Name: environmental modulations of the number of midbrain dopamine neurons in adult mice
Uploaded: 2015-01-20T14:00:00.000+00:00
Duration: 9 min 35 s
Description: Watch this Scientific Journal Video about Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice at JoVE.com

This study was supported by the National Health and Medical Research Council of Australia (NHMRC) Project grant 1022839. AJH is an Australian Research Council (ARC) FT3 Future Fellow (FT100100835). The Florey Institute of Neuroscience and Mental Health acknowledges support from the Victorian Government&#8217;s Operational Infrastructure Support Grant.

注:对动物的所有实验程序批准了神经科学和心理健康动物伦理委员会的弗洛里学院，并符合澳大利亚国家卫生和医学研究理事会公布的做法，动物科学目的的维护和使用的代码（ 第 7版， 2004年）。 1.环境手法<ol><li>性别配对<ol><li>使用性成熟（&gt; 8周龄），年龄匹配的雄性和雌性小鼠。 注意:我们通常使用C57BL / 6小鼠，但不得不使用Swiss小鼠在这个协议中相同的结果。我们通常使用每组8只雄性和n = 8的女性对每个实验，分成n = 2男男性对（N = 4男），N = 2女女对（N = 4女），和n = 4男女对（N = 4的男性和n = 4女）。 </li><li>抵达后进入动物圈养设施，集团内部的小鼠性别&gt; 3天适应环境。在这里，和通过量豪特性别配对，让外来的环境刺激（ 如环境温度，光照:黑暗循环，食物，水，处理和清洗）不变或平均分配给所有小鼠。 </li><li>随机每只小鼠分配给男性，男性对，女性对女性或男性，女性对。放置各对到干净笼子中隔离（2只小鼠/笼）与随意获得食物和水，并简单地连续地安置他们以这种方式为7天。 注:常规牧在此期间确定的，但一定要平均分配给所有小鼠。注意避免男女同窝的配对。 </li></ol></li><li>环境富集<ol><li>使用性成熟（&gt; 8周龄），年龄匹配的雄性或雌性小鼠。 注意:我们通常使用N = 18只小鼠的每个实验中，分成n = 6 /治疗组。 </li><li>抵达后进入动物圈养设施让他们组安置在同一封装标准（SH）（ 例如: </em&gt;见下文）的条件下，在这个阶段&gt; 3天适应环境。在这里和在整个实验中，保持外来环境刺激（ 如环境温度，灯:暗循环，食物，水，处理和清洗）恒定或平均分配给所有小鼠。 </li><li>开始环境富集，随机分配每只小鼠3组之一:SH;运行轮（RW）或环境富集的（EE），并放置各组小鼠的汇集成一个相同的干净笼子具有随意获取食物和水。 注:在这里，大老鼠拿着笼子所有群体，测量27厘米宽42厘米长，16厘米深的被使用。 <ol><li>此外，提供以下环境条件下各组:地板上的SH只包括垃圾（纸张颗粒或木屑）; RW包括SH加2转轮; EE包括RW加玩具（绳索，梯子，隧道，和对象，如空纸巾卷和张纸巾），用以前PLORE，玩，爬，隐藏和鸟巢。 </li><li>以这种方式连续地安置他们14天。 注:常规牧在此期间确定的，但一定要平均分配给所有小鼠。 </li></ol></li><li>受EE小鼠额外"超浓缩"（SE）通过将它们一起放入含新玩具1小时/天（同一时间的每一天），5天/周更大的笼子里。 注:在这里，宽46厘米，69厘米长，40厘米深的塑料桶被使用。 <ol><li>通过提供一组不同的玩具每个会话保持新鲜感。干净的玩具要被重新提交给小鼠用肥皂水和80％的乙醇以去除气味。 </li><li>在每次SE会话，返回的小鼠其EE笼子。手柄SH和RW老鼠一样SE小鼠（除SE本身）在此期间（ 例如删除和以及它的笼子里返回每个SH和RW鼠标）。 </li></ol></li></ol></li></ol><ol><li>准备<ol><li>使用无菌渗透泵和脑输注套件，其是市售的。植入前一天，使用无菌技术，首要的植入物通过填充每个泵，连接管和套管与无菌的药物或载体溶液（如在与泵提供的说明书所述）。将其连接在一起，并培育在无菌盐水中过夜，在37℃。分别孵育毒品和车辆充注式假以避免交叉污染。 </li></ol></li><li>手术治疗。 注:根据实验者的国家，特种动物实验许可或津贴必须追求这种类型的手术和术后实验。 <ol><li>消毒所有的手术设备，聘请整个无菌技术。麻醉鼠标（ 例如使用在空气中1-2％异氟烷），并将其放置在一个立方米otaxic井架。检查麻醉深度在整个过程和管理进一步麻醉剂作为必要（例如缺席肢撤回到有毒爪夹紧指示充足麻醉）。确保眼睛免受干燥通过润滑剂眼膏，和鼠标的体温保持正常的整个过程。 </li><li>通过从眼睛2之间整理开始厘米后路头骨的背面边缘的皮肤作正中切口。钝解剖皮肤远离底层筋膜按住鼠标的背面以创建皮下"口袋"足够大，以舒适地配合在泵和连接管，一旦插管植入（连接管不应该被完成时弯曲）。