Clinical and Translational Science Award program of NCATS UL1 TR0000427 and KL2 TR000428 (Cengiz P), UL1TR000427 to the UW ICTR from NIH/NCATS and funds from Waisman Center (Cengiz P), K08 NS088563-01A1 from NINDS (Cengiz P) and NIH P30 HD03352 (Waisman Center), NIH/NINDS 1K08NS078113 (Ferrazzano P). We would like to thank Albee Messing, PhD, for providing us the GFAP knockout mice.

注:此研究是按照指南的护理的美国国立卫生研究院的实验动物的建议，并进行使用。动物方案经威斯康星大学麦迪逊分校，机构动物护理和使用委员会的大学。即这里提出初级星形胶质细胞培养协议从由张ý19 等和Cengiz第20页等人提出的协议通过。有一些修改。 1.海马解剖和星形胶质细胞文化<ol><li>准备所有必需的试剂和材料，包括在冰上手术剪，光滑细镊子，平头镊子，纸巾，废物袋，70％乙醇和2解剖菜（3.5cm直径）填充用2ml Hank氏平衡盐溶液（HBSS）中的每个。确保手术设备的无菌（釜）前程序和使用所有其他材料（星形胶质细胞电镀液:DMEM + 5％马血清+1％青霉素-链霉素，马血清，HBSS，0.25％胰蛋白酶，盖玻片，培养皿15/50 mL管，T25烧瓶等 ）预先无菌的。 </li><li>轻轻握住和喷雾头和鼠标小狗颈部用70％的乙醇和使用锋利的无菌剪刀斩首[ 图1（1）]。 </li><li>从后执行正中切口沿头皮，露出大脑[ 图1（2-3）]小剪刀把前颅底。 </li><li>从颈部用小剪刀鼻子小心切开头盖骨。然后切头盖骨前方的嗅球和下小脑，断开颅底颅内。 </li><li>作一小切口的颅底和沿中线切开。使用无菌平头镊子剥离的头骨。具有弯曲镊子的帮助下除去脑干。从头骨移除脑并放置到第一解剖盘[ 图1（4-9）] 21。 </li><li>使用弯钳，翻转大脑，使大脑腹面朝上，然后两个半球用锋利的无菌手术刮刀[ 图1（10,11）]分离</li><li>剥开大脑半球并小心地取出膨出脑和丘脑组织用无菌平弯钳揭示海马，在内侧颞叶[ 图1（12-14）小，海马形结构。 </li><li>除去这两个海马裂片[ 图1（15,16）]，并通过与细镊子牵拉小心解剖从裂片脑膜。这一步骤避免了脑膜细胞和成纤维细胞的最终星形胶质细胞培养物污染。 </li><li>转移准备海马瓣进入第二盘填充用HBSS，并将其返回到冰[ 图1（17,18）。从一个单一的鼠标池都海马叶（P0 - P2）准备河马campal星形胶质细胞的文化。这使星形胶质细胞密度适当。肉末用锋利的无菌手术刀片（约4倍）海马叶。 </li><li>抽吸的HBSS从盘使用连接到1毫升吸移管中的无菌尖端和添加0.25％胰蛋白酶的3毫升，混合，转移到15毫升的无菌锥形管中，并在37°C孵育所述组织20分钟轻轻摇动。 </li><li>在300×g离心5分钟，离心管中。吸去上清液小心使用连接到真空管线的无菌玻璃吸管。加入10 mL星形胶质细胞电镀液及磨碎 - 用火抛光的玻璃吸管，直到组织块均匀（20 30倍）。 </li><li>在300×g离心5分钟，离心管中。吸上清液，加入新鲜预热星形胶质细胞电镀媒体2毫升。通过70微米网状过滤器（细胞过滤）细胞进入一个新的50 mL锥形管。 </li><li>在含有3毫升的聚-D-赖氨酸/层粘连蛋白包被的无菌的T25培养瓶中板整个细胞悬浮液星形胶质细胞电镀媒体，然后孵育该烧瓶在37℃在一个5％CO 2培养箱。 </li><li>在按日期抽吸整个媒体I N体外 （DIV）1，DIV 3和DIV 7，并用2毫升的新鲜星形电镀媒体的更换。每一次观察进展，星形细胞融合光镜下而更换星形胶质细胞电镀媒体。 注:在DIV 1中，所有的可行的星形胶质细胞附着在培养瓶的表面，并且包括神经元在上清液浮动死亡或正在死亡的细胞。在格3中，附着的细胞开始分裂，以形成星形细胞层。在缺乏支持性的条件下，文化是几乎没有任何神经细胞生长的。在DIV 7，星形胶质细胞层为约80 - 90％汇合和几个小胶质细胞以及少突胶质细胞前体细胞是存在于星形细胞层的顶部。 </li><li>吸从烧瓶电镀介质，加入新鲜预热的星形胶质细胞电镀液2毫升，漂洗在第二DIV 11再次删除，当星形胶质细胞汇合，准备子培养。 </li><li>加入2毫升0.25％的胰蛋白酶，轻轻转动该烧瓶几次并用无菌玻璃吸管吸出胰蛋白酶。 </li><li>加入2毫升0.25％胰蛋白酶，让瓶中坐4的组织培养罩 - 在室温5分钟。 </li><li>除去胰蛋白酶，并保持该烧瓶在37℃在一个5％CO 2培养箱10分钟。加入5毫升预热的星形胶质细胞电镀液，并通过点击烧瓶对你的手掌分离星形细胞层（3 - 4次），其次是温和的研磨实现完全分离和收集在一个15毫升的锥形管的星形胶质细胞。 </li><li>离心管中，在300×g离心5分钟，吸出上清液，并添加1毫升新鲜星形胶质电镀介质。 </li><li>添加细胞悬浮液10μL的对血球和使用倒置相差显微镜（10X物镜）计数在中央大网格面积（1mm 2中）的细胞。 MULtiply 10 4来估算每毫升的细胞数。一个T25培养瓶中将产生〜1×10 6个总解离细胞。种子〜1×10 5细胞中在24孔培养皿的孔12毫米直径的聚-D-赖氨酸预包被的玻璃盖玻片2mL的星形胶质细胞电镀介质。 </li><li>在一个5％CO 2培养箱在37℃。 14（第2节） - 在DIV 12进行OGD /再氧化。 </li><li>治疗星形细胞培养在DIV 3用5mM L-亮氨酸甲基酯（LME）的盐酸直到DIV 11如果缺乏的小胶质细胞的海马培养是期望的。 LME孵育后，受到文化晃动（1小时300转），以去除污染的小胶质细胞。 注:LME是已被广泛地用作一种方法来消除增殖胶质22上的小胶质细胞毒性剂。 </li></ol> 2. OGD /再氧化处理<ol><li>从盖玻片含胶质细胞贴壁吸文化传媒（DIV 12 - 14）和冲洗3次轻轻旋转24孔培养皿保持盖玻片用含有（以mM计）1毫升等渗OGD溶液（pH 7.4）几次:0葡萄糖，21的NaHCO 3，120氯化钠，5.36氯化钾，0.33 的 Na 2 HPO 4， 0.44 KH 2 PO 4，1.27 氯化钙 ，和0.81 硫酸镁 。 </li><li>添加0.2mL的OGD溶液很好覆盖盖玻片并在含有94％的N 2，1％O 2缺氧培养箱孵育2小时，和5％ 的 CO 2。与缺氧的前30分钟，以促进气体交换轨道摇床（50rpm下）轻轻混匀。 </li><li>孵育常氧对照细胞在5％CO 2和大气中相同，只是在加入5.5毫葡萄糖的OGD溶液的缓冲液2小时。 </li><li>对于再氧化，吸液OGD并添加星形胶质细胞的2毫升电镀液。孵育在5％CO 2和大气中以37℃进行5小时。 </li></ol> 3.免疫细胞化学染色&lt;/ P&gt; <ol><li>使用连接到真空管线中的无菌玻璃吸管吸出培养基，并通过添加的0.1M Tris缓冲盐水（TBS）的1mL中迅速漂洗盖玻片一次（154毫摩尔NaCl，16毫Trizma碱，84毫摩尔Tris盐酸，pH 7.4）中到孔中。吸并在1X磷酸盐缓冲液（PBS）加入2毫升4％多聚甲醛（PFA）的。在室温下孵育15分钟。 </li><li>抽吸并添加1毫升的0.1M的TBS，然后放置在摇床上2分钟。重复3次。添加1毫升封闭液（10％山羊血清，10毫克/毫升BSA，0.025％的Triton X-100的0.1M的TBS），在37℃的摇床上孵育30分钟。 </li><li>抽吸阻断溶液并添加初级小鼠单克隆抗GFAP抗体（0.2毫升1:500稀释在封闭溶液中）和兔多克隆抗S100B（1:500），或兔多克隆抗HIF1α（1:200），60分钟在上一个摇床上37℃。 </li><li>或者，一些星形细胞孵育用兔多克隆抗IBA1（1:200）和鼠问oclonal抗MAP2访问培养纯度。 </li><li>吸一次抗体，并通过添加1毫升的0.1M的TBS并放置在摇床上进行2分钟，然后吸出冲洗盖玻片。重复两次。 </li><li>在60分钟，在37℃的摇床上封闭溶液二次山羊抗兔488缀合的抗小鼠568缀合的抗体的1:200稀释:添加1 0.2毫升。 </li><li>抽吸的第二抗体，并通过添加1毫升的0.1M的TBS和地点上的摇床上进行2分钟，然后吸出冲洗盖玻片。重复两次。 </li><li>通过放置在位于上滑盖干燥幻灯片从井和干删除盖玻片。通过对反转的的Vectashield单滴一个新的幻灯片盖玻片山安装hardset培养基DAPI（1.5纳克/μL）。 </li><li>使用两种20X干燥或60X油浸物镜共聚焦显微镜图像盖玻片。获取图像（512×512）DAPI（405纳米EX / 450纳米EM）和荧光标记的488 GFAP抗体（488纳米EX / 515纳米EM）。保留采集参数的20X 60X和组内保持不变。 </li></ol> 4.性别决定采用PCR <ol><li>热小狗脚趾或在50mM的NaOH 95℃45分钟手指剪辑和中和等体积的1M Tris，pH值6.8的。 </li><li>添加1μL的提取的DNA溶液的19微升以下混合物的:5皮摩尔引物为生成素和Sry基因的基因，1×反应缓冲液，各0.2mM的脱氧核苷酸和8有用Taq聚合酶。 </li><li>使用的引物序列; Sry基因5&#39;TCATGAGACTGCCAACCACAG3&#39;，5&#39;CATGACCACCACCACCACCAA3&#39;和生成素5&#39;TTACGTCCATCGTGGACAGC3"，5&#39;TGGGCTGGGTGTTAGTCTTA3"23。 </li><li>在95℃95℃3分钟，然后30℃变性个循环15秒，退火58℃15秒，和延伸率在72℃1分钟:执行以下PCR方案。在此之后30个循环，将反应结束以最终72℃延伸1分钟。 </li><li>单独的PCR产品在紫外光照射下电泳在含溴化乙锭的2％琼脂糖凝胶和可视化。 （ 图2A） 注意！溴化乙锭是一种潜在的致癌物质，必须小心处理和妥善处置按机构的规定。 </li></ol>

