这项工作得到了加拿大卫生研究院、自然科学和工程研究理事会 (NSERC 发现补助金) 和萨伏依基金会的支持。OC 得到了来自 NSERC 的博士奖学金的支持。

所有的实验都是按照大学的动物保护委员会和加拿大动物保育理事会的动物福利准则进行的。
1. 初步准备 (可选: 提前1天准备)
<ol><li>准备三种类型的人工脑脊液 (ACSF) 溶液 (正常, 蔗糖和恢复解决方案, 1 升每一个; 请参见表 1)。调整溶液的渗透到 300 10 mOsm25和冷却 ACSF 蔗糖下降到一个接近冰点 (0-4 °c)。 注: 使用低钠切削液 (ACSF 蔗糖) 有助于保持急性切片的表面层神经元的生存能力26。</li>
	<li>准备0.75 毫升的补丁解决方案, 其中包含红色荧光 (例如, Alexa-594) 和绿色合成钙指示器 (例如, 俄勒冈绿色 BAPTA-1; 请参见表 1)。在修补程序解决方案中包括 biocytin (请参见表 1), 如果需要后端对记录的单元格进行形态学标识。</li>
	<li>将溶液的渗透压调整到 280, 5 mOsm 和 pH 值7.35 到0.5。随时把溶液放在冰箱或冰上。 注: 钙指示器的选择应基于其动态范围和调查的 Ca2 +事件的性质。</li>
	<li>使用微拉拔器, 从硼硅酸盐玻璃毛细血管 (参见材料表) 中制备贴片吸管, ≈2µm 尖 (3-5 MΩ的吸管阻力) 和硼硅酸盐θ玻璃毛细血管的刺激吸管 (见材料) 与2-5 µm 提示。 注: 为使给定直径和形状吸管的拉拔器设置将由拉拔灯丝类型和给定类型的玻璃的坡道测试结果决定。</li>
</ol>2. 海马切片制备
<ol><li>麻醉鼠标 (P13-20; 鼠标的应变和性别取决于正在处理的实验问题), 将1毫升异氟醚置于吸入麻醉腔内的纸巾上。当动物被深深的麻醉, 如缺乏运动和缓慢的呼吸的证明, 从麻醉室和斩首使用断头台删除。</li>
	<li>用一把小剪刀从头骨的后部切开到鼻子, 露出头骨。使用一小对迷你骨 rongeurs, 使一个洞在 bregma 水平。然后用剪刀做一个沿矢状缝合的延髓切口。与 rongeurs 抓住每个头骨皮瓣的背面和向上和向外抬起, 以消除头骨覆盖每个半球。</li>
	<li>当大脑完全暴露后, 用小铲子把视神经咬倒。从头骨中取出大脑, 将其放入含有连续氧、冷 ACSF 蔗糖的培养皿中 (请参阅表 1以了解蔗糖浓度)。<ol>
<li>如果需要大鼠的组织, 在脑提取前用注射器 (25G) 填充20毫升的冰冷 ACSF, 进行心内灌注, 以获得优质切片。</li>
	</ol>
</li>
	<li>从提取的动物大脑中制备海马切片。<ol>
<li>让大脑冷却大约2分钟. 将一张滤纸放在一个冰包装的培养皿上, 将大脑转移到滤纸上。解剖两个半球。</li>
		<li>胶水的解剖半球 (方向是由实验目标确定, 以获得冠状, 横向, 或水平切片) 到 vibratome 表, 并浸入到 vibratome 托盘充满冰冷氧 ACSF-蔗糖。使用 vibratome 冷却器或将冰放在 vibratome 托盘周围, 使300µm 厚片在大约1摄氏度的温度下。</li>
		<li>使用平漆刷, 在含有含氧 ACSF 的浴缸中积累含有海马体 (每半球多达6片) 的切片, 加热到37摄氏度。</li>
		<li>将切片放在 ACSF 回收浴中至少45分钟。</li>
	</ol>
</li>
</ol>3. 全细胞膜片钳录音
<ol><li>在目标 (放大: 40x, 数值孔径: 0.8) 的激光扫描双光子显微镜下放置一个海马片。持续灌注的氧 ACSF-正常加热30-32 摄氏度的浴缸, 速度为 2.5-3 毫升/分钟。</li>
	<li>使用红外差分干涉对比 (IR-DIC) 或 Dodt-SGC 显微镜, 以找到一个感兴趣的细胞使用其大小, 形状和位置。 注意: 根据目标神经元的数量, 转基因小鼠模型可以用来方便地定位感兴趣的细胞。在这种情况下, 这个步骤可以取代使用荧光光源。</li>