刮清除筋膜来自头骨的背侧。 </li><li>使用约1.5毫米直径的牙科毛刺，深入到颅骨在适当的立体坐标üNTIL骨的一个薄的，柔性层保留。撕下使用细镊子这层（这避免损坏底层硬脑膜及脑与牙科毛刺）。确保颅骨被清洁的任何血液和骨头碎片，而没有进一步的出血。 </li><li>与由一个套管夹持器保持的插管，放置在泵和连接管进入皮下"口袋"覆盖在小鼠的背部。接着，在适当的立体坐标和大脑的表面上定位插管的尖端。小心地将导管进入大脑，但停止大约为1毫米的短所需的深度。 注:其余深度将下列申请牙科丙烯酸的第一层的协商。 </li><li>确保再次头骨的表面清洁，干燥，然后准备一个小体积的牙科丙烯酸及用牙签来平滑它在头骨的整个暴露面积，包括入毛刺孔通过围绕日头骨Ë套管。前的丙烯酸类变硬，降低套管末端剩余深度到目标。 </li><li>准备另一个小体积的牙科丙烯酸和在第一层这一点，以及在该插管的白色塑料支撑，固定到位插管。重复与丙烯酸的附加层是必要的。 </li><li>一旦亚克力硬化，小心地取出导管架，切断用手术刀与丁烷火焰加热的白色塑料套管可拆卸标签。最后，缝合封闭在整个植入皮肤。 </li></ol></li><li>动物手术后的治疗<ol><li>应用防腐剂软膏在皮肤上边距，和管理抗炎到鼠标（ 例如美洛昔康，3毫克/千克SC）。从框架中取出鼠标，将其放置在加热灯下，观察，直到它恢复意识。不要返回鼠标已经动过手术，以其他动物的公司，直到完全康复。 注:EXPerimental操作如性别配对和环境富集可以启动或次日手术最早恢复。 </li><li>监测他们的体重，日常一般行为和外观。治疗感染周围的手术切口外用抗菌软膏迹象（ 如肿胀，充血，脓）。治疗任何疾病的迹象，疼痛，压力或不适（ 如丢失&gt; 10％的体重，社交退缩，缺乏疏导，"起毛"，运动功能障碍，癫痫发作）全身镇痛药和/或抗生素是必要的。 </li><li>安乐死的小鼠，如果&gt; 15％的重量损失或疾病，疼痛，压力或不适的症状是1周补救处理中不可恢复的。 </li></ol></li></ol> 3.脑组织制备，免疫组化处理和体视<ol><li>灌注<ol><li>紧接着环境/药物操作，准备的大脑进行研究。 <br/&gt;注意:在这里报导的实验致密TH +神经元的数目进行测定，在每一个不同的治疗组进行比较。 </li><li>一是管理的麻醉剂过量杀死小鼠（ 如 100毫克/公斤的IP戊巴比妥钠）。一旦麻醉，但前心脏停止跳动，躺在它的后面鼠标和领带或牵制前肢。 </li><li>使用手术刀切开皮肤覆盖在胸部，然后通过正下方的肋笼肌肉切开进入腹腔。放置夹在胸腔的剑突和肝脏暴露隔膜抬起胸腔起来了。 </li><li>切穿隔膜，并通过横向两侧使用大剪刀，直到胸腔可以折回在头上以暴露肺和心脏的肋骨。使用细剪刀作一小切口在右心房作为退出点为血液和灌注溶液，然后通过左ventri的基部切CLE并放置套管向上通过左心室，左心房，并进入主动脉。 </li><li>夹紧套管就位然后泵温热（37℃）肝素和0.1M磷酸盐缓冲通过脉管，直至溶液离开右心房生理盐水（PBS）是完全自由的血液。接着，通过脉管泵冷（4℃）固定液（在PBS中，如4％多聚甲醛），直到整个鼠标被固定好。取出大脑并放入PBS中，用30％的蔗糖为2-3天，直到大脑汇。 注:其它类型的组织制剂可能取决于在相应的实验室的专门知识被应用。 </li></ol></li><li>自由浮动的冷冻切片的制备<ol><li>通过感兴趣脑区切连续切片和在PBS中收集这些。 注:我们下调用低温恒温器40微米厚的部分。其它类型的组织切片可能根据在correspondi的专业知识应用吴的实验室。 </li></ol></li><li>免疫组化<ol><li>感兴趣的蛋白质进行免疫组化的标准。孵育在PBS中的5％正常山羊血清和0.3％的Triton X-100的部分在室温下30分钟，然后用免疫反应的多克隆兔抗TH（1:400），在4℃下48小时，然后生物素化的多克隆山羊抗 - 兔（1:1000），在室温下搅拌2小时。 </li><li>接着，孵育在抗生物素蛋白 - 过氧化物酶（1:500），在室温下搅拌1小时，然后在钴和镍的强化二氨基联苯胺（0.5毫克/毫升）在室温下搅拌18-20分钟;对二氨基联苯胺孵育的最后3-5分钟添加过氧化氢（0.01％）以催化发色沉淀。前，后，和上述各步骤之间的冲洗切片3次，10分钟的每个在PBS。 </li><li>安装部分的糊化显微镜载玻片，风干他们，那么尼氏染色（中性红），脱水酒精，清除（X-3B）和盖玻片。 </li></ol></li><lI&gt;体视<ol><li>估计TH +和TH-黑质致密（和VTA和LC）的神经元的使用无偏体视学方法的总数。确保stereologist是盲目的接受治疗。排除神经胶质细胞体直径&lt;5μm的基础上，并只计算那些细胞具有可见细胞核。 </li><li>通过TH +细胞，并根据Paxinos和Watson 7的脑图谱解剖标志/边界的空间位置识别黑质致密部（VTA和LC和）。计数的计数帧内TH +细胞（55×55微米= 3025微米2）以规则的预定的时间间隔（X = 140微米，Y = 140微米为黑质致密; X = 100微米，Y = 100微米为VTA和LC）贯穿在每一个第四部分中的每个细胞核。 </li></ol></li></ol>

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The authors have nothing to disclose.

