这项研究得到了 NSF (HBCU 研究发起奖, 人力资源开发1401426和 EPSCoR EPS-0814251) 和 NIH (COBRE 1P20GM103653-01A1) 的支持。K.S. 得到特拉华 INBRE-III. 6404 的奖学金支持。

下面给出的协议已经标准化, 以测试 BPA 暴露在 (早期) 胚胎发生期间的影响, 并可能被修改, 用于与 BPS, BPF, 或其他在小鸡胚胎神经元的 EDC。
该议定书遵循特拉华州立大学的体制政策和国家卫生研究院关于禽类胚胎的政策。《议定书》还确认了争端谅解书的材料和化学品安全准则。
1. 材料设置
<ol><li>将 BPA (m.w. 228.29) 溶于水中10% 乙醇 (v/v), 使1毫米的库存溶液 (0.23 毫克每毫升10% 乙醇)。在µM 介质中进行后续稀释 (0.1 µM、0.5 µM、1µM、5µM 和 10 neurobasal)。 注意: BPA 是一种环境毒素, 在处理粉末时应注意。</li>
	<li>在37摄氏度的商用台式鸡蛋孵化器孵化小鸡卵。为 E7 胚胎孵化7天的卵。</li>
	<li>准备多边环境协定. 用70% 乙醇浸泡3到4小时, 然后在 BSLII 罩中用大约10毫升的不育蒸馏水冲洗三次, 以杀菌所需的多边环境协定。 注: 多边环境协定包含 8 x 8 网格中的64个纳米孔铂电极。电极直径为30µm, 电极间距为200µm。不要使用变性乙醇杀菌, 因为这将导致对多边环境协定的腐蚀。</li>
	<li>将多边环境协定放在一个无菌容器内, 盖上 (以保持无菌条件), 并移动到工作台顶部孵化器。烘烤至少6小时在55°c。这一步骤对正确的灭菌至关重要, 温度不应超过摄氏60摄氏度。保持不孕, 将含有多边环境协定的碟子移至 BSLII 罩内存放, 直至进一步使用。 注: 我们使用烤箱安全玻璃烤盘与塑料盖子和表面消毒它与70% 乙醇和地方在 BSLII 敞篷为烘干。</li>
	<li>整除了额外的蜂窝矩阵 ECM 解决方案。解冻的瓶子在4°c 隔夜和冻结100µL 整除数在-20 摄氏度。 注: ECM 已被发运冻结。ECM 不应该被带到室温, 直到它在室温下聚合, 一旦聚合, 就不可能涂上涂层。没有生长因子的 ECM 更倾向于防止由于未知因素造成的额外影响。</li>
	<li>消毒所有解剖器械 (#5 钳、弯钳、小剪刀、弹簧剪刀) 和自密封材料, 涂上70% 乙醇, 让它们在解剖罩中干燥。</li>
</ol>2. 鸡胚神经元培养与网络活动
<ol><li>电镀当天, 从-20 °c 冰箱中取出一小瓶 ECM, 用70% 乙醇喷雾, 然后放在冰上。在 BSLII 罩内加入300µL 冷 neurobasal 培养基稀释至25%。</li>
	<li>使用 P200 pipetteman 将100µL 25% ECM 添加到多边环境协定中心, 注意不要触摸电极, 并立即去除表面上的薄膜。在 CO2孵化器 (37 ° C 和 5% CO2) 中覆盖多边环境协定和位置, 直到准备好对神经元进行板。</li>
	<li>对 E7 卵的外壳进行消毒 (胚胎日 7, 在37摄氏度孵化7天), 70% 乙醇。将胚胎斩首成一个 Sylgard 的底部解剖皿, 含有冷不育的汉克平衡盐溶液 (HBSS) 而不含钙。切开眼睛, 去掉眼球。使用 #5 细钳和弹簧剪刀在腹侧进行切口, 去除外层皮肤, 露出前脑和视神经顶盖。仔细剥离和去除脑膜膜。将前脑转移到另一个培养皿中, 用弹簧剪刀把它切成2毫米的小块。 注: 脑膜膜切除后, 前脑不应有任何血管附着。如果有的话, 最好是在无菌解剖罩进行解剖, 虽然我们取得了相当好的结果, 当解剖是在罩外进行, 只要仪器和解剖区彻底消毒70% 乙醇.</li>
	<li>使用无菌转移吸管, 将前脑片收集到15毫升离心管中, 并在让前脑片下沉到离心管底部后尽可能 HBSS。</li>
	<li>添加1毫升0.05% 胰蛋白酶/EDTA (预热到37°c) 和孵化在37°c 15 分钟。</li>
	<li>使用巴斯德吸管, 小心地去除胰蛋白酶, 不扰乱组织和添加1毫升 neurobasal 培养基。让组织的片断下沉到底部并且去除媒介。再重复一次这个洗涤。这一步是洗掉胰蛋白酶/EDTA。</li>
	<li>加入2毫升的 neurobasal 培养基, 开始研制过程。研磨包括服用无菌的火抛光的巴斯德吸管, 并通过它轻轻地通过它几次, 直到没有更多的组织被看到。如果在反复传球后留下任何碎片, 只要让它们沉淀到底部即可。 注: 避免起泡, 而研制过程-如果泡沫形成, 停止和消除的愿望。</li>