	<li>用含有红色荧光 (例如、Alexa-594) 的 ACSF 正常溶液填充刺激吸管。</li>
	<li>将刺激吸管 (连接到电刺激单元) 放在切片的顶端, 以便尖端与感兴趣的单元位于同一区域。</li>
	<li>在用补丁解决方案填充贴片吸管并将其附加到头部阶段后, 将其放在切片上, 使其尖端直接位于感兴趣的单元上方。</li>
	<li>将注射器放入三路旋塞阀, 通过塑料管将其连接到贴片吸管。使用注射器向贴片吸管 (≈0.1 毫升) 注入恒定的正压。保持旋塞阀在 "关闭" 位置。</li>
	<li>激活放大器控制软件模块, 并通过点击 "VC" 按钮切换到电压钳模式。打开电生理学数据采集软件 (例如, clampex) 获取电生理信号, 并点击 "膜测试" 图标, 使其不断发送一个平方电压脉冲 (5 mV, 10 毫秒), 这是必要的监测吸管阻力的变化。</li>
	<li>降低贴片吸管, 直到它正确地放在目标单元格的顶端。当与细胞膜接触时, 通过将旋塞阀转变为 "打开" 位置来消除压力。 注: 当发现吸管阻力 (≈ 0.2 MΩ) 小增加时, 检测到细胞膜的接触, 而细胞内的小凹痕是由正压引起的。</li>
	<li>使用一个空注射器安装在旋塞阀, 直到该软件提供的吸管阻力达到 1 GΩ, 对贴片吸管施加轻微的负压。在数据采集软件的 "膜测试" 窗口中, 将单元格夹紧-60 mV。</li>
	<li>继续应用负压, 直到打开单元格并实现全细胞配置。这种细胞状态的检测是基于在 "膜测试" 窗口中的吸管电阻 (从 1 GΩ到 70-500 MΩ的变化) 和大电容瞬态的出现而发生的突变。</li>
</ol>4. 双光子 Ca2 +成像
<ol><li>如果对兴奋后突触反应感兴趣, 添加 gabaA受体阻滞剂 gabazine (10 µM) 和 gabaB受体阻滞剂 CGP55845 (2 µM) 在 ACSF 浴。</li>
	<li>在放大器控制软件模块中, 通过点击 "CC" 按钮将补丁配置设置为电流钳, 并记录细胞的射击模式, 以响应去极化型电流 (0.8-1.0 nA, 1 s) 的体细胞注射。等待至少30分钟的单元格, 以填充修补程序解决方案中存在的指示器。 注: 细胞的射击模式和活动性质可用于识别细胞的亚型;例如, 快速峰值单元格。在开始记录猫 (请参见表 2进行故障排除) 之前, 必须通过扩散来稳定指示器浓度。</li>
	<li>AP 诱发的猫<ol>
<li>使用图像采集软件, 开始获取图像。使用红色荧光信号定位感兴趣的枝晶。为确保可见的响应, 首先选择近端枝晶 (≤50µm), 因为 AP 反向的效率可能会随着 GABAergic 中间神经元22中的距离显著下降.</li>
		<li>在图像采集软件中使用 "矩形工具", 放大目标区域并切换到 "xt" (linescan) 模式。将扫描定位在感兴趣的树枝状分支上。利用采集软件中的激光强度控制器, 将双光子激光器设置为最小的功率电平, 基线绿色荧光只是略微可见, 以避免毒性。</li>
		<li>在电生理学数据采集软件 "协议" 窗口和图像采集软件中, 创建所需持续时间的记录试验, 其中包含所需振幅的体细胞电流注入。<ol>
<li>使用图像采集软件, 单击 "开始记录" 按钮, 连续获取 1-2 s 的荧光. 重复图像采集3-10 次。在单扫描之间至少等待三十年代, 以避免光损伤。如果获得 linescans 沿枝晶的多个点, 采取随机顺序, 以避免秩序效应。</li>
		</ol>
</li>
	</ol>
</li>
	<li>Synaptically 诱发猫<ol>
<li>使用红色荧光信号定位感兴趣的枝晶。</li>
