这项研究是由院内研究计划部的国立精神卫生研究所，国立卫生研究院（DIRP）。

1。无菌密封的玻璃商会，与MEA长期的录音<ol><li>螺纹口玻璃瓶聚四氟乙烯塑料盖（ACE玻璃），稳固而紧密的文化室封闭所需，钢瓶被切断（Aceglass）从底部的螺纹（图1A，B）的约2毫米。水（3倍），5分钟200证明乙基酒精的沸点冲洗干净的玻璃环，让干。 </li><li>分装芯片解决方案需要附加玻璃戒指来测量表面。混合15毫升的部分A和乙彻底Sylgard 184有机硅弹性体包，让我们坐了15分钟，以去除气泡，在1毫升分装储存在-20 ° C </li><li>胶玻璃环，以“千年生态系统评估”（8x8网格W /内部接地电极，电极直径30微米，200/100微米电极间的距离为鼠/鼠标）（图1A，B）。小针头注射器取1毫升的硅（23℃）。应用硅玻璃环，中心MEA玻璃环，适用于硅周围构建一个更强有力的密封环外的其他层的切割面糙米，让治愈1 - 2小时〜60℃热板。 </li><li>在层流罩3倍去离子水，70％的酒精冲洗消毒MEA室和室盖（3 ×;最后冲洗让坐在酒精10分钟），室的10分钟的曝光和第内部紫外线灯。高压灭菌器MEA室（120 ° C，湿; 45分钟），并让干燥。 </li><li>文化室与D -聚赖氨酸的大衣内的多边环境协定的表面。对于新的多边环境协议，而亲脂性，外套从电极网格解决方案的反复液滴的愿望。对于使用过的多边环境协议，覆盖腔底部与解决方案，吸出多余的液体，使蒸发层流罩内的无菌条件下。附加盖密封存储和将来的使用测量室。 </li></ol> 2。器官型培养的制备及生长所需的成分<ol><li>溶于0.9％氯化钠无菌的琼脂培养基，倒入无菌培养皿（猎鹰100 X15〜5毫米的水平），放冷，用封口膜无菌包装储存。切从固体琼脂20 × 10 × 5毫米使用块。 </li><li>商店超级胶水，如DEVCON超级胶二，包装用70％乙醇擦拭开放前的层流罩内，以保持无菌。 </li><li>准备加入40克的葡萄糖40毫升去离子水养殖水体（Sigma公司）50％D -葡萄糖（SIGMA超，G7528）。在2毫升分装贮存于-20 ° C </li><li>添加4毫升50％D -葡萄糖Gey的平衡盐溶液500毫升，阴寒（混合液体/冰晶）在使用前冷冻贿赂。 </li><li>重组在5毫升去离子养殖水体（摇轻柔，避免形成泡沫）鸡血浆，让解决方案，解决5 - 10分钟，轻轻的漩涡，并decantate的无菌培养皿中明确的内容。无菌过滤器（0.22微米孔径过滤器;蛋白）等离子溶液，分装成cryotubes 350μL（NuncTM），储存在-20 ° C。 </li><li>因此，无菌过滤器（0.22微米孔径过滤器），分装40μL到cryotubes（NuncTM）从牛血浆的重组凝血酶，储存于-20 ° C对于工作的解决方案，Gey W / D -葡萄糖的平衡盐溶液在375μL稀释的凝血酶溶液40μL。 </li><li>准备混合100毫升200毫升基础培养基鹰，马血清，100毫升汉克的缓冲盐溶液4毫升50％葡萄糖2毫升200毫米L -谷氨酰胺补充400毫升培养液中。可存储4 - 8周100毫升PYREX瓶在4 ° C </li><li>准备通过混合0.3毫米阿糖胞苷胞嘧啶-β- D - arabinofuranoside，和0.3毫米0.3毫米尿苷，5 - 氟-2&#39; - 脱氧尿苷，无菌过滤，分装200μL，并储存在-20 ° C 6的有丝分裂抑制剂 - 12个月。 </li></ol> 3。皮层和腹侧被盖区（VTA）组织剥离（时间：&lt;1小时） <ol><li>议事产量皮层和腹侧被盖区〜12共同培养大鼠或小鼠，并准备在层流罩内，无菌条件下的组织切片。组织收集的总时间应超过1小时。 </li><li>以健康，营养良好的仔兔（产仔数〜10;腹部的“牛奶现货”的存在），在1 - 2天（PND）产后。保持轻轻地吻一个小狗，让它挂自由，并迅速在基地的脖子，用锋利的剪刀杀头。 </li><li>对大脑清除，清除皮肤（两个横向剪刀削减），切细眼的剪刀（1矢状中线切1冠状切开皮质/小脑交界）颅骨开放。翻转回所有4个颅骨瓣。一个削尖的锅铲，削减从正面通过嗅球，尾端下方的大脑提前锅铲。轻轻抬起的头骨和大脑让它滑入无菌，冷藏，Gey的快速冷却和临时存储解决方案。重复步骤3.2至3.3（总时间为2个多动脑：&lt;20分钟）。 </li><li>为了获得腹侧被盖区组织，转移到无菌，干燥的Petri使用的小铲菜的大脑。轻轻滑动每个大脑1厘米左右的横盘整理，进一步消除多余的液体。日冕，在使用刀片小脑的水平纵向切开取出脑干。 </li><li>机械稳定在大脑切片程序的安装光盘上胶琼脂块。