The neurotransmitter released via efflux through the transporter can move a large amount of dopamine to the extracellular space, and has been shown to play a major regulatory role in extracellular dopamine homeostasis6. Here we describe how simultaneous patch clamp and amperometry recording can be used to measure released dopamine via the efflux mechanism with millisecond time resolution when the membrane potential is controlled. For this, whole-cell current and oxidative (amperometric) signals are measured simultaneously using an Axopatch 200B amplifier (Molecular Devices, with a low-pass Bessel filter set at 1,000 Hz for whole-cell current recording). For amperometry recording a carbon fiber electrode is connected to a second amplifier (Axopatch 200B) and is placed adjacent to the plasma membrane and held at +700 mV. The whole-cell and oxidative (amperometric) currents can be recorded and the current-voltage relationship can be generated using a voltage step protocol. Unlike the usual amperometric calibration, which requires conversion to concentration, the current is reported directly without considering the effective volume7. Thus, the resulting data represent a lower limit to dopamine efflux because some transmitter is lost to the bulk solution.

The amperometric technique measures dopamine release from a single cell by detecting the oxidative current produced by spontaneous dopamine oxidization. Simultaneous voltage clamp and amperometry methodology reveal the mechanistic relationship between the overall "activity" of dopamine transporter and the regulatory role of this activity on the reverse transport of dopamine.

单细胞的多巴胺释放，同时电压钳和电流分析法测量

After its release into the synaptic cleft, dopamine exerts its biological properties via its pre- and post-synaptic targets1. The dopamine signal is ...

Comprehensive Profiling of Dopamine Regulation in Substantia Nigra and Ventral Tegmental Area

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five decades of inquiry into the molecular deficiencies associated with dopamine regulation. The majority of these inquiries of dopamine regulation in the brain focus upon the molecular basis for its regulation in the terminal field regions of the nigrostriatal and mesoaccumbens pathways; striatum and nucleus accumbens. Furthermore, such studies have concentrated on analysis of dopamine tissue content with normalization to only wet tissue weight. Investigation of the proteins that regulate dopamine, such as tyrosine hydroxylase (TH) protein, TH phosphorylation, dopamine transporter (DAT), and vesicular monoamine transporter 2 (VMAT2) protein often do not include analysis of dopamine tissue content in the same sample. The ability to analyze both dopamine tissue content and its regulating proteins (including post-translational modifications) not only gives inherent power to interpreting the relationship of dopamine with the protein level and function of TH, DAT, or VMAT2, but also extends sample economy. This translates into less cost, and yet produces insights into the molecular regulation of dopamine in virtually any paradigm of the investigators' choice.
We focus the analyses in the midbrain. Although the SN and VTA are typically neglected in most studies of dopamine regulation, these nuclei are easily dissected with practice. A comprehensive readout of dopamine tissue content and TH, DAT, or VMAT2 can be conducted. There is burgeoning literature on the impact of dopamine function in the SN and VTA on behavior, and the impingements of exogenous substances or disease processes therein 1-5. Furthermore, compounds such as growth factors have a profound effect on dopamine and dopamine-regulating proteins, to a comparatively greater extent in the SN or VTA 6-8. Therefore, this methodology is presented for reference to laboratories that want to extend their inquiries on how specific treatments modulate behaviour and dopamine regulation. Here, a multi-step method is presented for the analyses of dopamine tissue content, the protein levels of TH, DAT, or VMAT2, and TH phosphorylation from the substantia nigra and VTA from rodent midbrain. The analysis of TH phosphorylation can yield significant insights into not only how TH activity is regulated, but also the signaling cascades affected in the somatodendritic nuclei in a given paradigm.
We will illustrate the dissection technique to segregate these two nuclei and the sample processing of dissected tissue that produces a profile revealing molecular mechanisms of dopamine regulation in vivo, specific for each nuclei (Figure 1).

Dopamine is distinctly regulated in the midbrain nuclei, which contain the cell bodies and dendrites of the dopamine neurons. Here we describe a dissection and sample-handling approach to maximize results, and thus conclusions and insights, on dopamine regulation in the midbrain nuclei of the substantia nigra (SN) and ventral tegmental area (VTA) in rodents.

在黑质和腹侧被盖区多巴胺调控的综合性能分析

Dopamine is a vigorously studied neurotransmitter in the CNS. Indeed, its involvement in locomotor activity and reward-related behaviour has fostered five ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Simultaneous Recording of Electroretinography and Visual Evoked Potentials in Anesthetized Rats

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the electrical responses of the retina to light stimulation, while the VEP measures the corresponding functional integrity of the visual pathways from the retina to the primary visual cortex following the same light event. The ERG waveform can be broken down into components that reflect responses from different retinal neuronal and glial cell classes. The early components of the VEP waveform represent the integrity of the optic nerve and higher cortical centers. These recordings can be conducted in isolation or together, depending on the application. The methodology described in this paper allows simultaneous assessment of retinal and cortical visual evoked electrophysiology from both eyes and both hemispheres. This is a useful way to more comprehensively assess retinal function and the upstream effects that changes in retinal function can have on visual evoked cortical function.

This protocol describes simultaneous measurement of electroretinogram and visual evoked potentials in anesthetized rats.

电图同时记录和麻醉大鼠视觉诱发电位

The electroretinogram (ERG) and visual evoked potential (VEP) are commonly used to assess the integrity of the visual pathway. The ERG measures the ...

Assessment of Dopaminergic Homeostasis in Mice by Use of High-performance Liquid Chromatography Analysis and Synaptosomal Dopamine Uptake

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) neurotransmission is strongly associated with several central nervous system-associated diseases such as Parkinson&#39;s disease, attention-deficit-hyperactivity disorder and drug addiction1,2,3,4. Delineating disease mechanisms involving DA imbalance is critically dependent on animal models to mimic aspects of the diseases, and thus protocols that assess specific parts of the DA homeostasis are important to provide novel insights and possible therapeutic targets for these diseases.
Here, we present two useful experimental protocols that when combined provide a functional read-out of the DAergic system in mice. Biochemical and functional parameters on DA homeostasis are obtained through assessment of DA levels and dopamine transporter (DAT) functionality5. When investigating the DA system, the ability to reliably measure endogenous levels of DA from adult brain is essential. Therefore, we present how to perform high-performance liquid chromatography (HPLC) on brain tissue from mice to determine levels of DA. We perform the experiment on tissue from dorsal striatum (dStr) and nucleus accumbens (NAc), but the method is also suitable for other DA-innervated brain areas.
DAT is essential for reuptake of DA into the presynaptic terminal, thereby controlling the temporal and spatial activity of released DA. Knowing the levels and functionality of DAT in the striatum is of major importance when assessing DA homeostasis. Here, we provide a protocol that allows to simultaneously deduce information on surface levels and function using a synaptosomal6 DA uptake assay.
Current methods combined with standard immunoblotting protocols provide the researcher with relevant tools to characterize the DAergic system.

Synaptosomal dopamine uptake and high-performance liquid chromatography analysis represent experimental tools to investigate dopamine homeostasis in mice by assessing the function of the dopamine transporter and levels of dopamine in striatal tissue, respectively. Here we present protocols to measure dopamine tissue content and assess the functionality of the dopamine transporter.

高效液相色谱分析和触多巴胺摄取对小鼠多巴胺能稳态的评价

Dopamine (DA) is a modulatory neurotransmitter controlling motor activity, reward processes and cognitive function. Impairment of dopaminergic (DAergic) ...