	<li>用 neurobasal 培养基稀释重悬浮细胞 1:10, 用台盼蓝染料和 hemocytometer 计数活细胞。在0.5 毫升离心管中混合50µL 的稀释细胞与50µL 的台盼蓝溶液。添加10µL 到 hemacytometer 后放置在盖玻片和计数明亮的透明细胞 (蓝色细胞死了, 不应该计数)。 注意: 从单个 E7 光学顶盖的典型细胞数介于1和 5 x 10 7 细胞/mL 之间.</li>
	<li>将 ECM 上的离解细胞涂在2200细胞/平方毫米的密度上。对于我们的多边环境协定系统, 这相当于每个多边环境协定的大约13万个单元。添加 neurobasal 介质以将多边环境协定中的体积增加到1毫升, 并在 CO2孵化器中过夜以实现单元格附件和突起扩展 (图 1)。 注意: 对于不同区域和电极数的多边环境, 可以尝试不同的细胞密度--一般情况下, 较高的细胞密度会导致网络活动的增加, 但也会导致更短的生存文化。</li>
	<li>第二天, 添加 BPA 到最后浓度的10µM 在 neurobasal 培养基从1毫米的股票在步骤1.1 所处理的多边协定。对控制多边环境协定, 在水 (v/v) 溶液中加入相同体积的10% 乙醇。</li>
	<li>每隔一天用含有10µM BPA 的 neurobasal 培养基替换所用介质, 直到7天的体外(7DIV) 和此后的每一天, 随着网络活动的增加。 注意: 随着网络活动的增加, 新陈代谢活动增加, 媒体的变化需要更加频繁。不要让中号变成橙色。</li>
</ol>3. 记录神经元网络活动
<ol><li>在购置日, 在录制前将培养基与新鲜的 neurobasal 培养基交换, 并将多边环境协定返回 CO2孵化器, 至少2小时。通过单击温度图标 (补充图 3.1a-b), 启动录制软件并将多边环境协定的温度设置为37°c。 注: 记录可以早在3天开始电镀, 只要文化还活着就可以继续。</li>
	<li>通过右键单击 "缪斯" 图标 (在左侧窗口面板中的 "流" 下), 然后选择 "添加处理" 和 "尖峰检测器", 然后在弹出窗口中单击 "确定", 设置采集参数。"尖峰探测器 (6 x STD)" 将出现在文件名下面 (补充图 3.2a b)。</li>
	<li>下一步右击尖峰探测器, 选择 "添加处理" 和 "突发检测器", 并在弹出窗口中单击 "确定"。"爆裂探测器" (ISI) 将出现在缪斯图标的下面 (补充图 3.3a-b)。</li>
	<li>下一步右击突发检测器, 选择 "添加处理" 和 "神经统计编译器"。在弹出窗口中, 确保选择了文件头、聚合井统计信息和同步。单击 "确定"。"统计编译器" 将出现在下面 (补充图 3.4a-b)。 注意: 这将设置自适应阈值交叉 (6x STD 过滤器), 100 毫秒之间的穗间间隔, 最小峰值为 5, 平均发射率检测在十年代, 同步参数为20毫秒, 最小穗速率为每分钟0.083333 个峰值。</li>
	<li>通过单击底部的时钟图标, 设置录制时间为5分钟 (30万毫秒) 的定时录制。这是通过将设置节中的信息更改为每5.1 分钟记录一次并记录5分钟来实现的。这将立即启动, 并将执行一次 (补充图 3.5)。</li>
	<li>在移动多边环境协定之前, 确保多边环境协定的温度达到37摄氏度。从孵化器中移除多边环境协定, 将其放在记录单元上, 并锁定协定。通过单击 "文件播放" 窗口中 "计划录制" 部分或 "记录" 图标中的 "开始" 记录, 开始记录网络活动。通常情况下, 记录时间为5分钟 (30万毫秒) 是足够的, 虽然可以获得长达10分钟的更长的记录。</li>
	<li>录音后, 将多边环境协定归还孵化器。 注意: 必须注意保持不孕, 并尽量减少环境协定在孵化器之外的时间。</li>
</ol>4. 数据分析
<ol><li>对原始数据的分析可以使用记录软件离线进行。单击 "文件" 下拉菜单并打开录制。选择要分析的原始数据文件。重放原始录制以捕获所需的峰值参数。</li>
	<li>为了获得峰值的平均数, 使用尖峰检测模块和获取同步索引, 使用神经统计编译器模块。</li>
	<li>像以前一样加载峰值检测器、爆裂探测器和统计编译模块 (第3节)。</li>
	<li>从尖峰检测器、爆裂探测器和统计编译器模块窗口中的下拉菜单中, 分别选择 "尖峰列表"、"网络突发列表" 和 "高级度量"。</li>
	<li>单击 "记录" 按钮开始录制将在定向文件夹中创建. csv 文件的数据文件。峰值参数 (峰值和同步索引的总数) 将记录在. csv 文件中。 注意: 不建议在记录原始数据时进行实时分析, 因为这可能会消耗处理器时间和计算资源。</li>
</ol>