放置在磁盘上的琼脂块前一个几毫米（避免感人的琼脂胶），细线的强力胶。 </li><li>使用一个小铲，转让和挂载每个大脑额极下来。确保正面两极粘在安装光盘和腹侧双方接触没有任何胶水残留的琼脂，以达到合适的机械在切割和切割片升空。稳定</li><li>小心淹没和大脑在vibratome托盘组件（如莱卡VT1000）的安全安装磁盘充满无菌的，冷冻Gey的解决方案。一个仔细清洗刀片（90％乙醇），降低振动频​​率最高的脑冠状切片和前进速度相对较低，在400〜500微米的厚度。使用一个倒置的巴斯德吸管吸球，转让和收集片含有35 × 10毫米，用无菌的，冷冻Gey的解决方案的Petri充满菜的腹侧被盖区（图1C，看到也冠状板 18 - 15 20 E22）。 </li><li>皮质部分，重复步骤3.2 - 3.6，但适用于大脑皮层和小脑之间的纵向切开，并安装与额极forebrains。对未来的Cortex夹层约3冠状切片（厚度为350微米）的纹状体水平开始收集。 </li><li>用微型刀的碎刀片，解剖1〜2毫米宽的额叶皮层和中脑区包含立体显微镜下腹侧被盖区（图1C）的冠状切面。收集组织切片分别在小碟充满冷冻Gey的解决方案（如玻片）。 </li></ol> 4。安装在“千年生态系统评估”的皮层和腹侧被盖区组织切片（时间：&lt;1小时） <ol><li>放置于室温下一个重点的电极阵列的立体显微镜测量。血浆25μL液滴在干净，无粉尘，无菌电极阵列矩阵中央。用小铲刀，小心滑入一个皮层和腹侧被盖区部分血浆滴。 </li><li>放在散热板测量，重新调整视图，让阴寒为〜15秒，然后添加到等离子体液滴的凝血酶25μL。使用凝血酶枪头，小心地跨越千年生态系统评估“的小圆周运动的等离子体/凝血酶的混合物。不要直接接触脆电极阵列。轻轻的位置上皮层的背侧边境沿线数组数组的第二个电极行。通过这种方式，开发浅层最终将覆盖数组的提醒。腹侧被盖区被放在相邻的腹皮层部分的边界（图1D）。 </li><li>第松散关闭千年生态系统评估室保持高湿度，而坐在MEA /文化大会在室温〜5分钟罩内，让等离子/凝血酶凝血。同时，重复步骤4.1 - 4.3 3更多的文化。 </li><li>小心添加600μL文化中的小水滴中的文化室，使用25 × 5 / 8针1毫升注射器。 </li><li>紧紧贴着千年生态系统评估室和地方MEA /文化大会上的摇摆存储盘内的​​孵化器（图1B）。为了加快程序，3 - 4多边环境协定可在重叠的序列组装。大会时间为12多边环境协定应&lt;1小时。 </li><li> 在体外 （DIV），后2天，加10μL的有丝分裂抑制剂。文化传媒刷新了60％，在4格，以后每4天。 </li></ol> 5。电生理记录和激励生成<ol><li>在局部领域的潜力（LFP）和消防动作电位神经元的倾向显着变形之间的关系，建立后〜1 个星期5,6纪录在24 kHz〜10分钟的自发活动，从每个电极的MEA（五金：MEA1060 W /消隐电路，X1200增益，12位A / D，0-4096 mV的范围内，多声道系统;软件：MC_Rack）。理由是通过内部的接地电极，或外部加入的Ag / AgCl的半细胞。 </li><li>单独用一个带通滤波器1 LFP - 200赫兹（带通300 - 3000赫兹）从外秒杀活动。秒杀活动可以进一步分为单级和多单元使用脱线扣球的分拣机（如Plexon公司公司）的活动。计算油价平均为每个电极。皮质文化，最平均，在培养的神经元扣球的首选时间确定为负LFP挠度（nLFP）。 </li><li>从大幅面痕迹噪声-3标准偏差（SD）的阈值计算每个电极（图2）确定nLFPs的高峰时间和振幅，跨门槛（图2B，C）。选择一个时间斌ΔT（例如介于2 - 8毫秒），并识别连接所有电极在连续的时间长度为ΔTbines（图2D相同nLFPs的阵列上的时空nLFP集群;有关详细信息 ，看到2,4 ，5）。 </li><li>为了确定神经元雪崩，计算每个nLFP集群的大小，活跃电极或nLFP幅度的总和，例如，建造一个大小直方图，在双对数坐标图。对于神经元雪崩，粒度分布如下功法在双对数坐标2（图2E，F）的直线近似。法律对权力的统计测试，请参见16。 </li><li>引出选择电极，通过它的电流控制幅度小号刺激应用（STG 1008刺激发生器，多声道系统），在组织中引起的反应。为了减少电极损坏，使用范围有限，电中性刺激与双极方波单冲击：采用振幅S 50微秒100微秒幅度+ S / 2和S之间的10 - 200微安。见主人的进一步细节手册。 </li><li>要记录的动态范围记录LFP的反应，在4 kHz的采样率在所有电极刺激后500毫秒，记录刺激。消隐电路（多通道系统）​​断开时的刺激，减少刺激的文物和防止前级放大器饱和头级放大器使用。 </li></ol> 6。代表性的成果： 随着新的多边环境协定的约8 - 9 10文化生存的许多个星期。我们长期的录音大多培养箱内培养液中，这使我们能够遵循个别文化的发展，在许多个星期的课程5。根据我们的实验中，大幅面录音可以可靠地获得超过100天文化的多边环境协定。相比之下，外秒杀活动是更可靠的测量与相对新的多边环境协定（&lt;40天文化）。在一个典型的实验中，我们转移到存储盘（图1B，右）多边环境协定的托盘头阶段附加保持密封的文化室（图1B，左）。皮层5，皮质的腹侧被盖区共同文化6，以及在麻醉大鼠和清醒的猴子体内 7，在表面层的神经元放电神经元雪崩期间主要发生密切的磷酸锂铁的峰值负偏转（ nLFP ）。因此，本地同步的神经元群体的时空组织，可以通过测量发生在空间和时间上的阵列17 nLFPs的估计。 “千年生态系统评估”上的活动，往往出现在时间上的集群，这样，在一个电极的活动是伴有其他部位的活动。在这样的活动期间LFP典型波形如图2a所示，超过绘制3集群，除了发生几秒钟。对于每一个群集，负磁场偏转可以看出几个电极内窗口1号当提取nLFP高峰跨多个负面的SD阈值，nLFP高峰时段的形式的活动，是在“列”的点附近重合nLFPs各种焊条（图2B）的代表在其​​中一个光栅方便的可视化。这一活动的时空组织是相当复杂的，“列”，从而出现更多或更少的时间分辨率低的同质，组成独立的集群，在更高的时间分辨率等（图2C）。事实上，时空nLFP集群的出现，是高度组织化的皮质网络。更具体地说，该组织是神经元雪崩的规模不变。这表现在给定的时间分辨率ΔT计算的簇大小的概率。在这里，集群组成，在相同或连续的时间箱（图2D）发生的nLFPs。当这种集群的大小总数nLFPs，每个集群或综合nLFP每个集群振幅表示，簇的大小分布，揭示了一个功法，其斜坡已被证明是 -1.5 2,4,5,7（图2E，F）。请注意，这种分配标识的簇大小，规格有S kxs，其中k是一个常数因子的比例尺度不变的订购， 是 k -1.5，这是独立的S.此功法组织是独立的阵列2的大小，ΔT2，时间分辨率和阈值，以找出重要nLFP的偏差 。由于与神经元组的大小7 nLFP幅度尺度，nLFPs规模不变的组织反映了规模不变，即分形，订货Øf本地同步，包括各种规模的神经元组。 <img src="/files/ftp_upload/2949/2949fig1.jpg" alt="图1"/> 图1（A）“千年生态系统评估”的侧面和顶部的意见安装螺纹玻璃环，和相应的上限。 （二）孵化器的内部。左：探头安装孵化器的条件下，允许从一个单一的文化录制。右：托盘，文化的发展的许多多边环境协定。侧车轮：步进电机控制设备摇摆交替淹没，暴露于大气的阶段，文化的发展所需。 （三）用于皮质的腹侧被盖区共培养大鼠冠状片示意图。皮质部分（左）和中脑的部分（中，右）中含有的腹侧被盖腹侧被盖区（VTA;灰色），获得沿虚线切割。 CTX：皮质; WM：白色物质; CPU：纹状体; VTA：脑桥：脑桥区。也相应冠状板8，18和20 15。 （四）安置和一个单一的皮层腹侧被盖区上的多边环境协定和其对第9格文化发展联合培养增长。注意的扁平化的文化和阵列上逐步扩大。反光组织部分表明退化的细胞和组织碎片。健康的组织，是不透明的，可见光透射下的灰。 <img src="/files/ftp_upload/2949/2949fig2.jpg" alt="图2"/> 图（2）神经元在大脑皮层器官文化的雪崩。 （一）三个阵列上的自发活动期间Overplot，几秒钟分开。请注意，每个活动期间，许多电极阵列上的负面LFP挠度（每种颜色的标签，一个活动期）组成。 （二）每个电极nLFPs的负峰值时间是组装成一个活动的栅格。 “表示Column&#39;状结构附近的同步活动的时期。 （三）需要注意的是出现在一个时间尺度高度同步的列较高的时间尺度（时间尺度3）的多个列组成。 （四）神经元雪崩算法的示意图。在一个2 × 2电极阵列高峰时间和幅度的负LFP挠度过路- X SD的噪声阈值（nLFP）确定。 nLFPs时空组织聚成先后活跃的时间宽度ΔT箱。簇大小是确定的活性位点，即与nLFP电极（S = 4），或综合nLFP振幅总和（S = 130μV）。续航时间是衡量ΔT的倍数。 （E，F）簇大小分布的功法确定为神经元雪崩的集群。请注意，δD为阵列（这里为200微米）的一个特定的极间距离的选择，引入了一个特别的ΔT的动态时，应遵守。更具体地说，在其中的比例ΔD/ΔT接近平均在网络中的传播速度，在这功法的坡度α神经元雪崩 2,4,5接近-1.5 。请<a href="http://www.jove.com/files/ftp_upload/2949/2949largefig2.jpg">点击这里</a>看大图图2。