操作环境 这些环境的操纵（性别配对和丰富环境）的设计背后的动机是为了确定是否对环境，和/或行为的环境提示时，与变化的中脑多巴胺神经元的数量有关。因此，重点是提供环境和刺激行为有可能从事脑DA信号。这些措施包括配对异性和丰富环境，包括访问运行车轮，绳索，梯子，隧道，和对象的探索，玩耍，攀爬，隐藏和鸟巢。这两种环境都应该促进奖励激励的认知，情感和运动行为，已急性增加中脑多巴胺神经元的放电和DA释放有关（ 如:8）。我们的假设是，他们还与中脑多巴胺信号BR更持久的变化有关应该了解的变化中脑多巴胺神经元的数目。这里和其他地方6，证据展示，这是真实的情况。 我们的调查开始使用性别配对协议 ​​（1.1），因为它包括一个核心原始和基本的奖励激励的行为（ 即交配）。此外，生育，对粘结和社会行为被称为诱导（在催产素，抗利尿激素和促肾上腺皮质激素释放激素细胞的下丘脑9密度例如更改）在其他神经化学系统类似的变化。事实上，经过7天 ​​的连续配对与相对的性别，我们表明这里和其他地方6，男性有大约10％的更多的TH +中脑的神经元，而女性有大约少10％的TH +中脑的神经元。[注:这些影响包括致密（本研究和6）和VTA 6，但不是蓝斑6，其中TH帮助合成的去甲肾上腺素。这支持的概念，即脑多巴胺神经元在成人脑中的数目不是固定的，而是变化取决于环境，行为，或环境/行为的相互作用。其它证据认为这些变化所带来的变化，TH基因和蛋白在现存中脑神经元中的表达，相对于DA神经发生或变性。 （1）DA神经发生在成年小鼠脑发生或者没有在所有10-14，或者速率远低于可以占到一周15,16增加约500个新的黑质多巴胺神经元。 （2）研究定量的变化在黑质致密的神经元的数目以下靶向脑的神经元的电活动各种药物2周输注一贯显示TH +和TH-细胞相等（再次约10％），但相反的变化，从而导致在没有净变化细胞总数4,5。这是与TH蛋白水平的调控高于或低于免疫组织化学DET一致ectable水平不增加或神经元的减法。 （3）许多研究已经报道TH基因和蛋白表达的细胞内超过几个小时17-23时间刻度活性依赖性变化，同样已报道在脑4。 问题由此产生哪些潜在的变化TH基因和蛋白在现存的脑神经元中的表达机制。可能性包括性激素，它可依据两者中TH +在男性和女性中脑的神经元的数目的基线差异，以及性别特异性改变（增加雄性和雌性减小）以下的配对在这里和前面6的报道。另一种可能性是TH表达活动依赖的调节。黑质致密部和腹侧被盖区神经元（包括DA和非-DA）接收突触输入主要来自纹状体，下丘脑，丘脑底核，和额叶皮层24。在性别配对，这些输入上下文可以通过感觉运动行为，嗅觉，信息素，压力，新陈代谢，或再现调节。 TH表达的活性依赖性调节可以通过影响基因和蛋白表达的钙或神经肽信号传导途径介导的（ 例如，1）。 为了帮助对性别配对的影响底层上脑多巴胺神经元的数目活性依赖性机制，我们采用的环境富集协议（1.2），这更好的控制抗性类固醇，也许还有其他激素的潜在影响性激素区分（ 如信息素，应力，再现），以及用于传入驱动神经元活性的潜在影响。这里的相关因素是不同的群体都更类似的术语性别（男性全部），激素，社会地位和压力。虽然社会地位和压力可能仍然不同群体之间（ 如更占优势和积极的男性在一组），这是不可能，这将始终是相同的基团（ 例如:EE）和EE的效果现在已经复制多次6。在EE，增加中脑TH +神经元的大小是一样的，在性别配对协议 ​​（比较图1和2本研究的男性，以及图1和2中6）。此外，EE-诱导增加引起环境富集致密TH +神经元中脑GABA A受体（ 图2本研究以及图3中6），这在中脑多巴胺提供约70％的所有传入输入的并发封锁被完全取消神经元25。总之，这些数据表明传入驱动神经活性至少作为强有力的影响过中脑多巴胺神经元激素的数量。 进一步了解环保事实RS调节脑TH +神经元的数目是来自比较RW和EE的影响。 RW包括良好学习或陈腐运动活性（在车轮上运行的），而EE包括这样的活性的类似的量，尽管更大的品种（ 即各种玩具，但不变整个14天），再加上持续暴露于新颖的玩具在在SE组件（1.2.4）。我们此前公布的数据显示，6既没有或少量增加黑质致密和VTA TH +神经元RW相比，SH老鼠，但在EE（+ SE）小鼠更大的增加。这突出的感官刺激，认知，新颖性和/或学习和记忆的脑多巴胺神经元比单独陈腐的体力活动的数量更有效的调节剂。 EE已被广泛使用，以诱导治疗作用和增强脑修复在脑疾病，如阿尔茨海默氏症，亨廷顿氏和帕金森氏病26的多种动物模型。娜这样的环境操作的TURE的是，他们广泛不同实验室之间（如做"标准，安置"的条件），但也有对EE协议是值得注意的，并已先前所讨论的27个重点方面。 EE提供了增加的环境的新颖性和复杂性相对标准外壳，从而提高感官刺激，认知活动和体育锻炼的水平。在设计EE实验中，需要考虑行为学因素，包括动物的感官和认知能力和它们的本能的驱动器。由于小鼠（和老鼠）是夜间活动，以劣势的视觉能力人，但优异的体感和嗅觉acuities，浓缩物应反映实验动物的先天本能和能力。因此，这可能是特别突出到小鼠中的对象的新颖性，形状，质地和气味。的啮齿类动物的莫蒂相对强弱评估vations用于各种浓缩物导致的建议列入咀嚼物，以允许锻炼的机会根本，物种典型行为28,29。嵌套材料的规定被认为是丰富的协议和小鼠的一个基本组成部分会本能地撕由纸或纸板的任何对象，从而为感官，认知和运动活动30,31机会。它也是重要的实验与多个EE笼子，物体的等效新颖性和复杂性被提供，以最小化EE组内方差，以及类似的控制的环境参数应行使为SH基。理想的是，每笼小鼠的数目应该是EE和SH基（除非社会群体大小的影响被具体地研究）之间是相同的，并没有"食物款待"应当在EE使用，因为组之间任何饮食差异可能变乱吨他解释认知活动和体育锻炼是增强EE 12的效果。其他主要考虑是暴露的时间到丰富的环境和年龄，富集开始。而基因的差异表达已显示后仅3小时在富集环境32，以发生33,34可能需要结构和功能改变曝光的时间变得更长。此外，浓缩诱发塑性可能会在出生后发育的关键时期35得到加强。 渗透泵和脑灌注导管植入药物输注 渗透泵和插管植入物是非常有效的递送药物直接进入大脑。随着经验的积累，在植入手术需要每鼠约1小时。仅牙科丙烯酸足以修复到位套管长时间（注:我们已经长达28天的邻疗法实验）;不需要额外的安装硬件，诸如骨螺钉。是很重要的，以确保有足够的松弛在连接泵和插管，以允许最大的头部和颈部的屈曲不把太多的压力上的连接管。这尤其是对于较长期的输液，其中结缔组织的泵周围生长可代替锚它由此减少植入物的柔韧性。如果可能的话，老鼠也单设下手术，以防止损坏的室友，通过搔抓，拉扯或咬的植入物。药物的剂量在很大程度上是由加入到泵的量来确定。然而，在这方面还应该注意支付给泵流量，药物在37℃下的化学稳定性，和药物的扩散从套管通过大脑的速率。当前从供应商所提供的泵可在一个流率0.15微升/小时维持输注长达6周;快EST提供流量为10微升/小时，但只持续1周。较长的输注时间可以通过执行额外小手术以与在连接管的背面满泵更换耗尽的泵（ 即不干扰植入插管）来实现。亚甲蓝（1％亚甲基蓝，MW 320，在0.9％NaCl）中，从一个套管末端扩散的空间范围植入上面0.3mm左致密中途沿其中侧方面被测量为从脑的横向边缘延伸到刚刚横跨中线mediolaterally，并在整个脑的整个背腹程度（2周后输注）。这是比小核像致密得 ​​多普遍，药物的扩散可能会更大（或更小），这取决于它的分子量，化学稳定性，缓冲等的确，人们可以从本数据推断（ 图2），该无论印防己毒素和荷包牡丹到达对侧黑质致密的生物活性concentrati附件。