<ol>
	<li>Powell, E. M., Geller, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+astrocyte-mediated+cues+in+neuronal+guidance+and+process+extension.">Dissection of astrocyte-mediated cues in neuronal guidance and process extension.</a> Glia. 26, 73-83 (1999).</li><li>Koehler, R. C., Roman, R. J., Harder, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytes+and+the+regulation+of+cerebral+blood+flow.">Astrocytes and the regulation of cerebral blood flow.</a> Trends in neurosciences. 32, 160-169 (2009).</li><li>Seifert, G., Schilling, K., Steinhauser, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocyte+dysfunction+in+neurological+disorders:+a+molecular+perspective.">Astrocyte dysfunction in neurological disorders: a molecular perspective.</a> Nature reviews. Neuroscience. 7, 194-206 (2006).</li><li>Ballabh, P., Braun, A., Nedergaard, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+blood-brain+barrier:+an+overview:+structure,+regulation,+and+clinical+implications.">The blood-brain barrier: an overview: structure, regulation, and clinical implications.</a> Neurobiology of disease. 16, 1-13 (2004).</li><li>Araque, A., Parpura, V., Sanzgiri, R. P., Haydon, P. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tripartite+synapses:+glia,+the+unacknowledged+partner.">Tripartite synapses: glia, the unacknowledged partner.</a> Trends in neurosciences. 22, 208-215 (1999).</li><li>Anderson, M. A., Ao, Y., Sofroniew, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogeneity+of+reactive+astrocytes.">Heterogeneity of reactive astrocytes.</a> Neuroscience letters. 565, 23-29 (2014).</li><li>Cengiz, P., 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=Inhibition+of+Na+/H++exchanger+isoform+1+is+neuroprotective+in+neonatal+hypoxic+ischemic+brain+injury.">Inhibition of Na+/H+ exchanger isoform 1 is neuroprotective in neonatal hypoxic ischemic brain injury.</a> Antioxidants &amp; redox signaling. 14, 1803-1813 (2011).</li><li>Ferriero, 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=Neonatal+brain+injury.">Neonatal brain injury.</a> The New England journal of medicine. 351, 1985-1995 (2004).</li><li>Hill, C. A., Fitch, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sex+differences+in+mechanisms+and+outcome+of+neonatal+hypoxia-ischemia+in+rodent+models:+implications+for+sex-specific+neuroprotection+in+clinical+neonatal+practice.">Sex differences in mechanisms and outcome of neonatal hypoxia-ischemia in rodent models: implications for sex-specific neuroprotection in clinical neonatal practice.</a> Neurol Res Int. , 1-9 (2012).</li><li>Cikla, U., 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=ERalpha+Signaling+Is+Required+for+TrkB-Mediated+Hippocampal+Neuroprotection+in+Female+Neonatal+Mice+after+Hypoxic+Ischemic+Encephalopathy(1,2,3).">ERalpha Signaling Is Required for TrkB-Mediated Hippocampal Neuroprotection in Female Neonatal Mice after Hypoxic Ischemic Encephalopathy(1,2,3).</a> eNeuro. 3, (2016).</li><li>Uluc, 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=TrkB+receptor+agonist+7,+8+dihydroxyflavone+triggers+profound+gender-+dependent+neuroprotection+in+mice+after+perinatal+hypoxia+and+ischemia.">TrkB receptor agonist 7, 8 dihydroxyflavone triggers profound gender- dependent neuroprotection in mice after perinatal hypoxia and ischemia.</a> CNS Neurol Disord Drug Targets. 12, 360-370 (2013).</li><li>Cikla, U., 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=Suppression+of+microglia+activation+after+hypoxia-ischemia+results+in+age-dependent+improvements+in+neurologic+injury.">Suppression of microglia activation after hypoxia-ischemia results in age-dependent improvements in neurologic injury.</a> J Neuroimmunol. 291, 18-27 (2016).</li><li>McQuillen, P. S., Ferriero, 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=Selective+vulnerability+in+the+developing+central+nervous+system.">Selective vulnerability in the developing central nervous system.</a> Pediatr Neurol. 30, 227-235 (2004).</li><li>Morken, T. S., 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=Altered+astrocyte-neuronal+interactions+after+hypoxia-ischemia+in+the+neonatal+brain+in+female+and+male+rats.">Altered astrocyte-neuronal interactions after hypoxia-ischemia in the neonatal brain in female and male rats.</a> Stroke; a journal of cerebral circulation. 45, 2777-2785 (2014).</li><li>Chisholm, N. C., Sohrabji, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Astrocytic+response+to+cerebral+ischemia+is+influenced+by+sex+differences+and+impaired+by+aging.">Astrocytic response to cerebral ischemia is influenced by sex differences and impaired by aging.