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	<li>Spitzer, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+activity+in+early+neuronal+development.">Electrical activity in early neuronal development.</a> Nature. 444, 707-712 (2006).</li><li>Beggs, J. M., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+avalanches+in+neocortical+circuits.">Neuronal avalanches in neocortical circuits.</a> J. Neurosci. 23, 11167-11177 (2003).</li><li>Beggs, J. M., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+avalanches+are+diverse+and+precise+activity+patterns+that+are+stable+for+many+hours+in+cortical+slice+cultures.">Neuronal avalanches are diverse and precise activity patterns that are stable for many hours in cortical slice cultures.</a> J Neurosci. 24, 5216-5229 (2004).</li><li>Stewart, C. V., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inverted-U+profile+of+dopamine-NMDA-mediated+spontaneous+avalanche+recurrence+in+superficial+layers+of+rat+prefrontal+cortex.">Inverted-U profile of dopamine-NMDA-mediated spontaneous avalanche recurrence in superficial layers of rat prefrontal cortex.</a> J. Neurosci. 26, 8148-8159 (2006).</li><li>Stewart, C. V., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostasis+of+neuronal+avalanches+during+postnatal+cortex+development+in+vitro.">Homeostasis of neuronal avalanches during postnatal cortex development in vitro.</a> J. Neurosci. Meth. 169, 405-416 (2007).</li><li>Gireesh, E. D., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+avalanches+organize+as+nested+theta-+and+beta/gamma-oscillations+during+development+of+cortical+layer+2/3.">Neuronal avalanches organize as nested theta- and beta/gamma-oscillations during development of cortical layer 2/3.</a> Proc. Natl. Acad. Sci. U. S. A. 105, 7576-7581 (2008).</li><li>Petermann, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+cortical+activity+in+awake+monkeys+composed+of+neuronal+avalanches.">Spontaneous cortical activity in awake monkeys composed of neuronal avalanches.</a> Proc. Natl. Acad. Sci. U. S. A. 106, 15921-15926 (2009).</li><li>Kinouchi, O., Copelli, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+dynamical+range+of+excitable+networks+at+criticality.">Optimal dynamical range of excitable networks at criticality.</a> Nature Physics. 2, 348-351 (2006).</li><li>Shew, W. L., Yang, H., Petermann, T., Roy, R., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+avalanches+imply+maximum+dynamic+range+in+cortical+networks+at+criticality.">Neuronal avalanches imply maximum dynamic range in cortical networks at criticality.</a> J. Neurosci. 29, 15595-15600 (2009).</li><li>Shew, W. L., Yang, H., Yu, S., Roy, R., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Information+capacity+is+maximized+in+balanced+cortical+networks+with+neuronal+avalanches.">Information capacity is maximized in balanced cortical networks with neuronal avalanches.</a> J Neurosci. 5, 55-63 (2011).</li><li>Karpiak, V., Plenz, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+and+maintenance+of+organotypic+cultures+for+multi-electrode+array+recordings.">Preparation and maintenance of organotypic cultures for multi-electrode array recordings.</a> Current Protocols in Neuroscience. 1, 6-6 (2002).</li><li>Hammerle, H., Egert, U., Mohr, A., Nisch, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracellular+recording+in+neuronal+networks+with+substrate+integrated+microelectrode+arrays.">Extracellular recording in neuronal networks with substrate integrated microelectrode arrays.</a> Biosens. Bioelectron. 9, 691-696 (1994).</li><li>Nisch, W., Bock, J., Egert, U., Hammerle, H., Mohr, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+thin+film+microelectrode+array+for+monitoring+extracellular+neuronal+activity+in+vitro.">A thin film microelectrode array for monitoring extracellular neuronal activity in vitro.</a> Biosens. Bioelectron. 9, 737-741 (1994).</li><li>Egert, U. <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+organotypic+long-term+culture+of+the+rat+hippocampus+on+substrate-integrated+multielectrode+arrays.">A novel organotypic long-term culture of the rat hippocampus on substrate-integrated multielectrode arrays.</a> Brain Res. Protoc. 2, 229-242 (1998).</li><li>Altman, J., Bayer, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Atlas of Prenatal Rat Brain Development. , (1995).</li><li>Clauset, A., Shalizi, C. R., Newman, M. 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=Power-law+distributions+in+empirical+data.">Power-law distributions in empirical data.</a> arXiv. , (2009).</li><li>Plenz, D., Thiagarajan, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organizing+principles+of+neuronal+avalanches:+cell+assemblies+in+the+cortex?.">The organizing principles of neuronal avalanches: cell assemblies in the cortex?.</a> Trends Neurosci. 30, 101-110 (2007).</li><li>C&#228;ser, M., Bonhoeffer, T., Bolz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+organization+and+development+of+slice+cultures+from+rat+visual+cortex.">Cellular organization and development of slice cultures from rat visual cortex.</a> Exp. Brain Res. 477, 234-244 (1989).</li><li>C&#228;ser, M., Sch&#252;z, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maturation+of+neurons+in+neocortical+slice+cultures.