DArgic信号通过在中脑腹侧被盖区（VTA）和黑质致密部（SNC）的神经元被认为是为奖励激励的认知，情感和运动行为的重要。但是，过多或过少的中脑多巴胺信令引起各种神经病症的许多禁用症状（ 例如 ，帕金森氏病，注意力缺陷多动障碍，精神分裂症，和药物成瘾）。药物提高或降低的DA信令缓解这些症状，但它们也产生副作用可归因于失调信令和脱靶效应。药物功效也随时间下降，由于脑的代偿反应。因此面临的挑战是在一个更有针对性和生理方式恢复正常脑多巴胺信令，以及一个偏爱的方法是通过增加或减少中脑多巴胺神经元的数目。 证据已经积累了SEVERAL几十年来，参与代谢和贩运DA等儿茶酚胺类物质在成熟的成体细胞的基因和蛋白质的表达是可以修改的（1回顾）。在中脑，酪氨酸羟化酶免疫阳性的数量（TH + DA中合成的限速酶）的神经元减小然后增大以下的神经毒素给药2,3，而TH免疫阴性（TH-）的神经元的数量示出了相反的模式（ 即增加然后下降3）。这是与损失一致，那么获得了"DA型"的一些细胞。 TH +和TH-致密的神经元的数量也已表明下列即改变这些细胞4,5的电活动的各种处理中相等，但相反的方向发生变化。例如，输注小电导的钙活化的钾（SK）通道拮抗剂蜂毒明肽进入脑2周（以相同的量）减少TH +和增加的数目的NUTH-的黑质神经元MBER 4,5。与此相反，注入对SK通道激动剂1- EBIO的增加TH +的数量和减小（以相同的量）TH-致密的神经元4,5的数量。类似的变化被视为承接各种针对黑质神经元的活动，包括一些有针对性的传入输入4个处理。致密DArgic神经元的数目的由神经元活性和传入输入此表观调节引起该环境或行为可以影响黑质致密的神经元的数量的可能性。暴露在不同的环境中确实成年小鼠具有更多或更少的中脑（SNC和VTA）TH +神经元，并且至少其中的一些环境引起的变化由突触输入的中脑6并发封锁被废除。这种沟通的目的是:（1）提供有关如何实现我们的环保操作和药物输注进一步详情; （2）提供进一步的数据支持我们的论点，即电子nvironment调节中脑多巴胺神经元的数目， 经由传入输入。