</a> Neurobiology of disease. 85, 245-253 (2016).</li><li>Liu, M., Oyarzabal, E. A., Yang, R., Murphy, S. J., Hurn, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+assessing+sex-specific+and+genotype-specific+response+to+injury+in+astrocyte+culture.">A novel method for assessing sex-specific and genotype-specific response to injury in astrocyte culture.</a> Journal of neuroscience methods. 171, 214-217 (2008).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+the+biological+contributions+to+human+health:+does+sex+matter?.">Exploring the biological contributions to human health: does sex matter?.</a> Journal of women's health &amp; gender-based. 10, 433-439 (2001).</li><li>Collins, F. S., Tabak, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Policy:+NIH+plans+to+enhance+reproducibility.">Policy: NIH plans to enhance reproducibility.</a> Nature. 505, 612-613 (2014).</li><li>Zhang, Y., 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=Rapid+single-step+induction+of+functional+neurons+from+human+pluripotent+stem+cells.">Rapid single-step induction of functional neurons from human pluripotent stem cells.</a> Neuron. 78, 785-798 (2013).</li><li>Cengiz, P., 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=Sustained+Na+/H++exchanger+activation+promotes+gliotransmitter+release+from+reactive+hippocampal+astrocytes+following+oxygen-glucose+deprivation.">Sustained Na+/H+ exchanger activation promotes gliotransmitter release from reactive hippocampal astrocytes following oxygen-glucose deprivation.</a> PloS one. 9, e84294 (2014).</li><li>Landis, S. 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=A+call+for+transparent+reporting+to+optimize+the+predictive+value+of+preclinical+research.">A call for transparent reporting to optimize the predictive value of preclinical research.</a> Nature. 490, 187-191 (2012).</li><li>Hamby, M. E., Uliasz, T. F., Hewett, S. J., Hewett, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+an+improved+procedure+for+the+removal+of+microglia+from+confluent+monolayers+of+primary+astrocytes.">Characterization of an improved procedure for the removal of microglia from confluent monolayers of primary astrocytes.</a> J Neurosci Methods. 150, 128-137 (2006).</li><li>McClive, P. J., Sinclair, A. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+DNA+extraction+and+PCR-sexing+of+mouse+embryos.">Rapid DNA extraction and PCR-sexing of mouse embryos.</a> Molecular reproduction and development. 60, 225-226 (2001).</li><li>Wolterink-Donselaar, I. G., Meerding, J. M., Fernandes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+gender+determination+in+newborn+dark+pigmented+mice.">A method for gender determination in newborn dark pigmented mice.</a> Lab Anim (NY). 38, 35-38 (2009).</li><li>Uliasz, T. F., Hamby, M. E., Jackman, N. A., Hewett, J. A., Hewett, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+primary+astrocyte+cultures+devoid+of+contaminating+microglia.">Generation of primary astrocyte cultures devoid of contaminating microglia.</a> Methods Mol Biol. 814, 61-79 (2012).</li><li>Raponi, E., 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=S100B+expression+defines+a+state+in+which+GFAP-expressing+cells+lose+their+neural+stem+cell+potential+and+acquire+a+more+mature+developmental+stage.">S100B expression defines a state in which GFAP-expressing cells lose their neural stem cell potential and acquire a more mature developmental stage.</a> Glia. 55, 165-177 (2007).</li><li>Souza, D. G., Bellaver, B., Souza, D. O., Quincozes-Santos, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+adult+rat+astrocyte+cultures.">Characterization of adult rat astrocyte cultures.</a> PloS one. 8, e60282 (2013).</li><li>Puschmann, T. B., Dixon, K. J., Turnley, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species+differences+in+reactivity+of+mouse+and+rat+astrocytes+in+vitro.">Species differences in reactivity of mouse and rat astrocytes in vitro.</a> Neuro-Signals. 18, 152-163 (2010).</li><li>Schildge, S., Bohrer, C., Beck, K., Schachtrup, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+mouse+cortical+astrocytes.">Isolation and culture of mouse cortical astrocytes.</a> Journal of visualized experiments : JoVE. , (2013).</li><li>Saura, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglial+cells+in+astroglial+cultures:+a+cautionary+note.">Microglial cells in astroglial cultures: a cautionary note.</a> J Neuroinflammation. 4 (26), (2007).</li></ol>