+A+light+and+electron+microscopic+study+on+in+situ+and+in+vitro+material.">Maturation of neurons in neocortical slice cultures. A light and electron microscopic study on in situ and in vitro material.</a> J. Hirnforsch. 33, 429-443 (1992).</li><li>G&#246;tz, M., Bolz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+vasoactive+intestinal+polypeptide+(VIP)-containing+neurons+in+organotypic+slice+cultures+from+rat+visual+cortex.">Development of vasoactive intestinal polypeptide (VIP)-containing neurons in organotypic slice cultures from rat visual cortex.</a> Neurosci. Lett. 107, 6-11 (1989).</li><li>G&#246;tz, M., Bolz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+and+Preservation+of+cortical+layers+in+slice+cultures.">Formation and Preservation of cortical layers in slice cultures.</a> J. Neurobiol. 23, 783-802 (1992).</li><li>Bolz, J., Novak, N., G&#246;tz, M., Bonhoeffer, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+target-specific+neuronal+projections+in+organotypic+slice+cultures+from+rat+visual+cortex.">Formation of target-specific neuronal projections in organotypic slice cultures from rat visual cortex.</a> Nature. 346, 359-362 (1990).</li><li>Bolz, J., Novak, N., Staiger, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+specific+afferent+connections+in+organotypic+slice+cultures+from+rat+visual+cortex+cocultured+with+lateral+geniculate+nucleus.">Formation of specific afferent connections in organotypic slice cultures from rat visual cortex cocultured with lateral geniculate nucleus.</a> J. Neurosci. 12, 3054-3070 (1992).</li><li>Novak, N., Bolz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+specific+efferent+connections+in+organotypic+slice+cultures+from+rat+visual+cortex+cocultured+with+lateral+geniculate+nucleus+and+superior+colliculus.">Formation of specific efferent connections in organotypic slice cultures from rat visual cortex cocultured with lateral geniculate nucleus and superior colliculus.</a> Eur. J. Neurosci. 5, 15-24 (1993).</li><li>Plenz, D., Aertsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+dynamics+in+cortex-striatum+co-cultures.+I.+Anatomy+and+electrophysiology+of+neuronal+cell+types.">Neural dynamics in cortex-striatum co-cultures. I. Anatomy and electrophysiology of neuronal cell types.</a> Neurosci. 70, 861-891 (1996).</li><li>Plenz, D., Aertsen, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+dynamics+in+cortex-striatum+co-cultures.+II.+Spatio-temporal+characteristics+of+neuronal+activity.">Neuronal dynamics in cortex-striatum co-cultures. II. Spatio-temporal characteristics of neuronal activity.</a> Neurosci. 70, 893-924 (1996).</li><li>Plenz, D., Kitai, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+cortex-striatum-mesencephalon+cultures:+the+nigro-striatal+pathway.">Organotypic cortex-striatum-mesencephalon cultures: the nigro-striatal pathway.</a> Neurosci. Lett. 209, 177-180 (1996).</li><li>Plenz, D., Kitai, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Up'+and+'down'+states+in+striatal+medium+spiny+neurons+simultaneously+recorded+with+spontaneous+activity+in+fast-spiking+interneurons+studied+in+cortex-striatum-substantia+nigra+organotypic+cultures.">Up' and 'down' states in striatal medium spiny neurons simultaneously recorded with spontaneous activity in fast-spiking interneurons studied in cortex-striatum-substantia nigra organotypic cultures.</a> J. Neurosci. 18, 266-283 (1998).</li><li>Plenz, D., Herrera-Marschitz, M., Kitai, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphological+organization+of+the+globus+pallidus-subthalamic+nucleus+system+studied+in+organotypic+cultures.">Morphological organization of the globus pallidus-subthalamic nucleus system studied in organotypic cultures.</a> J. Comp. Neurol. 397, 437-457 (1998).</li><li>Plenz, D., Kitai, S. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+basal+ganglia+pacemaker+formed+by+the+subthalamic+nucleus+and+external+globus+pallidus+[see+comments].">A basal ganglia pacemaker formed by the subthalamic nucleus and external globus pallidus [see comments].</a> Nature. 400, 677-682 (1999).</li><li>Plenz, D., Kitai, S. T. <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+high-frequency+oscillations+in+local+circuits+of+rat+somatosensory+cortex+cultures.">Generation of high-frequency oscillations in local circuits of rat somatosensory cortex cultures.</a> J Neurophysiol. 76, 4180-4184 (1996).</li><li>Klostermann, O., Wahle, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+spontaneous+activity+and+morphology+of+interneuron+types+in+organotypic+cortex+and+thalamus-cortex+cultures.">Patterns of spontaneous activity and morphology of interneuron types in organotypic cortex and thalamus-cortex cultures.</a> Neurosci. 92, 1243-1259 (1999).</li><li>Cowan, R. L., Wilson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneous+firing+patterns+and+axonal+projections+of+single+cortico-striatal+neurons+in+the+rat+medial+agranular+cortex.">Spontaneous firing patterns and axonal projections of single cortico-striatal neurons in the rat medial agranular cortex.</a> J. Neurophysiol. 71, 17-32 (1994).</li><li>Porter, L. L., Rizzo, E., Hornung, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dopamine+affects+parvalbumin+expression+during+cortical+development+in+vitro.">Dopamine affects parvalbumin expression during cortical development in vitro.</a> J Neurosci. 19, 8990-9003 (1999).</li></ol>

