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>

<ol>
	<li>Aumann, T., Horne, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+regulation+of+the+dopamine+phenotype+in+substantia+nigra+neurons.">Activity-dependent regulation of the dopamine phenotype in substantia nigra neurons.</a> Journal of neurochemistry. 121, 497-515 (2012).</li><li>Sauer, H., Oertel, W. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progressive+degeneration+of+nigrostriatal+dopamine+neurons+following+intrastriatal+terminal+lesions+with+6-hydroxydopamine:+a+combined+retrograde+tracing+and+immunocytochemical+study+in+the+rat.">Progressive degeneration of nigrostriatal dopamine neurons following intrastriatal terminal lesions with 6-hydroxydopamine: a combined retrograde tracing and immunocytochemical study in the rat.</a> Neuroscience. 59, 401-415 (1994).</li><li>Stanic, D., Finkelstein, D. I., Bourke, D. W., Drago, J., Horne, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Timecourse+of+striatal+re-innervation+following+lesions+of+dopaminergic+SNpc+neurons+of+the+rat.">Timecourse of striatal re-innervation following lesions of dopaminergic SNpc neurons of the rat.</a> The European journal of neuroscience. 18, 1175-1188 (2003).</li><li>Aumann, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+activity+regulates+expression+of+tyrosine+hydroxylase+in+adult+mouse+substantia+nigra+pars+compacta+neurons.">Neuronal activity regulates expression of tyrosine hydroxylase in adult mouse substantia nigra pars compacta neurons.</a> Journal of neurochemistry. 116, 646-658 (2011).</li><li>Aumann, T. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SK+channel+function+regulates+the+dopamine+phenotype+of+neurons+in+the+substantia+nigra+pars+compacta.">SK channel function regulates the dopamine phenotype of neurons in the substantia nigra pars compacta.</a> Experimental neurology. 213, 419-430 (2008).</li><li>Aumann, T. D., Tomas, D., Horne, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+and+behavioral+modulation+of+the+number+of+substantia+nigra+dopamine+neurons+in+adult+mice.">Environmental and behavioral modulation of the number of substantia nigra dopamine neurons in adult mice.</a> Brain and behavior. 3, 617-625 (2013).</li><li>Paxinos, G., Franklin, K. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The mouse brain in stereotaxic coordinates. , (2001).</li><li>Schultz, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Behavioral+dopamine+signals.">Behavioral dopamine signals.</a> Trends in neurosciences. 30, 203-210 (2007).</li><li>Sun, P., Smith, A. S., Lei, K., Liu, Y., Wang, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+bonds+in+male+prairie+vole:+long-term+effects+on+emotional+and+social+behavior,+physiology,+and+neurochemistry.">Breaking bonds in male prairie vole: long-term effects on emotional and social behavior, physiology, and neurochemistry.</a> Behavioural brain research. 265, 22-31 (2014).</li><li>Aponso, P. M., Faull, R. L., Connor, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+progenitor+cell+proliferation+and+astrogenesis+in+the+partial+progressive+6-hydroxydopamine+model+of+Parkinson's+disease.">Increased progenitor cell proliferation and astrogenesis in the partial progressive 6-hydroxydopamine model of Parkinson's disease.</a> Neuroscience. 151, 1142-1153 (2008).</li><li>Frielingsdorf, H., Schwarz, K., Brundin, P., Mohapel, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=No+evidence+for+new+dopaminergic+neurons+in+the+adult+mammalian+substantia+nigra.">No evidence for new dopaminergic neurons in the adult mammalian substantia nigra.</a> Proc Natl Acad Sci U S A. 101, 10177-10182 (2004).</li><li>Lie, D. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+adult+substantia+nigra+contains+progenitor+cells+with+neurogenic+potential.">The adult substantia nigra contains progenitor cells with neurogenic potential.