<table><tbody><tr><td>Isofluorane</td><td>Baxter Healthcare Pty Ltd, Baxter Drive, NSW 2146, Australia</td><td>AHN3640</td><td></td></tr><tr><td>ALZET Osmotic pump 1002</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0004317</td><td></td></tr><tr><td>ALZET Brain infusion kit 1</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0004760</td><td></td></tr><tr><td>ALZET cannula holder 1</td><td>DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td><td>0008860</td><td></td></tr><tr><td>Vertex Monomer Self-curing (dental acrylic solvent)</td><td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td><td>n/a</td><td></td></tr><tr><td>Vertex Self Curing (dental acrylic powder)</td><td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td><td>n/a</td><td></td></tr><tr><td>METACAM (Meloxicam)</td><td>Troy Laboratories, 98 long Street, smithfield NSW 2164 Australia</td><td>L10100</td><td></td></tr><tr><td>Sodium Pentobarbitone</td><td>Lethabarb, Virbac, Milperra, NSW, Australia</td><td>571177</td><td></td></tr><tr><td>Normal goat serum</td><td>chemicon-temecula, CA</td><td>S26-Litre</td><td></td></tr><tr><td>Triton X-100</td><td>Merck Millipore Headquarters&amp;nbsp;, 290 Concord road, Billerica, MA 01821</td><td>1.08603.1000</td><td></td></tr><tr><td>Polyclonal rabbit anti-tyrosine hydroxylase</td><td>Merck Millipore Headquarters&amp;nbsp;, 290 Concord road, Billerica, MA 01821</td><td>AB152</td><td></td></tr><tr><td>Polyclonal biotinylated goat anti-rabbit</td><td>Dako Australia Pty. Ltd., Suite 4, Level 4, 56 Berry street, North Sydney, NSW, Australia 2060</td><td>EO432</td><td></td></tr><tr><td>Avidin peroxidase</td><td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td><td>A3151-1mg</td><td></td></tr><tr><td>Diamino-benzidine</td><td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td><td>D-5637</td><td></td></tr><tr><td>Stereo Investigator</td><td>MicroBrightField Bioscience, 185 Allen Brook Lane, Suite 101, Williston, VT 05495</td><td>n/a</td><td></td></tr></tbody></table>

environmental modulations of the number of midbrain dopamine neurons in adult mice

在大脑或"大脑可塑性"持久的变化背后以下疾病或损伤适应行为和大脑修复。此外，我们的环境之间的相互作用可以引起大脑的可塑性。越来越多的研究，试图找出其中的环境刺激大脑的可塑性有利于治疗大脑和行为障碍。两个环境的操作中，描述了增加或减少的酪氨酸羟化酶免疫阳性的数量（TH +中多巴胺（DA）的合成中的限速酶）的神经元，在成年小鼠中脑。第一配对包括雄性和雌性小鼠持续1周，其中约12％的男性增加中脑TH +神经元一起，但大约12％的女性降低中脑TH +神经元。所述第二壳体包括小鼠连续2周在含有运行车轮，玩具，绳索，排料等，"富集环境&#39;（EE），我通过在雄性约14％ncreases脑TH +神经元。此外，协议中描述的同时注入药物直接进入脑在这些环境的操作，以帮助识别潜在的环境引起的大脑可塑性的机制。例如，EE-诱导更多的脑TH +神经元的突触输入的同时封锁到脑神经元废止。总之，这些数据表明，有关环境的信息是通过突触输入中继到中脑的神经元，以打开或关闭的"DA"基因的表达。因此，适当的环境刺激，或药物的基本机制的靶向，可能是用于治疗与中脑多巴胺失衡有关的脑和行为障碍有用（ 例如，帕金森氏病，注意力缺陷多动障碍，精神分裂症，和药物成瘾）。