		<li>将刺激吸管放在感兴趣的树枝上面的薄片表面上。慢慢降低刺激吸管到位, 尽量减少运动, 以避免扰乱整个细胞的配置。将吸管放置在10-15 µm 的枝晶上。</li>
		<li>为了可视化突触 microdomains 在 aspiny 树突中的位置, 在图像采集软件中切换到 "xt" (linescan) 模式, 并将该线沿树突分支的兴趣点定位。</li>
		<li>在 "协议" 窗口 (电生理学数据采集软件) 和图像采集软件中, 创建一个记录试验, 在试用开始后触发激励单元。</li>
		<li>使用采集软件, 单击 "开始记录" 按钮, 连续沿枝晶扫描1-2 秒. 重复采集3-5 次, 在扫描之间等待三十年代以防止光损伤。 注意: 扫描的长度可以根据诱发事件的动力学进行调整, 但应尽量减少以防止光损伤 (请参见表2 进行故障排除)。</li>
		<li>重复步骤4.5.5 在不同的条件下 (例如, 不同的强度/长度的刺激, 引入药理阻滞剂,等) 取决于正在处理的具体问题。</li>
		<li>停止获取时, 细胞显示健康状况恶化的迹象: 退极化低于-45 mV, 增加基线 Ca2 +水平, 树突形态退化 (例如, 起泡, 碎片; 请参见表 2进行故障排除)。</li>
		<li>要获取细胞形态学的初步信息并保存刺激吸管位置的记录, 请获取红色通道中单元格的Z堆栈。使用 "xyz"模式中的捕获软件, 设置上下堆栈限制, 以将包含的所有进程镜像到整个单元格。将 "步长" 设置为1µm, 并使用 "开始记录" 按钮启动堆栈获取。</li>
		<li>当Z堆栈捕获完成时, 使用软件中的 "最大投影" 选项来叠加堆栈的所有焦点计划, 并验证获取的质量。然后, 慢慢地收回片中的补丁吸管。</li>
		<li>要修复自组后形态学标识的切片, 请使用平面画笔从浴缸中快速移除切片, 然后将其放在两个滤纸之间, ACSF 填充的培养皿中。更换 ACSF 与4% 多聚甲醛 (粉煤灰) 解决方案, 并把盘子放在4°c 房间或冰箱过夜。</li>
	</ol>
</li>
</ol>5. 免疫组化用于自组后形态学鉴定记录细胞
注: 在粉煤灰中固定1晚后, 切片可以储存在磷酸盐缓冲 (PB) 与叠氮化钠 (0.5%), 长达1月。
<ol><li>将该切片放入三缓冲盐水溶液 (TBS) 与海卫 X-100 (0.3%), 并执行4洗涤 (每5-10 分钟)。</li>
	<li>将切片中的0.3% 与10% 正常山羊血清 (1 h)。</li>
	<li>把切片在 TBS-海卫0.3% 与1% 和链亲和素共轭荧光(例如, Alexa 氟546共轭链亲和素, 1:200) 过夜孵化。</li>
	<li>在 tb 上洗4次 (5-10 分钟)。</li>
	<li>使用共焦显微镜在显微镜下的幻灯片和图像上安装切片。要进行完整的单元重建, 请使用20x 目标获取Z堆栈 (步骤大小: 1 µm)。对于成像 Alexa-546-conjugated 标签, 使用 543 nm 激光和 515-560 nm 检测过滤器集。</li>
</ol>6. 猫的分析
<ol><li>从红色和绿色通道提取 linescan 图像中的感兴趣区域 (ROIs) 中的荧光值, 并平均每个条件的多次重复值以减少噪音。 注意: 当沿枝晶进行 linescan 采集时, 可以从多个 ROIs 中提取数据, 这可能对应于树突 microdomains (2-5 µm), 从而允许对 Ca2 +信号划分进行研究。</li>
	<li>对于每个 ROI, 将 Ca2 +振幅中的更改表示为: <img alt="Equation" src="/files/ftp_upload/56776/56776eq1.jpg"> 注: 此处 G 为绿色通道的荧光值;G0是在预刺激期内绿色通道的基线荧光值;R 是红色通道18的荧光值。由于双光子激发是高度局部化的, 不产生显著的背景, 因此不需要对背景荧光进行减法运算。</li>
</ol>