没有利益冲突的声明。

1。技术问题： <ol><li> 无菌技术。多边环境协定和文化准备的准备工作都是层流罩内，在无菌条件下进行。不使用抗生素，从而影响神经元的活动，在任何时间，在编制和文化的过程中。 </li><li> 等离子/凝血酶凝血和组织上坚持对MEA 多边环境协定。组织的生存需要一个谨慎的平衡之间的等离子/凝血酶凝血和组织暴露在大气中时所需的时间。一个凝血时间短，风险过早支队从MEA部分，而长时间暴露到大气中的触发器组织变性。由于凝血酶解决方案的实力，决定了凝血过程的速度，这是一个非常重要的参数，成功，健康的文化MEA表面附着。我们取得最佳效果，使用1000个单位（1千片; 1美国国立卫生研究院单位= 0.324 ± 0.073毫克）。更重要的是，不完整的空间异质性凝血血浆/凝血酶的解决方案，促进在培养等离子体破裂的结果混合。这些等离子的“孔”文化健康造成严重影响，从微电极分离的文化，从而损害的电生理记录的质量。冷却板，在多边环境协定/组织装配工作，减缓凝血，并允许为均质混合血浆/凝血酶的解决方案和正确定位组织切片。 同样，加入单液滴中文化室淹没后仅5分钟凝固的文化，大大减少了由于表面张力的组织分队的风险。一个成功的文化，将扁平化“千年生态系统评估，并略有扩大显示周的健康成长，没有任何不完整的组织，多边环境协定的联系或组织变性（例如图1D）主要标志。 </li><li> 组织剥离。微型刀，极大地提高了我们的清扫过程。我们使用双重优势刀片（精细的科学工具 - 易碎的手术刀片 - 100050-00），从中我们分裂〜2毫米宽的“刀片”用钳子，和他们用手术刀持有人持有。从冠状切片用刀片垂直平稳，公司的议案解剖的组织样本，从而大大降低了由于机械应力拉动整个组织，提高文化健康。 </li><li>组织冷却。适当的散热片和组织切片在准备文化的成功至关重要。我们使用佩尔蒂埃元素连接到一个金属圆盘下方内置的定制冷板。佩尔蒂埃元素产生的热量是通过冷水灌注中删除。这大大减少了准备时间和标准化，在每个阶段的准备冷却（DOLD实验室与工程博士131种植塞甘，TX 78155，（830）560-1471））。 </li><li> 孵化条件。一个定制孵化器与精确的内部摇摆条件下，我们在培养上的多边环境协定的片的成功至关重要。内部的摇摆设备的基础上由Multichannelsystems原有的内部设计（目前市面上的），包括连接到两个大的一侧车轮的托盘。步进电机和电脑控制允许为一个精确的摇摆轨迹（摇摆角，摇摆速度，间歇性停顿）。最终，片文化需要公开的氛围和文化，慢交替的媒介。传统的方法是放置到狭窄的管片，慢慢地沿着有最长的轴旋转文化。在这里，自转速度慢不产生机械应力，由于旋转本身，和旋转速度足够高，以获得最佳的“进料/呼吸周期约5 - 10分钟的持续时间。千年生态系统评估室，其总量的〜2毫升，小的培养基量为中小型空调需要由组织本身更紧凑的内部，构成一个巨大的挑战。 ±〜70 °角（周期时间：200秒）之间的摇摆“千年生态系统评估”的液相和大气之间的文化转换的摇摆速度减慢，并​​停止在极端的角度，长时间暴露在大气中的摇摆已对于文化的生存至关重要。 </li></ol> 2。皮质文化的发育年龄来研究神经元雪崩通常采取的PND 0 - 1片从大鼠皮层，急性和培养“千年生态系统评估”的几个星期。早期的研究已清楚地表明，单皮质切片培养， 在体外培养的几个星期后，保持与识别细胞类型的层状结构，可以很容易地比较在体内细胞类18,18-21。分层组织已在体外系统在此方便地用于研究丘脑22日至24日在开发过程中，以及支配的皮层皮层下结构，如纹状体 25,26驾驶。事实上，在形成的大脑区域内和跨的神经元连接的特殊性， 允许复杂的体外系统，重新夺回了许多详细的投影系统的建设 ，如皮层基底节电路27-30。 经过4 - 6周在体外，单皮质片31和皮质切片培养与纹状体26或32丘脑显示自发向上和向下通常在33乌拉坦麻醉大鼠体内发现的状态这些最多的国家的优秀时间组织负有标志嵌套θ-和γ-振荡的锥体神经元和GABA能interneurons快速扣球31 electrophysiologically成熟的网络指标。重要的是，在多巴胺D2受体的刺激的情况下，成熟的小白蛋白阳性皮质interneurons延迟约2 周皮质切片文化34。嵌套在这些研究结果，发展的时间当然θ，β-和γ-振荡匹配体内的皮质切片时，腹侧被盖区（VTA），其中包含的多巴胺能神经元投射到联合培养皮质6。 这些研究表明，在研究神经雪崩，关键取决于成熟快的GABA能抑制，并在位于大脑皮层2,4的 ​​浅层时，应采取非常谨慎，以保证适当的皮层组织成熟。虽然神经元雪崩出现超过2的时间当然在单一皮质文化- 5 周 4，当需要一个发展的时间当然这是在体内发育匹配，皮质片需要适当的多巴胺受体刺激，例如共培养皮层切片腹侧被盖区6。

<table border="1"><tbody><tr><th>试剂名称</th><th>公司</th><th>目录编号</th><th>评论（可选） </th></tr><tr><td>综合平面多电极阵列</td><td>多声道系统（ <a href="www.multichannelsystems.com">www.multichannelsystems.com</a> ） ALA科学仪器 （www.alascience.com） </td><td> <a href="http://www.multichannelsystems.com/products-mea/product-details/products/158/20030ir-ito-wo.html">200/30iR-ITO-w/o</a> </td><td>钛硝酸盐（锡）电极（直径30毫米）大的表面，造成低阻抗（在1 kHz〜1.5kΩ）的和优秀的宽波段录音（W / O型 - 无环） </td></tr><tr><td>商会玻璃</td><td> www.aceglass.com </td><td> 7620-32 </td><td>螺纹口玻璃瓶缸</td></tr><tr><td>商会第</td><td> www.aceglass.com </td><td> 7622-114 </td><td>聚四氟乙烯塑料瓶盖插入</td></tr><tr><td> Sylgard 184 </td><td> www.wpiinc.com </td><td> SYLG184 </td><td>由两部分组成的有机硅弹性体</td></tr><tr><td>聚- D -赖氨酸</td><td> Sigma - Aldrich公司</td><td> P6407 - 5毫克</td><td> γ辐照，冻干粉，细胞培养测试。重组与5毫升去离子水在使用前。 </td></tr><tr><td> Gey的平衡盐溶液</td><td> Sigma - Aldrich公司</td><td> G9779 - 500ML </td><td>无菌过滤和培养测试</td></tr><tr><td>鸡血浆</td><td> Sigma - Aldrich公司</td><td> P3266 - 5ML </td><td>冻干重组在使用前用5毫升去离子水。 </td></tr><tr><td>凝血酶</td><td> Sigma - Aldrich公司</td><td> T6634 - 1千片</td><td>从牛血浆，冻干粉形式。 </td></tr><tr><td>马血清</td><td> Sigma - Aldrich公司</td><td> H1138 - 100ML </td><td>捐助者一哄而上，热灭活，细胞培养测试</td></tr><tr><td>基础培养基鹰</td><td> Invitrogen公司</td><td> 21010-046 </td><td> 1X，500毫升 - （+）厄尔的盐，（ - ）L -谷氨酰胺）， </td></tr><tr><td>汉克的缓冲盐溶液</td><td> Invitrogen公司</td><td> 24020-117 </td><td> 500毫升 - （+），镁，（+），钙，W /酚红） </td></tr><tr><td>玻片</td><td>商会幻灯片实验室TEK - W /盖（NUNC），无菌</td><td> 177429 </td><td></td></tr><tr><td>尿苷</td><td> Sigma - Aldrich公司</td><td> U3003 </td><td></td></tr><tr><td>阿糖胞苷胞嘧啶-β- D - arabinofuranoside </td><td> Sigma - Aldrich公司</td><td> C6645 </td><td></td></tr><tr><td> 5 - 氟-2&#39; - 脱氧尿苷</td><td> Sigma - Aldrich公司</td><td> F0503 </td><td></td></tr></tbody></table>