经受这些环境操纵成年小鼠已经改变脑的号码（SNC和VTA），但不LC，TH +神经元，和EE加要么印防己毒素或荷包牡丹碱并发脑灌注（GABA A受体拮抗剂）废除EE-诱导更致密TH +神经元。这些数据此前公布的6人。本资料汇编中如以前的研究的一部分进行重复实验，但还没有在其他地方发表。 具体来说，已配对的成年雌性小鼠成年雄性小鼠有更多的TH +黑质致密部和腹侧被盖区（脑）神经元比男性配对与男性，而女性搭配的男性比女性搭配女性（ 图1较少TH +黑质致密部和腹侧被盖区神经元6）。另一个例子是在这里提供在图1中，这立即显示平均值±TH +致密的神经元在雄性和雌性小鼠的SE的数目FOllowing性别配对。男性搭配女性连用7天有大约12％的TH +神经元黑质比男性配对的雄性（两个蓝色的柱图1）。相比之下，女性搭配男性有少约12％TH +黑质致密的神经元比女性搭配女性（两个红色柱图1）。 TH +致密和VTA的神经元的数量也增加了在成年雄性小鼠受到EE（ 图2中6），和EE-感应更致密TH +神经元是由脑GABA A受体（ 图3中6）的并发封锁废除。另一个例子是在这里提供在图2中，其中EE与车辆输注导致大约14％以上的TH +神经元致密除了SH与车辆输注（ 图2中左侧两列）。然而，EE与GABA 受体拮抗剂注射（或10μM印防己毒素或51，M荷包牡丹）完全消除这种增加（ 图2中右侧两列）。注意，这些数据是从SNC对侧输注插管，并且几乎是相同的，以在其中报告了6同侧致密观察到的影响。 <img alt="图1" src="/files/ftp_upload/52329/52329fig1highres.jpg" /> 图1:在黑质致密部TH +神经元的数目的影响性别配对紧接7天性别配对（协议1.1）成年小鼠灌注（3.1），它们的中脑被切开（3.2），将切片处理为酪氨酸羟化酶（TH ，幅度在DA合成限速酶）免疫组化（3.3），和TH +黑质致密部神经元数目使用体视学（3.4估计）。这里绘制的平均值±黑质致密部TH +神经元搭配雄性小鼠（n = 4雄性小鼠从N = 2男男配对，淡蓝色的山坳雄性小鼠SE数量UMN），男搭配女性（N = 6雄性小鼠从6男女配对，深蓝色列），女性搭配女性（N = 8雌性小鼠从N = 4女女配对，淡红色柱）和女性搭配的男性（N = 6雌性小鼠从每组6男女对，暗红色柱）。配对异性导致增加在黑质致密部TH +神经元中的男性数目但在黑质致密部TH +神经元的数量的减少，女性（P &lt;0.001单向ANOVA; * P &lt;0.05杜克成对多重比较）。 <img alt="图2" src="/files/ftp_upload/52329/52329fig2highres.jpg" /> 图2:影响环境富集与脑GABA在黑质致密部TH +神经元的数目A受体阻滞剂紧接着环境浓缩（协议1.2）并发输液两种车型，GABA 受体拮抗剂P的14天。icrotoxin（10μM），或与GABA A受体拮抗剂荷包牡丹碱（5μM）插入左中脑（方案2），成年雄性小鼠灌注（3.1），它们的中脑被切开（3.2），将切片处理为酪氨酸羟化酶（ TH，在DA合成）免疫组化（3.3）的限速酶，以及TH +神经元在正确的数量（健侧）黑质致密用体视学（3.4）估计。这里绘制的平均值±黑质致密部TH +神经元的标准SE数量安置（SH）雄性小鼠与车辆输液（N = 6，浅蓝色列），环境丰富（EE）与车辆输液男性（N = 6，第一深蓝色列），EE男性与印防己毒素注射（N = 6，第二个深蓝色列），而EE男性与荷包牡丹输液（N = 6，第三个深蓝色列）。 EE导致增加在黑质致密部TH +神经元的数目，但这个被废除通过输注印防己毒素，或荷包牡丹碱的成对侧脑（P &lt;0.001单向ANOVA; * P &lt;0.001杜克成对多重比较）。

Watch this Scientific Journal Video about Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice at JoVE.com

Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

中脑多巴胺神经元的数目在成年小鼠环境的调变

Long-lasting changes in the brain or &#8216;brain plasticity&#8217; underlie adaptive behavior and brain repair following disease or injury. Furthermore, ...

environmental-modulations-number-midbrain-dopamine-neurons-adult

Nanyang Technological University

Research

JoVE Journal

Neuroscience

8.7K 次观看.  The University of Melbourne. This protocol describes two different environmental manipulations and a concurrent brain infusion protocol to study environmentally-induced brain changes underlying adaptive behavior and brain repair in adult mice.

视频: Environmental Modulations of the Number of Midbrain Dopamine Neurons in Adult Mice

Assessment of Sensorimotor Function in Mouse Models of Parkinson's Disease

Sensitive and reliable behavioral outcome measures are essential to the evaluation of potential therapeutic treatments in preclinical trials for many neurodegenerative diseases. In Parkinson's disease, sensorimotor tests sensitive to varying degrees of nigrostriatal dysfunction are fundamental for testing the efficacy of potential therapeutics. Reliable and quite elegant sensorimotor measures exist for rats, however many of these tests measure sensorimotor asymmetry within the rat and are not entirely suitable for the newer genetic mouse models of PD. We have put together a battery of sensorimotor tests inspired by the sensitive tests in rats and adapted for mice. The test battery highlighted in this study is chosen for a) its sensitivity in a wide variety of mouse models of PD, b) its ease in implementing into a study, and c) its low expense. These tests have proven useful in characterizing novel genetic mouse models of PD as well as in testing potential disease-modifying therapies.