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 22, 6639-6649 (2002).</li><li>Chen, Y., Ai, Y., Slevin, J. R., Maley, B. E., Gash, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+proliferation+in+the+adult+hippocampus+and+substantia+nigra+induced+by+glial+cell+line-derived+neurotrophic+factor.">Progenitor proliferation in the adult hippocampus and substantia nigra induced by glial cell line-derived neurotrophic factor.</a> Experimental neurology. 196, 87-95 (2005).</li><li>Yoshimi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Possibility+for+neurogenesis+in+substantia+nigra+of+parkinsonian+brain.">Possibility for neurogenesis in substantia nigra of parkinsonian brain.</a> Ann Neurol. 58, 31-40 (2005).</li><li>Shan, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+de+novo+neurogenesis+and+dopaminergic+neurogenesis+in+the+substantia+nigra+of+1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced+Parkinson's+disease-like+mice.">Enhanced de novo neurogenesis and dopaminergic neurogenesis in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced Parkinson's disease-like mice.</a> Stem Cells. 24, 1280-1287 (2006).</li><li>Zhao, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+for+neurogenesis+in+the+adult+mammalian+substantia+nigra.">Evidence for neurogenesis in the adult mammalian substantia nigra.</a> Proc Natl Acad Sci U S A. 100, 7925-7930 (2003).</li><li>Baker, H., Kawano, T., Margolis, F. L., Joh, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transneuronal+regulation+of+tyrosine+hydroxylase+expression+in+olfactory+bulb+of+mouse+and+rat.">Transneuronal regulation of tyrosine hydroxylase expression in olfactory bulb of mouse and rat.</a> The Journal of neuroscience : the official journal of the Society for Neuroscience. 3, 69-78 (1983).</li><li>Black, I. B., Chikaraishi, D. M., Lewis, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trans-synaptic+increase+in+RNA+coding+for+tyrosine+hydroxylase+in+a+rat+sympathetic+ganglion.">Trans-synaptic increase in RNA coding for tyrosine hydroxylase in a rat sympathetic ganglion.</a> Brain research. 339, 151-153 (1985).</li><li>Zigmond, R. E., Chalazonitis, A., Joh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preganglionic+nerve+stimulation+increases+the+amount+of+tyrosine+hydroxylase+in+the+rat+superior+cervical+ganglion.">Preganglionic nerve stimulation increases the amount of tyrosine hydroxylase in the rat superior cervical ganglion.</a> Neuroscience letters. 20, 61-65 (1980).</li><li>Biguet, N. F., Rittenhouse, A. R., Mallet, J., Zigmond, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preganglionic+nerve+stimulation+increases+mRNA+levels+for+tyrosine+hydroxylase+in+the+rat+superior+cervical+ganglion.">Preganglionic nerve stimulation increases mRNA levels for tyrosine hydroxylase in the rat superior cervical ganglion.</a> Neuroscience letters. 104, 189-194 (1989).</li><li>Richard, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+tyrosine+hydroxylase+gene+expression+in+rat+brain+and+adrenals+by+exposure+to+cold.">Modulation of tyrosine hydroxylase gene expression in rat brain and adrenals by exposure to cold.</a> Journal of neuroscience research. 20, 32-37 (1988).</li><li>Schalling, M., Stieg, P. E., Lindquist, C., Goldstein, M., Hokfelt, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+increase+in+enzyme+and+peptide+mRNA+in+sympathetic+ganglia+after+electrical+stimulation+in+humans.">Rapid increase in enzyme and peptide mRNA in sympathetic ganglia after electrical stimulation in humans.</a> Proc Natl Acad Sci U S A. 86, 4302-4305 (1989).</li><li>Liaw, J. J., He, J. R., Barraclough, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+changes+in+tyrosine+hydroxylase+mRNA+levels+in+A1,+A2+and+locus+ceruleus+neurons+following+electrical+stimulation+of+A1+noradrenergic+neurons.">Temporal changes in tyrosine hydroxylase mRNA levels in A1, A2 and locus ceruleus neurons following electrical stimulation of A1 noradrenergic neurons.