multi-electrode array recordings of neuronal avalanches in organotypic cultures

皮层是自发活跃，即使在没有任何特定的输入或电机输出。在发展过程中，这个活动是非常重要的皮层细胞类型，迁移和分化形成的神经元连接1。在成熟的动物，正在进行的活动，反映过去和目前的状态到感官刺激的无缝集成，计算未来行动​​的动物。因此，一个清醒的认识即正在进行的自发活动的组织是一个先决条件，以了解皮质功能。 众多的录音技术透露，正在进行的活动是在大脑皮层的个别活动的瞬时总结较大的事件，与细胞外微电极可以在局部领域的潜力（LFP）检测，或在脑电图（EEG），magnetoencephalogram（MEG的许多神经元组成），并从功能磁共振成像（fMRI）的大胆信号。磷酸锂铁是目前的首选方法，在学习时，在介观尺度的高时空分辨率（数千神经元）神经元群活动。在细胞外微电极，在本地同步活动空间相邻的神经元在LFP数百毫伏的快速变形的结果。当使用微电极阵列，这种变形的组织可以很方便地监控在时间和空间。 神经元雪崩描述持续2,3大脑中的神经活动的规模不变的时空组织。它们是针对4,5 体外成立6在麻醉大鼠体内的，肤浅的的皮质层，并在清醒的猴子 7 。更重要的是，理论和实证研究2,8-10表明，神经元雪崩指示的皮质精美平衡的临界状态动态，优化信息传输和信息处理。 为了研究神经元雪崩的发展，维护和调节机制， 在体外的准备工作非常有益，因为它们允许精确控制的条件下稳定的雪崩活动录音。目前的协议，介绍了如何在体外培养的神经元雪崩研究器官皮质文化，即片文化上的平面，集成的微电极阵列（MEA;见11-14）增长，利用浅层发展优势。

Watch this Scientific Journal Video about Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures at JoVE.com

Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

一个可靠的方法来研究神经元雪崩，即规模不变的时空活动突发，在大脑皮层的临界状态动力学指示。雪崩出现自发的发展培养皮层的表面层与平面集成多电极阵列（MEA）的精确控制的条件下允许活动的长期测量。

器官型培养的神经元雪崩多电极阵列记录

The cortex is spontaneously active, even in the absence of any particular input or motor output.  During development, this activity is important for the ...

multi-electrode-array-recordings-neuronal-avalanches-organotypic

Research

JoVE Journal

Neuroscience

26.3K 次观看.  National Institute of Mental Health. 一个可靠的方法来研究神经元雪崩，即规模不变的时空活动突发，在大脑皮层的临界状态动力学指示。雪崩出现自发的发展培养皮层的表面层与平面集成多电极阵列（MEA）的精确控制的条件下允许活动的长期测量。

视频: Multi-electrode Array Recordings of Neuronal Avalanches in Organotypic Cultures

Multielectrode Array Recordings of the Vomeronasal Epithelium

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is planar multielectrode array (MEA) recording. Here we document the use of MEAs to study the mouse vomeronasal organ (VNO), which plays an essential role in the detection of pheromones and social cues via a diverse population of sensory neurons expressing hundreds of types of receptors. Combining MEA recording with a robotic liquid handler to deliver chemical stimuli, the sensory responses of a large and diverse population of neurons can be recorded. The preparation allows us to remove the intact neuroepithelium of the VNO from the mouse and stimulate with a battery of chemicals or potential ligands while monitoring the electrical activity of the neurons for several hours. Therefore, this technique serves as a useful method for assessing ligand activity as well as exploring the properties of receptor neurons. We present the techniques needed to prepare the vomeronasal epithelium, MEA recording, and chemical stimulation.

Multielectrode array (MEA) recordings provide a method for studying the electrical activity of large populations of neurons. Here, we present the details of a MEA preparation to record from the mouse vomeronasal epithelium while simultaneously stimulating the tissue.

多电极阵列的犁鼻上皮录音

Understanding neural circuits requires methods to record from many neurons simultaneously. For in vitro studies, one currently available technology is ...

科研

神经科学

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

器官型海马切片培养

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

The mouse is an excellent model organism to study mammalian brain development due to the abundance of molecular and genetic data. However, the developing mouse brain is not suitable for easy manipulation and imaging in vivo since the mouse embryo is inaccessible and opaque. Organotypic slice cultures of embryonic brains are therefore widely used to study murine brain development in vitro. Ex-vivo manipulation or the use of transgenic mice allows the modification of gene expression so that subpopulations of neuronal or glial cells can be labeled with fluorescent proteins. The behavior of labeled cells can then be observed using time-lapse imaging. Time-lapse imaging has been particularly successful for studying cell behaviors that underlie the development of the cerebral cortex at late embryonic stages 1-2. Embryonic organotypic slice culture systems in brain regions outside of the forebrain are less well established. Therefore, the wealth of time-lapse imaging data describing neuronal cell migration is restricted to the forebrain 3,4. It is still not known, whether the principles discovered for the dorsal brain hold true for ventral brain areas. In the ventral brain, neurons are organized in neuronal clusters rather than layers and they often have to undergo complicated migratory trajectories to reach their final position. The ventral midbrain is not only a good model system for ventral brain development, but also contains neuronal populations such as dopaminergic neurons that are relevant in disease processes. While the function and degeneration of dopaminergic neurons has been investigated in great detail in the adult and ageing brain, little is known about the behavior of these neurons during their differentiation and migration phase 5. We describe here the generation of slice cultures from the embryonic day (E) 12.5 mouse ventral midbrain. These slice cultures are potentially suitable for monitoring dopaminergic neuron development over several days in vitro. We highlight the critical steps in generating brain slices at these early stages of embryonic development and discuss the conditions necessary for maintaining normal development of dopaminergic neurons in vitro. We also present results from time lapse imaging experiments. In these experiments, ventral midbrain precursors (including dopaminergic precursors) and their descendants were labeled in a mosaic manner using a Cre/loxP based inducible fate mapping system 6.