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<li>Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Functional+imaging+of+single+synapses+in+brain+slices%5BTitle%5D)+AND+%22Exp+Physiol%22%5BJournal%5D)">Functional imaging of single synapses in brain slices.</a> Exp Physiol. 87, 733-736 (2002).</li>
<li>Yasuda, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+calcium+concentration+dynamics+in+small+neuronal+compartments%5BTitle%5D)+AND+%22Sci+STKE%22%5BJournal%5D)">Imaging calcium concentration dynamics in small neuronal compartments.</a> Sci STKE. 219, pl5(2004).</li>
<li>Maravall, M., Mainen, Z. F., Sabatini, B. L., Svoboda, K. <a target="_blank" href="https://www.ncbi.nlm.nih.gov/pubmed/10777761">Estimating intracellular calcium concentrations and buffering without wavelength rationing.</a> Biophys J. 78, 2655-2667 (2000).</li>
<li>Camiré, O., Topolnik, L. <a target="_blank" href="https://www.researchgate.net/publication/225093837_Functional_compartmentalisation_and_regulation_of_postsynaptic_Ca2_transients_in_inhibitory_interneurons">Functional compartmentalization and regulation of postsynaptic CaTs in inhibitory interneurons.</a> Cell Calcium. 52, 339-346 (2012).</li>
<li>Guet-McCreight, A., Camiré, O., Topolnik, L., Skinner, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Using+a+semi-automated+strategy+to+develop+multi-compartment+models+that+predict+biophysical+properties+of+interneuron-specific+3+%28IS3%29+cells+in+hippocampus%5BTitle%5D)+AND+%22eNeuro%22%5BJournal%5D)">Using a semi-automated strategy to develop multi-compartment models that predict biophysical properties of interneuron-specific 3 (IS3) cells in hippocampus.</a> eNeuro. 3, 1-26 (2016).</li>
<li>Evstratova, A., Chamberland, S., Topolnik, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell+type-specific+and+activity-dependent+dynamics+of+action+potential-evoked+Ca2%2B+signals+in+dendrites+of+hippocampal+inhibitory+interneurons%5BTitle%5D)+AND+%22J+Physiol%22%5BJournal%5D)">Cell type-specific and activity-dependent dynamics of action potential-evoked Ca2+ signals in dendrites of hippocampal inhibitory interneurons.</a> J Physiol. 589, 1957-1977 (2011).</li>
<li>Topolnik, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dendritic+calcium+mechanisms+and+long-term+potentiation+in+cortical+inhibitory+interneurons%5BTitle%5D)+AND+%22Eur+J+Neurosci%22%5BJournal%5D)">Dendritic calcium mechanisms and long-term potentiation in cortical inhibitory interneurons.</a> Eur J Neurosci. 35, 496-506 (2012).</li>
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</ol>