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作者声明他们没有竞争的兴趣。

快速生长和发育的阶段, 如胚胎发生特别容易受到各种内分泌干扰化合物, 包括 BPA 的有害影响。我们提供详细的协议, 以研究 BPA 暴露在一个体外脊椎动物神经网络模型的影响。通过使用该协议, 我们建立了 BPA 降低了发展中神经网络 (图 2a b) 的峰值速率和同步索引。
我们的方法是设计来研究 BPA 暴露在早期胚胎发生, 可以很容易地适应研究其他 EDCs 的影响。与其他脊椎动物模型 (包括老鼠或老鼠) 相比, 小鸡的胚胎神经元培养相对容易建立。此外, 没有要求单独的动物室-一个简单的孵化器在实验室的长凳上足以进行这些化验。我们描述了使用多电极系统 (多边环境协定), 以评估的影响, 该公司, BPA, 对网络活动。此处描述的协议可应用于其他系统。这项议定书的一个关键方面是维持不孕。这包括在无菌条件下对胚胎进行解剖--用70% 乙醇杀菌的仪器和使用无菌汉克的平衡盐溶液是足够的。由于这些数据是在几周内收集的, 因此保持对多边环境协定的无菌处理至关重要。曾经建立的神经元培养可以长期培养到三个月, 因此对于研究神经网络上的 EDCs 和其他化合物的长期接触是有用的。该协议的另一个重要方面是从解剖到电镀的时间。最佳时间为30分钟, 包括用胰蛋白酶培养组织。所花的时间越长, 细胞的存活率就越差。
我们这里描述了一个基本的协议, 可用于评估任何影响行为和神经功能的化学药品或药物。在这里, 我们使用了2200个细胞密度每毫米2;然而, 这可以修改和其他细胞密度可以使用。总的来说, 我们发现, 细胞密度的增加增加了网络活动-在较短的时间内更多的峰值。所描述的方法虽然在评估化学品对网络活动的影响方面非常有用, 但也有局限性。该方法的最大限制之一是这些体外培养是二维的, 可能无法反映脊椎动物大脑的三维结构。这可以通过使用切片录制来克服。另一种选择是将治疗方法应用于发育中的小鸡胚胎, 方法是在卵子的宽端切开一个小窗口, 在治疗方案的末尾解剖大脑, 在 vibratome 上做厚的切片, 并将其放置到多边环境协定中, 以记录网络活动。
我们的议定书具有重要意义, 因为它能够检查 EDCs 对网络活动发展的影响, 并为这些化学物质的作用提供了一个机械基础的探索。