</a> Brain research. Molecular brain research. 13, 171-174 (1992).</li><li>Watabe-Uchida, M., Zhu, L., Ogawa, S. K., Vamanrao, A., Uchida, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-brain+mapping+of+direct+inputs+to+midbrain+dopamine+neurons.">Whole-brain mapping of direct inputs to midbrain dopamine neurons.</a> Neuron. 74, 858-873 (2012).</li><li>Tepper, J. M., Lee, C. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GABAergic+control+of+substantia+nigra+dopaminergic+neurons.">GABAergic control of substantia nigra dopaminergic neurons.</a> Progress in brain research. 160, 189-208 (2007).</li><li>Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environmental+enrichment+and+brain+repair:+harnessing+the+therapeutic+effects+of+cognitive+stimulation+and+physical+activity+to+enhance+experience-dependent+plasticity.">Environmental enrichment and brain repair: harnessing the therapeutic effects of cognitive stimulation and physical activity to enhance experience-dependent plasticity.</a> Neuropathology and applied neurobiology. 40, 13-25 (2014).</li><li>Nithianantharajah, J., Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enriched+environments,+experience-dependent+plasticity+and+disorders+of+the+nervous+system.">Enriched environments, experience-dependent plasticity and disorders of the nervous system.</a> Nature reviews. Neuroscience. 7, 697-709 (2006).</li><li>Chmiel, D. J., Noonan, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preference+of+laboratory+rats+for+potentially+enriching+stimulus+objects.">Preference of laboratory rats for potentially enriching stimulus objects.</a> Laboratory animals. 30, 97-101 (1996).</li><li>Hanmer, L. A., Riddell, P. M., Williams, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+runway+paradigm+to+assess+the+relative+strength+of+rats'+motivations+for+enrichment+objects.">Using a runway paradigm to assess the relative strength of rats' motivations for enrichment objects.</a> Behavior research methods. 42, 517-524 (2010).</li><li>Burrows, E. L., McOmish, C. E., Hannan, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene-environment+interactions+and+construct+validity+in+preclinical+models+of+psychiatric+disorders.">Gene-environment interactions and construct validity in preclinical models of psychiatric disorders.</a> Progress in neuro-psychopharmacology &amp; biological psychiatry. 35, 1376-1382 (2011).</li><li>Van de Weerd, H. A., Van Loo, P. L. P., Van Zutphen, L. F. M., Koolhaas, J. M., Baumans, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strength+of+preference+for+nesting+material+as+environmental+enrichment+for+laboratory+mice.">Strength of preference for nesting material as environmental enrichment for laboratory mice.</a> Applied Animal Behaviour Science. 55, 369-382 (1998).</li><li>Rampon, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+enrichment+on+gene+expression+in+the+brain.">Effects of environmental enrichment on gene expression in the brain.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12880-12884 (2000).</li><li>Eckert, M. J., Abraham, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+environmental+enrichment+exposure+on+synaptic+transmission+and+plasticity+in+the+hippocampus.">Effects of environmental enrichment exposure on synaptic transmission and plasticity in the hippocampus.</a> Current topics in behavioral neurosciences. 15, 165-187 (2013).</li><li>Sztainberg, Y., Chen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+environmental+enrichment+model+for+mice.">An environmental enrichment model for mice.</a> Nature protocols. 5, 1535-1539 (2010).</li><li>Sale, A., Berardi, N., Maffei, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Environment+and+brain+plasticity:+towards+an+endogenous+pharmacotherapy.">Environment and brain plasticity: towards an endogenous pharmacotherapy.</a> Physiological reviews. 94, 189-234 (2014).</li></ol>