作者没有竞争的金融利益或其他利益冲突。

此处所示的方法演示了如何将双光子 Ca2 +成像和补丁钳电生理学结合在一起, 研究急性脑切片神经元树突状的树形 ca2 +信号。此方法允许对由电刺激或 backpropagating AP 在树突状段诱发的局部 Ca2 +海拔和细胞的躯体反应进行监测。这使得它成为研究树突树各部分如何集成输入和与体细胞通信的绝佳工具。但是, 考虑到实验的长度, 必须特别注意细胞的健康, 限制使用激光功率进...

神经元对网络活动的贡献在很大程度上取决于它所接受的突触输入的动态性质。传统上, 神经元突触活动特征的主要方法依赖于通过电刺激通道轴突引起的突触后电流的体细胞全细胞膜片钳记录。但是, 在这种情况下, 只有下部定位突触的活动才会如实地报告为1。此外, 为了评估突触特定机制, 在特定位置对神经元和树突的记录被用来瞄准感兴趣的突触和树突整合的机制。通过光学和电生理技术的联姻, 在突触生理学领域取得了重大突破。双光子励磁激光扫描显微镜 (2PLSM) 结合 Ca2 +成像和 optogenetic 工具可以揭示在脑切片中特定神经元连接的突触活动动态组织的巨大细节在体外和在体内。
2PLSM 从传统的单光子励磁显微镜中脱颖而出的几个关键优势: (1) 由于双光子激发的非线性性质, 荧光只在焦体积中产生, 所有发射的光子都代表了有用的信号 (不需要针孔);(2) 较长的波长, 用于 2PLSM, 更有效地穿透散射组织;此外, 分散的光子太稀, 无法产生双光子激发和背景荧光;(3) 光损伤和毒性也限于焦平面。因此, 尽管与传统共焦显微镜相比, 2 光子系统的成本很高, 但2PLSM 仍然是高分辨率研究厚活组织神经元结构和功能的一种选择方法。
2PLSM 在散射组织中的第一次应用是对树突状棘的结构和功能的图像3。2PLSM 结合 Ca2 +影像显示, 棘状功能作为隔离的生物化学隔间。由于在许多神经元类型中, 棘和单个突触之间存在一对一的对应关系4, 双光子 Ca2 +成像很快成为报告完整组织5中单个突触活动的有用工具, 6,7,8。此外, 2 plsm Ca2 +成像成功地用于监测单钙通道的活动和内在和突触电导之间的非线性相互作用, 以及评估状态和活动依赖性管理 Ca2 +信号在神经元树突5,6,7,8,9,10,11。
钙是一种无处不在的胞内第二信使, 其亚细胞时空组织决定了生理反应的方向, 从突触强度的变化到离子通道、枝晶和脊柱生长的调节, 作为以及细胞死亡和生存。树突状 Ca2 +海拔通过激活多个通路而发生。动作电位 (APs), backpropagating 到树突, 开放电压门控钙通道12和产生相对全局 Ca2 +瞬变 (猫) 在树突和刺13。突触传输与突触后 Ca 的激活相关联2 +-渗透性受体 (NMDA, Ca2 +渗透 AMPA 和 kainate), 触发突触猫6,14,15。最后, supralinear Ca2 +事件可在特定条件下的树突中生成11,12,13,16。
双光子 Ca2 +成像与膜片钳电生理记录结合使用合成 Ca2 +敏感荧光指示器, 通常通过补丁电极在整个细胞记录中传递.用于量化 Ca2 +动态的标准方法基于双指示器方法1718。它使用两个具有良好分离发射光谱的显影 (例如), 将红色 ca2 +不敏感染料与绿色 ca2 +指示器 (如俄勒冈州绿色 BAPTA-1 或 Fluo-4) 的组合, 与单指示器方法。首先, ca2 +不敏感的染料用于定位小的兴趣结构 (树突状分支和刺), 其中将执行 ca2 +成像。其次, 绿色和红色荧光 (ΔG) 的变化之间的比值被计算为 [ca2 +] 的度量值, 对于基线荧光的变化, 由于 [ca2 +]017中的波动, 它基本上不敏感。,18. 此外, 荧光变化可以用绝对 Ca2 +浓度19来校准。
当运行双光子 Ca2 +成像实验的急性切片是一个普遍关注的是细胞健康和图像获取的稳定性, 由于高激光功率通常使用。此外, 在 ca2 +成像实验中, 由于 ca2 +指示器充当高度移动外源 ca2 +的事实, 对亚细胞 Ca2 +动态的摄动和大量高估感到担忧。缓冲区.因此, Ca2 +指示器的选择及其浓度取决于神经元类型、猫的预期振幅和实验问题。
我们 修改了双光子 Ca2 +成像方法, 以调查 GABAergic 中间神经元9、10、11、20、21的树突中 ca2 + 的波动情况.,22. 虽然早期 Ca2 +成像研究的大部分都是在主神经元中进行的, 但抑制性中间神经元展示了不同于锥体细胞20的功能 Ca2 + 机制.,23,24. 这些 interneuron 特定的机制 (例如、Ca2 +可渗透 AMPA 受体) 可能在调节细胞活动方面发挥特定作用。虽然树突状 Ca2 +信号在中间神经元是一个诱人的目标进一步调查, 双光子 Ca2 + 成像在这些细胞的树突提出了额外的挑战, 从更薄的树枝直径和缺乏刺一个特别高的内生 Ca2 +绑定容量。由于我们的研究兴趣集中在研究海马中间神经元, 下面的协议, 虽然适用于不同的神经元群体, 适应于应对这些挑战。
该协议是用商业共焦双光子显微镜执行的, 配备两个外部的非 descanned 探测器 (NDDs)、电光调制器 (EOM) 和 Dodt 扫描梯度对比 (SGC), 并安装在光学表上。显微镜与钛: 蓝宝石多光子激光模式锁定在 800 nm (> 3 W, 140 fs 脉冲, 80 赫兹重复率)。该成像系统配备了一个标准的电生理平台, 包括一个温度控制的灌注室, 一个两个并联的翻译平台, 一个电脑控制的微电极放大器, 数字化仪, 刺激单元和数据采集软件。