In Parkinson's disease and movement disorders in general, sensitive and reliable behavioral assays are essential for testing novel potential therapeutics. Here, we describe a manageable battery of sensorimotor tests for mice that are sensitive to varying degrees of injury to the nigrostriatal system and useful for preclinical studies.

帕金森氏症小鼠模型的感觉功能评估

Sensitive and  reliable behavioral outcome measures are essential to the evaluation of  potential therapeutic treatments in preclinical trials for many  ...

科研

行为学

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

生物学

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 ...

神经科学

A Laser-induced Mouse Model of Chronic Ocular Hypertension to Characterize Visual Defects

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model of ocular hypertension to mimic human high-tension glaucoma. Here laser illumination is applied to the corneal limbus to photocoagulate the aqueous outflow, inducing angle closure. The changes of IOP are monitored using a rebound tonometer before and after the laser treatment. An optomotor behavioral test is used to measure corresponding changes in visual capacity. The representative result from one mouse which developed sustained IOP elevation after laser illumination is shown. A decreased visual acuity and contrast sensitivity is observed in this ocular hypertensive mouse. Together, our study introduces a valuable model system to investigate neuronal degeneration and the underlying molecular mechanisms in glaucomatous mice.

Chronic ocular hypertension is induced using laser photocoagulation of the trabecular meshwork in mouse eyes. The intraocular pressure (IOP) is elevated for several months after laser treatment. The decrease of visual acuity and contrast sensitivity of experimental animals are monitored using the optomotor test.

激光诱导的慢性高眼压表征视觉缺陷的小鼠模型

Glaucoma, frequently associated with elevated intraocular pressure (IOP), is one of the leading causes of blindness. We sought to establish a mouse model ...

医学

Murine Model for Parkinson's Disease: from 6-OH Dopamine Lesion to Behavioral Test

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key symptoms, such as tremor, rigidity and bradikinesia, develop when about 3/4 of dopaminergic cells are lost in the substantia nigra, and fail to provide for the smooth, coordinated regulation of striatal motor circuits. Depression and hallucinations are common, and dementia eventually occurs in 20% of patients. At this time, there is no treatment to delay or stop the progression of PD. Rather, the medications currently available aim more towards the alleviation of these symptoms. New surgical strategies may reversibly switch on the functionally damaged circuits through the electrical stimulation of deep brain structures, but although deep brain stimulation is a major advance, it is not suitable for all patients. It remains therefore necessary to test new cell therapy approaches in preclinical models.
Selective neurotoxic disruption of dopaminergic pathways can be reproduced by injection of 6-hydroxydopamine (6-OHDA) or MPTP (1-methyl-4-phenyl-1,2,3,6-tertahydropyridine) whereas depleting drugs and oxidative-damaging chemicals may also reproduce specific features of PD in rodents. Unlike MPTP, 6-OHDA lesions cause massive irreversible neuronal loss, and can be uni- or bilateral. The 6-OHDA lesion model is reliable, leads to robust motor deficits, and is the most widely used after 40 years of research in rats1. As interactions between grafted cells and host can now be studied more thoroughly in mice rather than in rats, the model has been transposed to mice2,3, where it has been recently characterized4.
In this video, we demonstrate how to lesion the left nigro-striatal pathway of anesthetized mice by slowly delivering 2.0 &mu;L of 6-OHDA through a stereotaxically inserted micro-syringe needle. The loss of dopaminergic input occurs within days, and the functional impairments can be monitored over post-operative weeks and months by rating animal rotations induced by dopaminergic agents5. Here, we show full-body contralateral rotations occurring 10 minutes after a single subcutaneous administration of apomorphine, measured one month after the lesion. Outcomes and drawbacks are discussed below.

Parkinson disease is caused by loss of dopaminergic innervation to the striatum, which can be experimentally induced by 6-OH-dopamine. We describe how to perform a stereotaxic lesion and to monitor apomorphine-induced rotational behavior in mice. This model is useful and reliable for testing new therapies for Parkinson disease.

帕金森氏病的小鼠模型：从6 - OH多巴胺病变行为测试

Parkinson's disease (PD) affects at least 6.5 million people worldwide, irrespective of gender, social, ethnic, economic, or geographic boundaries. Key ...

Gene-environment Interaction Models to Unmask Susceptibility Mechanisms in Parkinson's Disease

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease (PD) pathogenesis are less understood. Gene-environment interaction models have utility in unmasking the impact of specific cellular pathways in toxicity that may not be observed using a solely genetic or toxicant disease model alone.&#160;To evaluate if distinct LOX isozymes selectively contribute to PD-related neurodegeneration, transgenic (i.e. 5-LOX and 12/15-LOX deficient) mice can be challenged with a toxin that mimics cell injury and death in the disorder.&#160;Here we describe the use of a neurotoxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), which produces a nigrostriatal lesion to elucidate the distinct contributions of LOX isozymes to neurodegeneration related to PD. The use of MPTP in mouse, and nonhuman primate, is well-established to recapitulate the nigrostriatal damage in PD.&#160;The extent of MPTP-induced lesioning is measured by HPLC analysis of dopamine and its metabolites and semi-quantitative Western blot analysis of striatum for tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine. To assess inflammatory markers, which may demonstrate LOX isozyme-selective sensitivity, glial fibrillary acidic protein (GFAP) and Iba-1 immunohistochemistry are performed on brain sections containing substantia nigra, and GFAP Western blot analysis is performed on striatal homogenates.&#160;This experimental approach can provide novel insights into gene-environment interactions underlying nigrostriatal degeneration and PD.