4,4′--Isopropylidenediphenol, 通常称为双酚 a 或 BPA, 是一种抗氧化剂添加剂, 发现广泛的聚碳酸酯和环氧树脂为基础的产品, 从热纸, CDs, 粉碎玻璃, 到内涂层饮料罐。虽然 bpa 已经被认为是一种模拟雌激素1的内分泌干扰剂, 但由于每天对 bpa 的偶然接触而导致的不良影响的研究大多在过去10–15年中出现。甚至低水平的 BPA 暴露的后果是最深刻的早期发育期, 包括在胚胎发生期间通过暴露母亲2,3。在子宫内暴露女性胚胎对内分泌干扰化学物质也与增加的疾病易感性从阴道到乳腺癌4,5。动物研究表明, 暴露于 BPA 导致非典型的大脑结构和功能异常表现在行为6。因此, 欧洲和北美洲的大多数监管机构, 包括 FDA, 都禁止在婴儿奶瓶中使用 BPA。为了遵守法规, 许多制造商已改用 44 '-sulfonyldiphenol 或双酚 S (BPS)。根据 bpa 和 bps 是结构类似物的事实, 最近的报告显示, 在雌激素转录7中 bps 的可比效力, 研究这种化合物相对于 BPA 的毒性是很重要的。
在这里, 我们描述了一个协议, 以测试 BPA (和其他 EDCs) 对神经元网络的影响, 使用脊椎动物模型, 小鸡胚胎。体外神经元的培养形成突触接触并产生动作电位 (也称为峰值)。这些文化的峰值活动可以用多电极阵列 (多边环境协定) 系统记录下来。当它们在5毫秒内发生时, 峰值被认为是同步的。最终同步的初始随机峰值活动是开发神经网络8、9的关键特征。同步可以使用各种方法进行测量, 并且在文献9、10、11中描述了几种算法。在我们的分析中, 我们使用由 Paiva 和同事12开发的算法, 它集成到了驱动多边环境协定采集系统的记录软件中。胚胎小鸡神经元的鲁棒峰值活动为研究 BPA 对神经网络活动的影响提供了一个原型, 即13。利用多电极阵列记录峰值活动, 我们观察到 BPA 暴露抑制神经元峰值同步的发展9,14。在这里, 我们提供了一个详细的方法来研究 BPA 暴露在鸡胚神经元的文化, 并具有代表性的结果, 发展的神经元峰值同步性的鸡胚培养。

<table style="border-collapse:collapse;width:903pt" width="1204">
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		<col style="width:258pt" width="344" />
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			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">#5 foreceps</td>
			<td class="xl22" style="width:164pt" width="218">Fine Science Tools</td>
			<td class="xl20" style="width:258pt" width="344">11251-10</td>
			<td class="xl20" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">Axion Muse MEA</td>
			<td class="xl22" style="width:164pt" width="218">Axion Biosystems</td>
			<td class="xl22" style="width:258pt" width="344">M64-GL1-30Pt200</td>
			<td class="xl22" style="width:237pt" width="316">Will be called MEA system in manuscript</td>
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		<tr height="42" style="height:31.5pt">
			<td class="xl19" height="42" style="width:245pt;height:31.5pt" width="326">Axis Software</td>
			<td class="xl22" style="width:164pt" width="218">Axion Biosystems</td>
			<td class="xl19" style="width:258pt" width="344"></td>
			<td class="xl19" style="width:237pt" width="316">Will be called recording software in the manuscript</td>
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			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">BPA</td>
			<td class="xl22" style="width:164pt" width="218">Sigma-Aldrich</td>
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			<td class="xl22" style="width:237pt" width="316"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">curved forceps</td>
			<td class="xl22" style="width:164pt" width="218">Fine Science Tools</td>
			<td class="xl22" style="width:258pt" width="344">11272-50</td>
			<td class="xl22" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">EtOH</td>
			<td class="xl22" style="width:164pt" width="218">Sigma-Aldrich</td>
			<td class="xl22" style="width:258pt" width="344">64-17-5</td>
			<td class="xl22" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">Fertilized chicken eggs</td>
			<td class="xl22" style="width:164pt" width="218">from any local farm or Spafas</td>
			<td class="xl22" style="width:258pt" width="344"></td>
			<td class="xl22" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">HBSS</td>
			<td class="xl22" style="width:164pt" width="218">Fisher</td>
			<td class="xl23" style="width:258pt" width="344">14170112</td>
			<td class="xl22" style="width:237pt" width="316"></td>
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		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">Hemacytometer</td>
			<td class="xl22" style="width:164pt" width="218">Fisher</td>
			<td class="xl22" style="width:258pt" width="344">&#160;02-671-6</td>
			<td class="xl22" style="width:237pt" width="316"></td>
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		<tr height="40" style="height:30.0pt">
			<td class="xl22" height="40" style="width:245pt;height:30.0pt" width="326">Matrigel Growth Factor Reduced, Phenol Red-Free</td>
			<td class="xl22" style="width:164pt" width="218">BD Biosciences</td>
			<td class="xl23" style="width:258pt" width="344">356231</td>
			<td class="xl22" style="width:237pt" width="316">Will be called Extra Cellular Matrix (ECM) in the manuscript</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">Neurobasal medium</td>
			<td class="xl22" style="width:164pt" width="218">BrainBits</td>
			<td class="xl22" style="width:258pt" width="344">Nb4-500</td>
			<td class="xl22" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">Neuroexplorer statistical software</td>
			<td class="xl22" style="width:164pt" width="218">Nex Technologies</td>
			<td class="xl22" style="width:258pt" width="344">Neuroexplorer version 5</td>
			<td class="xl22" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">Pasteur pipettes</td>
			<td class="xl22" style="width:164pt" width="218">Fisher</td>
			<td class="xl22" style="width:258pt" width="344">&#160;13-678-20A</td>
			<td class="xl22" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">spring scissors</td>
			<td class="xl22" style="width:164pt" width="218">Fine Science Tools</td>
			<td class="xl22" style="width:258pt" width="344">15514-12</td>
			<td class="xl22" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl19" height="21" style="width:245pt;height:15.75pt" width="326">Sylgard bottom dissection dishes</td>
			<td class="xl19" style="width:164pt" width="218">Living Systems Instrumentaion</td>
			<td class="xl19" style="width:258pt" width="344">DD-90-S-BLK-3PK</td>
			<td class="xl19" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">Trypan Blue dye</td>
			<td class="xl22" style="width:164pt" width="218">Fisher</td>
			<td class="xl22" style="width:258pt" width="344">15-250-061</td>
			<td class="xl22" style="width:237pt" width="316"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl22" height="21" style="width:245pt;height:15.75pt" width="326">Trypsin-EDTA</td>
			<td class="xl22" style="width:164pt" width="218">Fisher</td>
			<td class="xl23" style="width:258pt" width="344">15400054</td>
			<td class="xl22" style="width:237pt" width="316"></td>
		</tr>
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assessment of the effects of endocrine disrupting compounds on the development of vertebrate neural network function using multi-electrode arrays