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?...

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。

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			<td style="width:167px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:379px;">Isofluorane</td>
			<td style="width:636px;">Baxter Healthcare Pty Ltd, Baxter Drive, NSW 2146, Australia</td>
			<td style="width:119px;">AHN3640</td>
			<td></td>
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			<td height="21" style="height:21px;width:379px;">ALZET Osmotic pump 1002</td>
			<td style="width:636px;">DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td>
			<td>0004317</td>
			<td></td>
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			<td height="21" style="height:21px;width:379px;">ALZET Brain infusion kit 1</td>
			<td style="width:636px;">DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td>
			<td>0004760</td>
			<td></td>
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			<td height="21" style="height:21px;width:379px;">ALZET cannula holder 1</td>
			<td style="width:636px;">DURECT Corporation, PO Box 530 Cupertino, CA 95015-0530</td>
			<td>0008860</td>
			<td></td>
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			<td height="21" style="height:21px;width:379px;">Vertex Monomer Self-curing (dental acrylic solvent)</td>
			<td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td>
			<td style="width:119px;">n/a</td>
			<td></td>
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			<td height="21" style="height:21px;width:379px;">Vertex Self Curing (dental acrylic powder)</td>
			<td>Vertex Dental, Postbus 10, 3700 AA ZEIST, The Netherlands</td>
			<td style="width:119px;">n/a</td>
			<td></td>
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			<td height="21" style="height:21px;width:379px;">METACAM (Meloxicam)</td>
			<td>Troy Laboratories, 98 long Street, smithfield NSW 2164 Australia</td>
			<td style="width:119px;">L10100</td>
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			<td height="21" style="height:21px;width:379px;">Sodium Pentobarbitone</td>
			<td>Lethabarb, Virbac, Milperra, NSW, Australia</td>
			<td style="width:119px;">571177</td>
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			<td height="21" style="height:21px;width:379px;">Normal goat serum</td>
			<td>chemicon-temecula, CA</td>
			<td style="width:119px;">S26-Litre</td>
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		</tr>
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			<td height="21" style="height:21px;width:379px;">Triton X-100</td>
			<td>Merck Millipore Headquarters&#160;, 290 Concord road, Billerica, MA 01821</td>
			<td style="width:119px;">1.08603.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:379px;">Polyclonal rabbit anti-tyrosine hydroxylase</td>
			<td>Merck Millipore Headquarters&#160;, 290 Concord road, Billerica, MA 01821</td>
			<td style="width:119px;">AB152</td>
			<td></td>
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			<td height="21" style="height:21px;">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 style="width:119px;">EO432</td>
			<td></td>
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			<td height="21" style="height:21px;">Avidin peroxidase</td>
			<td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td>
			<td style="width:119px;">A3151-1mg</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diamino-benzidine</td>
			<td>Sigma-aldrich, Castle Hill, NSW 1765 AU</td>
			<td style="width:119px;">D-5637</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stereo Investigator</td>
			<td>MicroBrightField Bioscience, 185 Allen Brook Lane, Suite 101, Williston, VT 05495</td>
			<td style="width:119px;">n/a</td>
			<td></td>
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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?...

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

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

Application of a NMDA Receptor Conductance in Rat Midbrain Dopaminergic Neurons Using the Dynamic Clamp Technique

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the electrical activity of one or more neurons in response to an experimentally-controlled input. The electrical activity of neurons can be recorded in isolated brain slices using patch clamp techniques with glass micropipettes. Traditionally, experimenters can mimic neuronal input by direct injection of current through the pipette, electrical stimulation of the other cells or remaining axonal connections in the slice, or pharmacological manipulation by receptors located on the neuronal membrane of the recorded cell.
Direct current injection has the advantages of passing a predetermined current waveform with high temporal precision at the site of the recording (usually the soma). However, it does not change the resistance of the neuronal membrane as no ion channels are physically opened. Current injection usually employs rectangular pulses and thus does not model the kinetics of ion channels. Finally, current injection cannot mimic the chemical changes in the cell that occurs with the opening of ion channels.
Receptors can be physically activated by electrical or pharmacological stimulation. The experimenter has good temporal precision of receptor activation with electrical stimulation of the slice. However, there is limited spatial precision of receptor activation and the exact nature of what is activated upon stimulation is unknown. This latter problem can be partially alleviated by specific pharmacological agents. Unfortunately, the time course of activation of pharmacological agents is typically slow and the spatial precision of inputs onto the recorded cell is unknown.
The dynamic clamp technique allows an experimenter to change the current passed directly into the cell based on real-time feedback of the membrane potential of the cell (Robinson and Kawai 1993, Sharp et al., 1993a,b; for review, see Prinz et al. 2004). This allows an experimenter to mimic the electrical changes that occur at the site of the recording in response to activation of a receptor. Real-time changes in applied current are determined by a mathematical equation implemented in hardware.
We have recently used the dynamic clamp technique to investigate the generation of bursts of action potentials by phasic activation of NMDA receptors in dopaminergic neurons of the substantia nigra pars compacta (Deister et al., 2009; Lobb et al., 2010). In this video, we demonstrate the procedures needed to apply a NMDA receptor conductance into a dopaminergic neuron.