<table><tbody><tr><td>Animal Strain: Mouse CD1</td><td>Charles River</td><td>022</td><td></td></tr><tr><td>Isoflurane</td><td>AbbVie Corporation</td><td>0B506-099</td><td></td></tr><tr><td>CGP 55845 hydrochloride</td><td>Abcam</td><td>ab120337</td><td></td></tr><tr><td>Calcium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>C4901</td><td></td></tr><tr><td>D-(+)-Glucose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>G8270</td><td></td></tr><tr><td>HEPES</td><td>Sigma-Aldrich</td><td>H3375</td><td></td></tr><tr><td>Magnesium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>M8266</td><td></td></tr><tr><td>Magnesium sulfate heptahydrate</td><td>Sigma-Aldrich</td><td>230391</td><td></td></tr><tr><td>Paraformaldehyde powder, 95%</td><td>Sigma-Aldrich</td><td>158127</td><td></td></tr><tr><td>Potassium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P3911</td><td></td></tr><tr><td>Potassium gluconate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P1847</td><td></td></tr><tr><td>Sodium azide&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S2002</td><td></td></tr><tr><td>Sodium bicarbonate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S8875</td><td></td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S5886</td><td></td></tr><tr><td>Sucrose&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S9378</td><td></td></tr><tr><td>Triton X-100&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>Trizma base&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T1503</td><td></td></tr><tr><td>Trizma hydrochloride&amp;nbsp;</td><td>Sigma-Aldrich</td><td>T3253</td><td></td></tr><tr><td>Sodium phosphate dibasic dihydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>71643</td><td></td></tr><tr><td>&lt;br/&gt; Sodium phosphate monobasic&lt;br/&gt; monohydrate&amp;nbsp;</td><td>Sigma-Aldrich</td><td>S9638</td><td></td></tr><tr><td>Biocytin</td><td>Sigma-Aldrich</td><td>B4261</td><td></td></tr><tr><td>Alexa Fluor 594 Hydrazide</td><td>ThermoFisher Scientific</td><td>A10438</td><td></td></tr><tr><td>SR95531 (Gabazine)&amp;nbsp;</td><td>Abcam</td><td>ab120042</td><td></td></tr><tr><td>&lt;br/&gt; Adenosine triphosphate (ATP)-Tris</td><td>Sigma-Aldrich</td><td>A9062</td><td></td></tr><tr><td>&lt;br/&gt; Guanosine (GTP)-Na&lt;sup&gt;+&lt;/sup&gt;</td><td>Sigma-Aldrich</td><td>G8877</td><td></td></tr><tr><td>&lt;br/&gt; Oregon Green BAPTA-1</td><td>ThermoFisher Scientific</td><td>O6812</td><td></td></tr><tr><td>&lt;br/&gt; Phosphocreatine di(tris) salt</td><td>Sigma-Aldrich</td><td>P1937</td><td></td></tr><tr><td>&lt;br/&gt; Streptavidin-conjugated Alexa-546</td><td>ThermoFisher Scientific</td><td>S11225</td><td></td></tr><tr><td>&lt;br/&gt; Patch Borosilicate Glass Capillaries</td><td>World Precision Instruments</td><td>1B100F-4</td><td></td></tr><tr><td>&lt;br/&gt; Theta Borosilicate Glass Capillaries</td><td>Sutter Instrument</td><td>BT-150-10</td><td></td></tr><tr><td>&lt;br/&gt; P-97 Flaming/Brown Micropipette puller</td><td>Sutter Instrument</td><td></td><td></td></tr><tr><td>&lt;br/&gt; TCS SP5 Confocal Multiphoton Microscope</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Chameleon Ultra II Ti:Sapphire multiphoton laser&amp;nbsp;</td><td>Coherent</td><td></td><td></td></tr><tr><td>LAS AF Imaging Acquisition Software</td><td>Leica Microsystems</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Temperature Controller TC-324B</td><td>Warner Instruments</td><td></td><td></td></tr><tr><td>&lt;br/&gt; MultiClamp 700B Amplifier</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Digidata 1440A Digitizer</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Confocal Translator</td><td>Siskiyou</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Micromanipulator</td><td>Siskiyou</td><td></td><td></td></tr><tr><td>&lt;br/&gt; pClamp Data Acquisition Software</td><td>Molecular Devices</td><td></td><td></td></tr><tr><td>&lt;br/&gt; A365 Constant Current Stimulus Isolator</td><td>World Precision Instruments</td><td></td><td></td></tr><tr><td>&lt;br/&gt; Vibraplane Optical Table</td><td>Kinetic Systems</td><td></td><td></td></tr></tbody></table>