双酚, 如双酚 a (BPA) 和双酚 S (BPS), 是聚合剂广泛用于生产塑料和许多日常产品。它们被归类为内分泌干扰化合物 (EDC) 与雌二醇样的性质。长期暴露于 EDCs, 即使在低剂量, 已与各种健康缺陷, 包括癌症, 行为紊乱, 和不孕, 在早期发育期更大的脆弱性。为了研究 BPA 对神经元功能发育的影响, 我们采用了从早期鸡胚脑中提取的体外神经网络作为模型。我们发现, 对 BPA 的接触影响了网络活动的发展, 特别是峰值活动和同步。网络活动的变化是药物或化合物分子靶的关键环节及其对行为结果的影响。多电极阵列越来越成为研究药物对网络活动的影响的有用工具,体外。市场上有几个可用的系统, 尽管电极数量有变化, 电极阵列的类型和质量以及分析软件, 基本的基本原理, 以及获得的数据在整个不同的系统。虽然目前仅限于对二维体外区域性进行分析, 但正在改进这些多边环境协定系统, 以便在脑切片中启用体内网络活动。在这里, 我们提供了一个详细的协议, 胚胎暴露和记录神经元网络活动和同步, 以及具有代表性的结果。

我们研究了 BPA 对鸡胚神经元培养同步发育的影响。峰值活动同步是神经网络体外正常发育的指标。当两个峰值发生在5毫秒之间时, 它们被认为是同步的。体外神经元培养最初显示随机峰值活动-峰值随机发生, 穗间间隔显示正常分布。随着培养的成熟, 峰值活动变得更加同步, 穗间间隔是恒定的。通过使用记录软件12生成同步指数 (SI), 量化的同步峰值活动, 通过交叉相关分析, 对某一区域性进行了脉冲峰值时间的相互相关分析。我们发现 BPA 暴露通过减少峰值活动和同步索引 (图 2a和2b) 对网络活动开发产生不利影响。总之, 我们已经表明, BPA 暴露对胚胎的有害影响它的发展, 包括神经元功能, 可以评估通过同步发展的小鸡神经元的文化。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56300/56300fig1.jpg"> 图 1:描述从小鸡前脑解剖到离解、电镀和记录网络活动步骤的协议示意图.<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56300/56300fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56300/56300fig2.jpg"> 图 2:(A)与控制文化相比, BPA 治疗 E7 鸡前脑培养物的平均穗数明显低。使用记录软件提取峰值的平均数量。方法 (控制, 14890 949.0, N = 15 和 BPA, 5624 @ 465.9, N = 22) 有显著差异 (配对 t 检验, p < 0.0001)。(B)与控制文化相比, BPA 治疗 E7 鸡前脑培养物的平均同步指数 (SI) 显著降低。使用记录软件中的神经统计编译器模块提取 SI。SI 手段 (控制, 0.5159 0.06547 N = 15 和 BPA, 0.1140 @ 0.01840 N = 22) 是明显地不同的 (未配对的 t 试验, p < 0.0001)。误差线描述标准偏差。<a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56300/56300fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>