的作者都没有竞争或利益冲突。

为了研究生理和病理条件下，在属性的性别差异和功能星形胶质细胞，在细胞培养物中制备性控初级星形胶质细胞是利用一个重要工具。在本研究中，我们报道了高效和可再现的方法来培养新生性控海马星形胶质细胞的高度富集的同质群体（P0-P2）的C57BL / 6（野生型）或K19F（GFAP空）小鼠幼仔体外 。建立这种方法可以帮助研究者了解性控海马星形胶质细胞的生理和病理功能体外缺血?...

星形胶质细胞是中枢神经系统（CNS）最重要的关键球员之一。越来越多的证据表明，星形胶质细胞的作用是比提供支持神经元多。事实上，在生理条件下的星形胶质细胞的作用可以是非常复杂的，如引导显影轴突1的迁移，调节CNS血流2中，并保持突触组织液3的pH稳态，和参与血脑屏障4和突触传递5。在病理条件下，星形细胞损伤响应与被叫其中形态，数量，位置，地形（相对于从损伤的距离）的星形胶质细胞和功能可在异构方式6,7-改变反应性星形胶质细胞增生的过程。看到下面的星形胶质细胞增生新生儿缺氧缺血性脑病，也许有利于新生儿的发病率和死亡率<s达&gt; 8。 最近的临床和实验研究表明，大脑损伤的严重程度似乎是性别依赖性和，作为相对于男性新生儿对缺氧/缺血（HI）相关的脑损伤的影响更加敏感，导致更严重的神经成果女性具有可比性脑损伤9-11。虽然损伤的定位取决于胎龄和持续时间和损伤的严重程度，海马是足月HI后在CNS中最常见的影响区域中的一个，并增加海马星形胶质细胞增生被上调的确认胶质纤维酸性蛋白（GFAP）新生儿HI 7,10,12,13后3天。星形胶质细胞功能的性别差异被证明在这两个新生儿和成人啮齿动物脑缺血后14,15。另外，男性星形细胞易感性的体外缺血通过增加CEL所示升死亡相比，在文化16女性皮层星形胶质细胞。 性别差异在子宫内开始并持续直至死亡17。在过去十年中，包括在细胞培养和体内研究，在实验条件下男女的重要性一直医学和NIH研究所的重点寻求基础知识，在生理和病理条件17,18见过的性别差异。可靠的方法开发分离和维护绝育手术海马星形胶质细胞的人口有必要了解的新生儿HI的病理后果的性别差异的细胞学基础。本研究的目的是提供的技术准备，以评估以下氧气/葡萄糖剥夺（OGD）和复氧（再氧化），诱导GFAP免疫反应阳性星形胶质细胞的作用，从新生小鼠丰富的性别特异性海马星形胶质细胞培养HI在细胞培养环境。这种技术可以用来测试有关含氧量正常和缺血性的条件下在新生儿男性和女性海马星形胶质细胞的任何假设。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Astrocyte culture media</td>
		</tr>
		<tr>
			<td>DMEM, high glucose</td>
			<td>cellgro</td>
			<td>10-013-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Horse serum</td>
			<td>Gibco</td>
			<td>26050-070</td>
			<td>Final Concentration: 10%</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Cellgro&#160;</td>
			<td>30-002-CI</td>
			<td>Final Concentration: 1%</td>
		</tr>
		<tr>
			<td rowspan="2">L-Leucine methyl ester hydrochloride</td>
			<td rowspan="2">Aldrich</td>
			<td rowspan="2">L1002-25G</td>
			<td rowspan="2">Final Concentration: 5 mM</td>
		</tr>
		<tr>
		</tr>
		<tr>
			<td colspan="4">Solution for brain tissue digestion</td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Life Technologies</td>
			<td>14170-088</td>
			<td></td>
		</tr>
		<tr>
			<td>0.25% Trypsin</td>
			<td>cellgro</td>
			<td>25-050-CI</td>
			<td>Final Concentration: 0.25%</td>
		</tr>
		<tr>
			<td colspan="4">Other</td>
		</tr>
		<tr>
			<td>70% (vol/vol) ethanol</td>
			<td>Roth</td>
			<td>9065.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-D-Lysine 12mm round coverslips&#160;</td>
			<td>Corning</td>
			<td>354087</td>
			<td></td>
		</tr>
		<tr>
			<td>Water</td>
			<td>Sigma</td>
			<td>W3500</td>
			<td>cell culture grade</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>cellgro</td>
			<td>21-040-CV</td>
			<td>cell culture grade</td>
		</tr>
		<tr>
			<td>0.05% Trypsin-EDTA&#160;</td>
			<td>Life Technologies</td>