In this video, we demonstrate how to apply a conductance into a dopaminergic neuron recorded in the whole cell configuration in rat brain slices. This technique is called the dynamic clamp.

一个NMDA受体电导的大鼠中脑多巴胺能神经元的动态钳技术中的应用

Neuroscientists study the function of the brain by investigating how neurons in the brain communicate. Many investigators look at changes in the ...

科研

神经科学

Studying the Integration of Adult-born Neurons

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the hippocampal dentate gyrus throughout life. Previous reports have shown that adult hippocampal neurogenesis is associated with diverse brain disorders, including epilepsy, schizophrenia, depression and anxiety (1). Deciphering the process of normal and aberrant adult-born neuron integration may shed light on the etiology of these diseases and inform the development of new therapies.
SGZ adult neurogenesis mirrors embryonic and post-natal neuronal development, including stages of fate specification, migration, synaptic integration, and maturation. However, full integration occurs over a prolonged, 6-week period. Initial synaptic input to adult-born SGZ dentate granule cells (DGCs) is GABAergic, followed by glutamatergic input at 14 days (2). The specific factors which regulate circuit formation of adult-born neurons in the dentate gyrus are currently unknown.
Our laboratory uses a replication-deficient retroviral vector based on the Moloney murine leukemia virus to deliver fluorescent proteins and hypothesized regulatory genes to these proliferating cells. This viral technique provides high specificity and resolution for analysis of cell birth date, lineage, morphology, and synaptogenesis.
A typical experiment often employs two or three viruses containing unique label, transgene, and promoter elements for single-cell analysis of a desired developmental process in vivo. The following protocol describes a method for analyzing functional newborn neuron integration using a single green (GFP) or red (dTomato) fluorescent protein retrovirus and patch-clamp electrophysiology.

A way to study the integration of newborn dentate granule cells in adult animals is described. This technique uses an engineered retrovirus to label newborn neurons, followed by electrophysiological recordings to determine in vivo functional integration.

成人出生的神经元的整合研究

Neurogenesis occurs in adult mammalian brains in the sub-ventricular zone (SVZ) of the lateral ventricle and in the sub-granular zone (SGZ) of the ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

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 ...

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

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

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

谱系重编程的成人大脑的周细胞源性细胞诱导成神经细胞

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

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

激光捕获显微切割 - 个人多巴胺神经元的分离和整个腹侧被盖区的示范

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

从黑质多巴胺神经元的Somatodendritic域亚细胞的膜片钳记录

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

使用荧光探针定量培养的神经元中的内体和溶酶体运动

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

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) ...

Use of Synaptic Zinc Histochemistry to Reveal Different Regions and Laminae in the Developing and Adult Brain

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions in the immature and adult brain. Here we describe a zinc histochemical staining procedure that reveals differences in staining patterns among different layers and brain regions. Others have utilized this procedure not only to reveal the distribution of zinc-containing neurons and circuits in the brain, but also to successfully delineate areal and laminar boundaries in the developing and adult brain in several species. Here we illustrate this staining procedure with images from developing and adult ferret brains. We reveal a zinc-staining pattern that serves as an anatomical marker of areas and layers, and can be reliably used to distinguish visual cortical areas in the developing and adult visual cortex. The main goal of this protocol is to present a histochemical method that allows the accurate identification of layers and regions in the developing and adult brain where other methods fail to do so. Secondarily, in conjunction with densitometric image analysis, this method allows one to assess the distribution of synaptic zinc to reveal potential changes throughout development. This protocol describes in detail the reagents, tools, and steps necessary to successively stain frozen brain sections. Although this protocol is described using ferret brain tissue, it can easily be adapted for use in rodents, cats, or monkeys as well as in other brain regions.

We describe a histochemical procedure that reveals characteristic laminar and areal zinc staining patterns in different brain regions. The zinc-staining pattern may be used in conjunction with other anatomical markers to reliably distinguish layers and regions in the developing and adult brain.

利用突触锌组织化学在发育和成人大脑中揭示不同的区域和叶片

Characterization of anatomical and functional brain organization and development requires accurate identification of distinct neural circuits and regions ...