Watch this Scientific Journal Video about Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays at JoVE.com

使用多电极阵列评估内分泌干扰化合物对脊椎动物神经网络功能发育的影响发育生物学|视频

Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

暴露在环境中的毒素会对发育中的胚胎产生剧烈影响。内分泌干扰的化学物质, 如 bisphenols, 对神经系统有负面影响。在这里, 我们描述了一个协议使用体外脊椎动物 (小鸡胚) 神经元网络模型来研究毒素暴露对早期胚胎的功能影响。

应用多电极阵列评价内分泌干扰物对脊椎动物神经网络功能发育的影响

Bis-phenols, such as bis-phenol A (BPA) and bis-phenol-S (BPS), are polymerizing agents widely used in the production of plastics and numerous everyday ...

assessment-effects-endocrine-disrupting-compounds-on-development

Nanyang Technological University

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SCIENTISTS IN ACTION

5.9K 次观看.  Delaware State University. 暴露在环境中的毒素会对发育中的胚胎产生剧烈影响。内分泌干扰的化学物质, 如 bisphenols, 对神经系统有负面影响。在这里, 我们描述了一个协议使用体外脊椎动物 (小鸡胚) 神经元网络模型来研究毒素暴露对早期胚胎的功能影响。

视频: Assessment of the Effects of Endocrine Disrupting Compounds on the Development of Vertebrate Neural Network Function Using Multi-electrode Arrays

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the field of environmental health. This is due in part to the paucity of model systems that can (1) accurately recapitulate keys features of reproductive processes and (2) do so in a medium- to high-throughput fashion, without the need for a high number of vertebrate animals.
We describe here an assay in the nematode C. elegans that allows the rapid identification of germline toxicants by monitoring the induction of aneuploid embryos. By making use of a GFP reporter line, errors in chromosome segregation resulting from germline disruption are easily visualized and quantified by automated fluorescence microscopy. Thus the screening of a particular set of compounds for its toxicity can be performed in a 96- to 384-well plate format in a matter of days. Secondary analysis of positive hits can be performed to determine whether the chromosome abnormalities originated from meiotic disruption or from early embryonic chromosome segregation errors. Altogether, this assay represents a fast first-pass strategy for the rapid assessment of germline dysfunction following chemical exposure.

Comprehensive Assessment of Germline Chemical Toxicity Using the Nematode Caenorhabditis elegans

We describe the detailed steps of a high-throughput chemical assay in the nematode Caenorhabditis elegans used to assess germline toxicity. In this assay, disruption of germline function following chemical exposure is monitored using a fluorescent reporter specific to aneuploid embryos.

生殖系化学毒性的利用线虫综合评估<em&gt;秀丽隐杆线虫</em

Identifying the reproductive toxicity of the thousands of chemicals present in our environment has been one of the most tantalizing challenges in the ...

科研

发育生物学

Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

Genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system have greatly improved gene targeting efficiency in human induced pluripotent stem cells (hiPSCs). This manuscript describes a protocol for generating lineage specific hiPSC reporter using CRISPR/Cas system assisted homologous recombination.