			<td>25300-062</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m Sterile cell strainer&#160;</td>
			<td>Fisher scientific</td>
			<td>22363548</td>
			<td></td>
		</tr>
		<tr>
			<td>3.5 cm petri dish</td>
			<td>BD Falcon</td>
			<td>353001</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml Falcon tube</td>
			<td>BD Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr>
			<td>50 ml Falcon tube</td>
			<td>BD Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps, fine&#160;</td>
			<td>Dumont</td>
			<td>2-1032; 2-1033</td>
			<td># 3c; # 5</td>
		</tr>
		<tr>
			<td>Forceps, flat tip</td>
			<td>KLS Martin</td>
			<td>12-120-11</td>
			<td></td>
		</tr>
		<tr>
			<td>13 cm surgical scissors</td>
			<td>Aesculap</td>
			<td>BC-140-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocal Microscope</td>
			<td>Nikon</td>
			<td>A1RSi&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5805000.017</td>
			<td>Centrifuge5804R</td>
		</tr>
		<tr>
			<td>Orbital Shaker</td>
			<td>Thermo Scientific</td>
			<td>SHKE 4450-1CE</td>
			<td>MaxQ 4450&#160;</td>
		</tr>
		<tr>
			<td>Anti-IBA1</td>
			<td>Wako</td>
			<td>019-19741</td>
			<td>Rabbit monoclonal</td>
		</tr>
		<tr>
			<td>Anti-MAP2</td>
			<td>Sigma</td>
			<td>M2320</td>
			<td>Mouse monoclonal</td>
		</tr>
		<tr>
			<td>Anti-HIF1alpha</td>
			<td>abcam</td>
			<td>ab179483</td>
			<td>rabbit monoclonal</td>
		</tr>
		<tr>
			<td>Anti-S100B</td>
			<td>Sigma</td>
			<td>HPA015768</td>
			<td>Rabbit polyclonal</td>
		</tr>
		<tr>
			<td>Anti-GFAP (cocktail)</td>
			<td>Biolegend</td>
			<td>837602</td>
			<td></td>
		</tr>
		<tr>
			<td>VECTASTAIN Elite ABC Kit (Rabbit IgG)</td>
			<td>Vector Labs</td>
			<td>PK-6101</td>
			<td>Contains 4 Reagents&#160;</td>
		</tr>
		<tr>
			<td>Goat Anti Rabbit Alexa-Fluor 488</td>
			<td>Invitrogen</td>
			<td>A11070</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat Anti Mouse Alexa-Fluor 568</td>
			<td>Invitrogen</td>
			<td>A11004</td>
			<td></td>
		</tr>
	</tbody>
</table>

sex differences in mouse hippocampal astrocytes after in-vitro ischemia

星形胶质细胞增生缺氧/缺血（HI）相关的脑损伤起着新生儿增加发病率和死亡率的作用。最近的临床研究表明，大脑损伤的严重程度似乎是性别依赖性的，并且该男性新生儿是HI有关的脑损伤的影响更加敏感，导致更严重的神经系统的结果相比，女性具有可比的脑损伤。可靠的方法来隔离和维护绝育手术海马星形胶质细胞的高度浓缩的人口的发展是至关重要的了解新生儿HI的病理后果的性别差异细胞基础。在这项研究中，我们描述了创建经受体外缺血，缺氧缺糖模型，随后复氧性具体海马星形细胞培养的方法。随后的反应性星形胶质被immunostai检查宁为胶质纤维酸性蛋白（GFAP）和S100B。这种方法提供了一个有用的工具来研究男性和女性的海马星形胶质细胞的新生之后HI的作用，分别。

理解的生理或病理生理条件下性控星形细胞机能的作用已经通过体外的条件下培养这些细胞被极大地阐明。执行性控培养的重要方面是确定鼠标小狗的性别其使用前。我们的基因通过PCR和通过视觉评价（ 图2）16所确定的小鼠的性别。采用PCR性别鉴定的方法与McClive和辛克莱修改获得通过，快速，简便和高重现性的方法23。 图2A</stron...