Lipoxygenase (LOX) isozymes can generate products that may increase or decrease neuroinflammation and neurodegeneration.&#160;A gene-environment interaction study could identify LOX isozyme-specific effects. Using the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) model of nigrostriatal damage in two LOX isozyme-deficient transgenic lines allows for comparison of the contribution of LOX isozymes on dopaminergic integrity and inflammation.

基因-环境相互作用模型揭开帕金森病的隐形易感机制

Lipoxygenase (LOX) activity has been implicated in neurodegenerative disorders such as Alzheimer&#39;s disease, but its effects in Parkinson&#39;s disease ...

Membrane Potential Dye Imaging of Ventromedial Hypothalamus Neurons From Adult Mice to Study Glucose Sensing

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with increasing connective tissue and decreased neuronal viability that occur with age. Here, we describe a methodology for the dissociation of healthy hypothalamic neurons from adult-aged mice. The ability to study neurons from adult-aged mice allows the use of disease models that manifest at a later age and might be more developmentally accurate for certain studies. Fluorescence imaging of dissociated neurons can be used to study the activity of a population of neurons, as opposed to using electrophysiology to study a single neuron. This is particularly useful when studying a heterogeneous neuronal population in which the desired neuronal type is rare such as for hypothalamic glucose sensing neurons. We utilized membrane potential dye imaging of adult ventromedial hypothalamic neurons to study their responses to changes in extracellular glucose. Glucose sensing neurons are believed to play a role in central regulation of energy balance. The ability to study glucose sensing in adult rodents is particularly useful since the predominance of diseases related to dysfunctional energy balance (e.g.&#160;obesity) increase with age.

The activity of single neurons from adult-aged mice can be studied by dissociating neurons from specific brain regions and using fluorescent membrane potential dye imaging. By testing responses to changes in glucose, this technique can be used to study the glucose sensitivity of adult ventromedial hypothalamic neurons.

膜下丘脑腹内侧神经元的电位染料成像从成年小鼠为研究葡萄糖传感

Studies of neuronal activity are often performed using neurons from rodents less than 2 months of age due to the technical difficulties associated with ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

方法对NF-κB依赖的成年神经发生的调制与分析

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

Primary Culture of Mouse Dopaminergic Neurons

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions such as motor control, motivation, and working memory. Nigrostriatal dopaminergic neurons selectively degenerate in Parkinson&#39;s disease (PD). This progressive neuronal loss is unequivocally associated with the motors symptoms of the pathology (bradykinesia, resting tremor, and muscular rigidity). The main agent responsible of dopaminergic neuron degeneration is still unknown. However, these neurons appear to be extremely vulnerable in diverse conditions. Primary cultures constitute one of the most relevant models to investigate properties and characteristics of dopaminergic neurons. These cultures can be submitted to various stress agents that mimic PD pathology and to neuroprotective compounds in order to stop or slow down neuronal degeneration. The numerous transgenic mouse models of PD that have been generated during the last decade further increased the interest of researchers for dopaminergic neuron cultures. Here, the video protocol focuses on the delicate dissection of embryonic mouse brains. Precise excision of ventral mesencephalon is crucial to obtain neuronal cultures sufficiently rich in dopaminergic cells to allow subsequent studies. This protocol can be realized with embryonic transgenic mice and is suitable for immunofluorescence staining, quantitative PCR, second messenger quantification, or neuronal death/survival assessment.

Dopaminergic neurons play a vital regulatory role in the brain. Their loss is associated with Parkinson's disease. In this video, we show how to generate primary cultures of central dopaminergic neurons from embryonic mouse mesencephalon. Such cultures are useful to study the extreme vulnerability of these neurons to various stresses.

小鼠多巴胺能神经元的原代培养

Dopaminergic neurons represent less than 1% of the total number of neurons in the brain. This low amount of neurons regulates important brain functions ...

Isolation, Culture and Long-Term Maintenance of Primary Mesencephalic Dopaminergic Neurons From Embryonic Rodent Brains

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes involved in physiological functions and vulnerability and death of these neurons is imparative to understanding the underlying causes and unraveling the cure for this common neurodegenerative disorder. Primary cultures of mesDA neurons provide a tool for investigation of the molecular, biochemical and electrophysiological properties, in order to understand the development, long-term survival and degeneration of these neurons during the course of disease. Here we present a detailed method for the isolation, culturing and maintenance of midbrain dopaminergic neurons from E12.5 mouse (or E14.5 rat) embryos. Optimized cell culture conditions in this protocol result in presence of axonal and dendritic projections, synaptic connections and other neuronal morphological properties, which make the cultures suitable for study of the physiological, cell biological and molecular characteristics of this neuronal population.

The causes of degeneration of midbrain dopaminergic neurons during Parkinson&#8217;s disease are not fully understood. Cellular culture systems provide an essential tool for study of the neurophysiological properties of these neurons. Here we present an optimized protocol, which can be utilized for in vitro modeling of neurodegeneration.

原发性脑多巴胺能神经元的分离，培养和长期维持胚胎脑鼠类

Degeneration of mesencephalic dopaminergic (mesDA) neurons is the pathological hallmark of Parkinson&#8217;s diseae. Study of the biological processes ...