的hiPSC神经细胞系特异性敲开记者代高效使用CRISPR / Cas9和Cas9双切口酶系统

Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

从脑室周围地区的成年大鼠和人类脊髓神经分离干/祖细胞

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Alginate Encapsulation of Pluripotent Stem Cells Using a Co-axial Nozzle

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening. However, the development of a mass culture system would be required for using PS cells in these applications. Suspension culture is one promising culture method for the mass production of PS cells, although some issues such as controlling aggregation and limiting shear stress from the culture medium are still unsolved. In order to solve these problems, we developed a method of calcium alginate (Alg-Ca) encapsulation using a co-axial nozzle. This method can control the size of the capsules easily by co-flowing N2 gas. The controllable capsule diameter must be larger than 500 &#181;m because too high a flow rate of N2 gas causes the breakdown of droplets and thus heterogeneous-sized capsules. Moreover, a low concentration of Alg-Na and CaCl2 causes non-spherical capsules. Although an Alg-Ca capsule without a coating of Alg-PLL easily dissolves enabling the collection of cells, they can also potentially leak out from capsules lacking an Alg-PLL coating. Indeed, an alginate-PLL coating can prevent cellular leakage but is also hard to break. This technology can be used to research the stem cell niche as well as the mass production of PS cells because encapsulation can modify the micro-environment surrounding cells including the extracellular matrix and the concentration of secreted factors.

We established a method of encapsulating pluripotent stem cells (PS cells) into alginate hydrogel capsules using a co-axial nozzle. This prevents cells from aggregating excessively and limits the shear stress experienced by cells in suspension culture. The technique is applicable to the mass production of PS cells as well as research on stem cell niche.

多能干细胞的海藻酸钠封装使用同轴喷嘴

Pluripotent stem cells (PS cells) are the focus of intense research due to their role in regenerative medicine and drug screening.  However, the ...

Geomagnetic Field (Gmf) and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) and the moment of the radiation of Angiosperms. This led to the hypothesis that alterations in GMF polarity may play a role in plant evolution. Here, we describe a method to test this hypothesis by exposing Arabidopsis thaliana to artificially reversed GMF conditions. We used a three-axis magnetometer and the collected data were used to calculate the magnitude of the GMF. Three DC power supplies were connected to three Helmholtz coil pairs and were controlled by a computer to alter the GMF conditions. Plants grown in Petri plates were exposed to both normal and reversed GMF conditions. Sham exposure experiments were also performed. Exposed plants were photographed during the experiment and images were analyzed to calculate root length and leaf areas. Arabidopsis total RNA was extracted and Quantitative Real Time-PCR (qPCR) analyses were performed on gene expression of CRUCIFERIN 3 (CRU3), copper transport protein1 (COTP1), Redox Responsive Transcription Factor1 (RRTF1), Fe Superoxide Dismutase 1, (FSD1), Catalase3 (CAT3), Thylakoidal Ascorbate Peroxidase (TAPX), a cytosolic Ascorbate Peroxidase1 (APX1), and NADPH/respiratory burst oxidase protein D (RbohD). Four different reference genes were analysed to normalize the results of the qPCR. The best of the four genes was selected and the most stable gene for normalization was used. Our data show for the first time that reversing the GMF polarity using triaxial coils has significant effects on plant growth and gene expression. This supports the hypothesis that GMF reversal contributes to inducing changes in plant development that might justify a higher selective pressure, eventually leading to plant evolution.

Geomagnetic Field Gmf and Plant Evolution: Investigating the Effects of Gmf Reversal on Arabidopsis thaliana Development and Gene Expression

This protocol describes a method to test the hypothesis that the reversal of the effective geomagnetic field (GMF) induces differential gene expression and alters the morphology of Arabidopsis thaliana. A triaxial octagonal system of Helmholtz coil-pairs was used to artificially generate reversed GMF conditions in the plant growth chamber.

地磁场（GMF）和植物进化:调查逆转基因食品对影响<em&gt;拟南芥</em&gt;开发与基因表达

One of the most stimulating observations in plant evolution is a correlation between the occurrence of geomagnetic field (GMF) reversals (or excursions) ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is the formation of fibroblastic foci, which is associated with the disease severity. Mesenchymal cells consisting of the fibroblastic foci are proposed to be derived from several cell sources, including originally resident intrapulmonary fibroblasts and circulating fibrocytes from bone marrow. Recently, mesenchymal cells that underwent epithelial-mesenchymal transition (EMT) have been also supposed to contribute to the pathogenesis of fibrosis. In addition, EMT can be induced by transforming growth factor &#946;, and EMT can be enhanced by pro-inflammatory cytokines like tumor necrosis factor &#945;. The gel contraction assay is an ideal in vitro model for the evaluation of contractility, which is one of the characteristic functions of fibroblasts and contributes to wound repair and fibrosis. Here, the development of a gel contraction assay is demonstrated for evaluating contractile ability of mesenchymal cells that underwent EMT.

Development of an In Vitro Assay to Evaluate Contractile Function of Mesenchymal Cells that Underwent Epithelial-Mesenchymal Transition

Here, we describe the development and application of a gel contraction assay for evaluating contractile function in mesenchymal cells that underwent epithelial-mesenchymal transition.