Watch this Scientific Journal Video about Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia at JoVE.com

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

星形胶质细胞是中枢神经系统（CNS）最重要的关键球员之一。在这里，我们报告，以研究男性和女性新生儿幼崽的星形胶质细胞功能的体外缺血后背后的机制性控海马星形胶质细胞培养协议的实用方法。

性别差异在小鼠海马星形胶质细胞<em&gt;在体外</em&gt;缺血

Astrogliosis following hypoxia/ischemia (HI)-related brain injury plays a role in increased morbidity and mortality in neonates. Recent clinical studies ...

sex-differences-mouse-hippocampal-astrocytes-after-vitro

Research

JoVE Journal

Neuroscience

Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

13.3K 次观看.  University of Wisconsin. 星形胶质细胞是中枢神经系统（CNS）最重要的关键球员之一。在这里，我们报告，以研究男性和女性新生儿幼崽的星形胶质细胞功能的体外缺血后背后的机制性控海马星形胶质细胞培养协议的实用方法。 

视频: Sex Differences in Mouse Hippocampal Astrocytes after In-Vitro Ischemia

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

漂白后荧光标记蛋白的荧光恢复（FRAP）在培养海马神经元的树突棘

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

科研

神经科学

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Isolation and Culture of Mouse Cortical Astrocytes

Astrocytes are an abundant cell type in the mammalian brain, yet much remains to be learned about their molecular and functional characteristics. In vitro astrocyte cell culture systems can be used to study the biological functions of these glial cells in detail. This video protocol shows how to obtain pure astrocytes by isolation and culture of mixed cortical cells of mouse pups. The method is based on the absence of viable neurons and the separation of astrocytes, oligodendrocytes and microglia, the three main glial cell populations of the central nervous system, in culture. Representative images during the first days of culture demonstrate the presence of a mixed cell population and indicate the timepoint, when astrocytes become confluent and should be separated from microglia and oligodendrocytes. Moreover, we demonstrate purity and astrocytic morphology of cultured astrocytes using immunocytochemical stainings for well established and newly described astrocyte markers. This culture system can be easily used to obtain pure mouse astrocytes and astrocyte-conditioned medium for studying various aspects of astrocyte biology.

Astrocytes have been recognized to be versatile cells participating in fundamental biological processes that are essential for normal brain development and function, and central nervous system repair. Here we present a rapid procedure to obtain pure mouse astrocyte cultures to study the biology of this major class of central nervous system cells.

小鼠脑皮层星形胶质细胞的分离和培养

Astrocytes  are an abundant cell type in the mammalian brain, yet much remains to be  learned about their molecular and functional characteristics. In ...

Sex Stratified Neuronal Cultures to Study Ischemic Cell Death Pathways

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms responsible for those differences remain unclear. Primary disassociated embryonic neuronal culture provides a simplified experimental model with which to investigate the neuronal cell signaling involved in cell death as a result of ischemia or disease; however, most neuronal cultures used in research today are mixed sex. Researchers can and do test the effects of sex steroid treatment in mixed sex neuronal cultures in models of neuronal injury and disease, but accumulating evidence suggests that the female brain responds to androgens, estrogens, and progesterone differently than the male brain. Furthermore, neonate male and female rodents respond differently to ischemic injury, with males experiencing greater injury following cerebral ischemia than females. Thus, mixed sex neuronal cultures might obscure and confound the experimental results; important information might be missed. For this reason, the Herson Lab at the University of Colorado School of Medicine routinely prepares sex-stratified primary disassociated embryonic neuronal cultures from both hippocampus and cortex. Embryos are sexed before harvesting of brain tissue and male and female tissue are disassociated separately, plated separately, and maintained separately. Using this method, the Herson Lab has demonstrated a male-specific role for the ion channel TRPM2 in ischemic cell death. In this manuscript, we share and discuss our protocol for sexing embryonic mice and preparing sex-stratified hippocampal primary disassociated neuron cultures. This method can be adapted to prepare sex-stratified cortical cultures and the method for embryo sexing can be used in conjunction with other protocols for any study in which sex is thought to be an important determinant of outcome.

Primary disassociated embryonic hippocampal neuronal cultures are useful for investigating the signaling mechanisms involved in neuron death. Sexing the embryos before the isolation and dissociation of the hippocampus allows the preparation of separate male and female cultures, which enables the researcher to identify and investigate sex-specific cell signaling.

Sex differences in neuronal susceptibility to ischemic injury and neurodegenerative disease have long been observed, but the signaling mechanisms ...