two-photon calcium imaging in neuronal dendrites in brain slices

钙 (Ca2 +) 成像是研究神经元树突内胞内 Ca2 +信号的时空动力学的有力工具。Ca2 +的波动可以通过多种膜和胞内机制进行, 并在诱导突触可塑性和调控树突状兴奋性方面起着关键作用。因此, 在树突状分支中记录不同类型的 Ca2 +信号的能力对于研究树突的集成信息的小组很有价值。双光子显微镜的出现使这种研究更容易通过解决活体组织中成像的固有问题, 如光散射和光损伤。此外, 通过将常规的电生理技术与双光子 Ca2 +成像相结合, 可以对神经元树突中的局部 Ca2 +的波动进行并行研究, 并记录突触活动在soma.在这里, 我们描述如何使用此方法来研究 GABAergic 抑制中间神经元树突中局部 Ca2 + 瞬态 (猫) 的动力学。该方法还可用于研究急性脑切片中不同神经元类型的树突状 Ca2 +信号。

利用本文提出的协议, 我们获得了由体细胞电流注入诱发的猫, 并通过电刺激在海马 CA1 区 oriens/alveus 中间神经元树突。在对神经元进行修补后, 根据其形状和位置进行识别, 我们在给定距离的 linescans 上的多点处获得了一个近端枝晶 (图 1A)。我们观察到 backpropagating APs 引起的猫的振幅减少, 因为躯体的距离增加 (指示 AP 振幅减少或钙通道分布的距离依?...

Watch this Scientific Journal Video about Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices at JoVE.com

Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

脑切片神经元树突的双光子钙成像

本文提出了一种将全细胞贴钳记录和双光子成像相结合的方法, 记录急性脑切片神经元树突中的 Ca2 +瞬态。

Calcium (Ca2+) imaging is a powerful tool to investigate the spatiotemporal dynamics of intracellular Ca2+ signals in neuronal dendrites. Ca2+ ...

two-photon-calcium-imaging-in-neuronal-dendrites-in-brain-slices

Research

JoVE Journal

Neuroscience

10.7K 次观看.  Université Laval. 本文提出了一种将全细胞贴钳记录和双光子成像相结合的方法, 记录急性脑切片神经元树突中的 Ca2 +瞬态。

视频: Two-photon Calcium Imaging in Neuronal Dendrites in Brain Slices

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation从啮齿动物脑切片的神经元，研究突触传递和图像突触前的码头

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

科研

神经科学

In Vivo 2-Photon Calcium Imaging in Layer 2/3 of Mice

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution . The method consists in implanting a cranial imaging window, injecting a fluorescent calcium indicator dye that can be taken up by large numbers of neurons and finally recording the activity of neurons with time lapse calcium imaging using an in-vivo two photon microscope. Co-injection of astrocyte-specific dyes allows one to differentiate neurons from astrocytes. The technique can be performed in mice expressing fluorescent molecules in specific subpopulations of neurons to better understand the network interactions of different groups of cells.