的发展<em&gt;体外</em&gt;检测以确定间充质细胞将经历上皮 - 间质转化的收缩功能

Fibrosis is often involved in the pathogenesis of various chronic progressive diseases such as interstitial pulmonary disease. Pathological hallmark is ...

Rapid Neuronal Differentiation of Induced Pluripotent Stem Cells for Measuring Network Activity on Micro-electrode Arrays

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, many protocols for differentiating hiPSCs into neurons have been developed. However, these protocols are often slow with high variability, low reproducibility, and low efficiency. In addition, the neurons obtained with these protocols are often immature and lack adequate functional activity both at the single-cell and network levels unless the neurons are cultured for several months. Partially due to these limitations, the functional properties of hiPSC-derived neuronal networks are still not well characterized. Here, we adapt a recently published protocol that describes production of human neurons from hiPSCs by forced expression of the transcription factor neurogenin-212. This protocol is rapid (yielding mature neurons within 3 weeks) and efficient, with nearly 100% conversion efficiency of transduced cells (&#62;95% of DAPI-positive cells are MAP2 positive). Furthermore, the protocol yields a homogeneous population of excitatory neurons that would allow the investigation of cell-type specific contributions to neurological disorders. We modified the original protocol by generating stably transduced hiPSC cells, giving us explicit control over the total number of neurons. These cells are then used to generate hiPSC-derived neuronal networks on micro-electrode arrays. In this way, the spontaneous electrophysiological activity of hiPSC-derived neuronal networks can be measured and characterized, while retaining interexperimental consistency in terms of cell density. The presented protocol is broadly applicable, especially for mechanistic and pharmacological studies on human neuronal networks.

We modify and implement a previously published protocol describing the rapid, reproducible, and efficient differentiation of human&#160;induced&#160;Pluripotent Stem Cells (hiPSCs) into excitatory cortical neurons12. Specifically, our modification allows for control of neuronal cell density and use on micro-electrode arrays to measure electrophysiological properties at the network level.

诱导多能干细胞的快速神经元分化有关微电极阵列测量网络活动

Neurons derived from human induced Pluripotent Stem Cells (hiPSCs) provide a promising new tool for studying neurological disorders. In the past decade, ...

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes (hPSC-CMs) Using Multi-electrode Arrays (MEAs)

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived cardiomyocytes (hPSC-CMs) are increasingly recognized as having great value for modeling cardiovascular diseases in humans, especially arrhythmia syndromes. They have also demonstrated relevance as in vitro systems for predicting drug responses, which makes them potentially useful for drug-screening and discovery, safety pharmacology and perhaps eventually for personalized medicine. This would be facilitated by deriving hPSC-CMs from patients or susceptible individuals as hiPSCs. For all applications, however, precise measurement and analysis of hPSC-CM electrical properties are essential for identifying changes due to cardiac ion channel mutations and/or drugs that target ion channels and can cause sudden cardiac death. Compared with manual patch-clamp, multi-electrode array (MEA) devices offer the advantage of allowing medium- to high-throughput recordings. This protocol describes how to dissociate 2D cell cultures of hPSC-CMs to small aggregates and single cells and plate them on MEAs to record their spontaneous electrical activity as field potential. Methods for analyzing the recorded data to extract specific parameters, such as the QT and the RR intervals, are also described here. Changes in these parameters would be expected in hPSC-CMs carrying mutations responsible for cardiac arrhythmias and following addition of specific drugs, allowing detection of those that carry a cardiotoxic risk.

Electrophysiological Analysis of human Pluripotent Stem Cell-derived Cardiomyocytes hPSC-CMs Using Multi-electrode Arrays MEAs

Electrophysiological characterization of cardiomyocytes derived from human Pluripotent Stem Cells (hPSC-CMs) is crucial for cardiac disease modeling and for determining drug responses. This protocol provides the necessary information to dissociate and plate hPSC-CMs on multi-electrode arrays, measure their field potential, and a method for analyzing QT and RR intervals.

使用多电极阵列（MEA）的人多能干细胞来源的心肌细胞（hPSC-CM）的电生理分析

Cardiomyocytes can now be derived with high efficiency from both human embryonic and human induced-Pluripotent Stem Cells (hPSC). hPSC-derived ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

秀丽线虫生殖的计算分析研究细胞核、蛋白质和细胞骨架的分布

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...