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

建模星形细胞瘤发病机制<em&gt;在体外</em&gt;和<em&gt;体内</em&gt;使用星形胶质细胞或条件，遗传工程小鼠神经干细胞

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

Immunohistochemical Visualization of Hippocampal Neuron Activity After Spatial Learning in a Mouse Model of Neurodevelopmental Disorders

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we describe a pERK immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. Specifically, we used pERK immunostaining to study neuronal activation following Morris water maze (MWM, a classical hippocampal-dependent learning task) in Engrailed-2 knockout (En2-/-) mice, a model of autism spectrum disorders (ASD). As compared to wild-type (WT) controls, En2-/- mice showed significant spatial learning deficits in the MWM. After MWM, significant differences in the number of pERK-positive neurons were detected in specific hippocampal subfields of En2-/- mice, as compared to WT animals. Thus, our protocol can robustly detect differences in pERK-positive neurons associated to hippocampal-dependent learning impairment in a mouse model of ASD. More generally, our protocol can be applied to investigate the profile of hippocampal neuron activation in both genetic or pharmacological mouse models characterized by cognitive deficits.

We describe an immunohistochemistry protocol to study the profile of hippocampal neuron activation following exposure to a spatial learning task in a mouse model characterized by cognitive deficits of neurodevelopmental origin. This protocol can be applied to both genetic or pharmacological mouse models characterized by cognitive deficits.

海马神经元活动的可视化免疫组化空间后学习神经发育障碍的小鼠模型

Induction of phosphorylated extracellular-regulated kinase (pERK) is a reliable molecular readout of learning-dependent neuronal activation. Here, we ...

Protocol for Three-dimensional Confocal Morphometric Analysis of Astrocytes

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify their functional and structural properties, a condition called reactive astrogliosis. Here, a protocol for assessment of the morphological properties of astrocytes is presented. This protocol includes quantification of 12 different parameters i.e. the surface area and volume of the tissue covered by an astrocyte (astrocyte territory), the entire astrocyte including branches, cell body, and nucleus, as well as total length and number of branches, the intensity of fluorescence immunoreactivity of antibodies used for astrocyte detection, and astrocyte density (number/1,000 &#181;m2). For this purpose three-dimensional (3D) confocal microscopic images were created, and 3D image analysis software such as Volocity 6.3 was used for measurements. Rat brain tissue exposed to amyloid beta1-40 (A&#946;1-40) with or without a therapeutic&#160;intervention was used to present the method. This protocol can also be used for 3D morphometric analysis of other cells from either in vivo or in vitro conditions.

Astrocytes in the CNS change their functional and structural properties in response to harmful stimuli. This report presents a protocol for assessment of three-dimensional astrocyte morphology in diseased conditions or after therapeutic interventions.

协议星形胶质细胞的三维共聚焦形态分析

As glial cells in the brain, astrocytes have diverse functional roles in the central nervous system. In the presence of harmful stimuli, astrocytes modify ...

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent improvements in methodologies and applications for the study of theta oscillations. A simple characterization of the isolated hippocampal preparation is presented whereby the relationship between internal hippocampal theta oscillators is examined together with the activity of pyramidal cells, and GABAergic interneurons, of the cornu ammonis-1 (CA1) and subiculum (SUB) areas. Overall, we show that the isolated hippocampus is capable of generating intrinsic theta oscillations in vitro and that rhythmicity generated within the hippocampus can be precisely manipulated by optogenetic stimulation of parvalbumin-positive (PV) interneurons. The in vitro isolated hippocampal preparation offers a unique opportunity to use simultaneous field and intracellular patch-clamp recordings from visually-identified neurons to better understand the mechanisms underlying theta rhythm generation.

Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit

Here, we present a protocol for recording rhythmic neuronal network theta and gamma oscillations from an isolated whole hippocampal preparation. We describe the experimental steps from extraction of the hippocampus to details of field, unitary and whole-cell patch clamp recordings as well as optogenetic pacing of the theta rhythm.

调整在海马Theta乐队<em&gt;体外</em&gt;:从孤立的啮齿动物皮下注射电路记录的方法

This protocol outlines the procedures for preparing and recording from the isolated whole hippocampus, of WT and transgenic mice, along with recent ...

In Utero Electroporation of Multiaddressable Genome-Integrating Color (MAGIC) Markers to Individualize Cortical Mouse Astrocytes

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

In Utero Electroporation of Multiaddressable Genome-Integrating Color MAGIC Markers to Individualize Cortical Mouse Astrocytes

Astrocytes tile the cerebral cortex uniformly, making the analysis of their complex morphology challenging at the cellular level. The protocol provided here uses multicolor labeling based on in utero electroporation to single out cortical astrocytes and analyze their volume and morphology with a user-friendly image analysis pipeline.

在多地址基因组集成颜色 （MAGIC） 标记的 Utero 电冲中， 以个性化皮质小鼠星形细胞

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

小鼠胚胎干细胞体外神经元分化

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...