Organotypic Slice Cultures of Embryonic Ventral Midbrain: A System to Study Dopaminergic Neuronal Development in vitro

A method to generate organotypic slices from the E12.5 murine embryonic midbrain is described. The organotypic slice cultures can be used to observe the behavior of dopaminergic neurons or other ventral midbrain neurons.

胚胎腹侧中脑器官型片文化：一个系统来研究多巴胺能神经元发展在体外</em

The  mouse is an excellent model organism to study mammalian brain development due  to the abundance of molecular and genetic data. However, the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

浦肯野细胞树突形态的器官切片培养分析

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

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 ...

Design and Fabrication of Ultralight Weight, Adjustable Multi-electrode Probes for Electrophysiological Recordings in Mice

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic targeting for microcircuit dissection and disease modeling. The introduction of optogenetics, for example, has allowed for bidirectional manipulation of genetically-identified neurons, at an unprecedented temporal resolution. To capitalize on these tools and gain insight into dynamic interactions among brain microcircuits, it is essential that one has the ability to record from ensembles of neurons deep within the brain of this small rodent, in both head-fixed and freely behaving preparations. To record from deep structures and distinct cell layers requires a preparation that allows precise advancement of electrodes towards desired brain regions. To record neural ensembles, it is necessary that each electrode be independently movable, allowing the experimenter to resolve individual cells while leaving neighboring electrodes undisturbed. To do both in a freely behaving mouse requires an electrode drive that is lightweight, resilient, and highly customizable for targeting specific brain structures.
A technique for designing and fabricating miniature, ultralight weight, microdrive electrode arrays that are individually customizable and easily assembled from commercially available parts is presented. These devices are easily scalable and can be customized to the structure being targeted; it has been used successfully to record from thalamic and cortical regions in a freely behaving animal during natural behavior.

Understanding the neural substrates of behavior requires brain circuit ensemble recording. Because of its genetic tractability, the mouse offers a model for circuit dissection and disease mimicry. Here, a method of designing and fabricating miniaturized probes is described that is suitable for targeting deep brain structure in the mouse.

设计和超轻重量的制作，可调节的多电极电生理记录小鼠探头

The number of physiological investigations in the mouse, mus musculus, has experienced a recent surge, paralleling the growth in methods of genetic ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

在器官型海马脑片搭配全细胞记录

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Investigating Functional Regeneration in Organotypic Spinal Cord Co-cultures Grown on Multi-electrode Arrays

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a promising target for intervention to improve functional regeneration. So far, no in vitro model exists that grants the possibility to examine functional recovery of propriospinal fibers. Therefore, a representative model that is based on two organotypic spinal cord sections of embryonic rat, cultured next to each other on multi-electrode arrays (MEAs) was developed. These slices grow and, within a few days in vitro, fuse along the sides facing each other. The design of the used MEAs permits the performance of lesions with a scalpel blade through this fusion site without inflicting damage on the MEAs. The slices show spontaneous activity, usually organized in network activity bursts, and spatial and temporal activity parameters such as the location of burst origins, speed and direction of their propagation and latencies between bursts can be characterized. Using these features, it is also possible to assess functional connection of the slices by calculating the amount of synchronized bursts between the two sides. Furthermore, the slices can be morphologically analyzed by performing immunohistochemical stainings after the recordings. 
Several advantages of the used techniques are combined in this model: the slices largely preserve the original tissue architecture with intact local synaptic circuitry, the tissue is easily and repeatedly accessible and neuronal activity can be detected simultaneously and non-invasively in a large number of spots at high temporal resolution. These features allow the investigation of functional regeneration of intraspinal connections in isolation in vitro in a sophisticated and efficient way.

Here we present a protocol that is based on spinal cord slices cultured on multi-electrode arrays to study functional regeneration of propriospinal connections in vitro.

调查器官脊髓联合培养物中生长多电极阵列功能再生

Adult higher vertebrates have a limited potential to recover from spinal cord injury. Recently, evidence emerged that propriospinal connections are a ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

小鼠脑切片准备扣押样活性的直接电流刺激和多电极阵列记录

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

Purkinje Cell Survival in Organotypic Cerebellar Slice Cultures

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early postnatal development, cerebellar Purkinje cells are known to go through a vulnerable period, during which they undergo programmed cell death. Here, we provide a detailed protocol to perform mouse organotypic cerebellar slice culture during this critical time. The slices are further labeled to assess Purkinje cell survival and the efficacy of neuroprotective treatments. This method can be extremely valuable to screen for new neuroactive molecules.

Organotypic slice cultures are a powerful tool to study neurodevelopmental or degenerative/regenerative processes. Here, we describe a protocol that models the neurodevelopmental death of Purkinje cells in mouse cerebellar slice cultures. This method may benefit research in neuroprotective drug discovery.

器官细胞生存在器官细胞切片培养

Organotypic slice culture is a powerful in vitro model that mimicks in vivo conditions more closely than dissociated primary cell cultures. In early ...