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons . In-vivo two-photon calcium imaging is the only method that allows one to record the activity of a dense neuronal population with single-cell resolution .

在体内2光子层钙成像对小鼠的2 / 3

To understand network dynamics of microcircuits in the neocortex, it is essential to simultaneously record the activity of a large number of neurons .  ...

生物学

Functional Calcium Imaging in Developing Cortical Networks

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in intact spinal cord, brainstem, retina, cortex and dissociated neuronal culture preparations. During periods of spontaneous activity, neurons depolarize to fire single or bursts of action potentials, activating many ion channels. Depolarization activates voltage-gated calcium channels on dendrites and spines that mediate calcium influx. Highly synchronized electrical activity has been measured from local neuronal networks using field electrodes. This technique enables high temporal sampling rates but lower spatial resolution due to integrated read-out of multiple neurons at one electrode. Single cell resolution of neuronal activity is possible using patch-clamp electrophysiology on single neurons to measure firing activity. However, the ability to measure from a network is limited to the number of neurons patched simultaneously, and typically is only one or two neurons. The use of calcium-dependent fluorescent indicator dyes has enabled the measurement of synchronized activity across a network of cells. This technique gives both high spatial resolution and sufficient temporal sampling to record spontaneous activity of the developing network.
A key feature of newly-forming cortical and hippocampal networks during pre- and early postnatal development is spontaneous, synchronized neuronal activity (Katz &amp; Shatz, 1996; Khaziphov &amp; Luhmann, 2006). This correlated network activity is believed to be essential for the generation of functional circuits in the developing nervous system (Spitzer, 2006). In both primate and rodent brain, early electrical and calcium network waves are observed pre- and postnatally in vivo and in vitro (Adelsberger et al., 2005; Garaschuk et al., 2000; Lamblin et al., 1999). These early activity patterns, which are known to control several developmental processes including neuronal differentiation, synaptogenesis and plasticity (Rakic &amp; Komuro, 1995; Spitzer et al., 2004) are of critical importance for the correct development and maturation of the cortical circuitry.
In this JoVE video, we demonstrate the methods used to image spontaneous activity in developing cortical networks. Calcium-sensitive indicators, such as Fura 2-AM ester diffuse across the cell membrane where intracellular esterase activity cleaves the AM esters to leave the cell-impermeant form of indicator dye. The impermeant form of indicator has carboxylic acid groups which are able to then detect and bind calcium ions intracellularly.. The fluorescence of the calcium-sensitive dye is transiently altered upon binding to calcium. Single or multi-photon imaging techniques are used to measure the change in photons being emitted from the dye, and thus indicate an alteration in intracellular calcium. Furthermore, these calcium-dependent indicators can be combined with other fluorescent markers to investigate cell types within the active network.

Spontaneous activity of developing neuronal networks can be measured using AM-ester forms of calcium-sensitive indicator dyes. Changes in intracellular calcium, indicating neuronal activation, are detected as transient changes in indicator fluorescence with one- or two-photon imaging. This protocol can be adapted for a range of developmentally-dependent neuronal networks in vitro.

在开发皮层网络功能钙成像

A hallmark pattern of activity in developing nervous systems is spontaneous, synchronized network activity. Synchronized activity has been observed in ...

Imaging Calcium Responses in GFP-tagged Neurons of Hypothalamic Mouse Brain Slices

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the precise molecular steps as well as the activity of specific neurons in multi-functional nuclei of brain areas such as the hypothalamus remain. This problem includes identification of the molecular components involved in the regulation of various neurohormone signal transduction cascades. Elevations of intracellular Ca2+ play an important role in regulating the sensitivity of neurons, both at the level of signal transduction and at synaptic sites.
New tools have emerged to help identify neurons in the myriad of brain neurons by expressing green fluorescent protein (GFP) under the control of a particular promoter. To monitor both spatially and temporally stimulus-induced Ca2+ responses in GFP-tagged neurons, a non-green fluorescent Ca2+ indicator dye needs to be used. In addition, confocal microscopy is a favorite method of imaging individual neurons in tissue slices due to its ability to visualize neurons in distinct planes of depth within the tissue and to limit out-of-focus fluorescence. The ratiometric Ca2+ indicator fura-2 has been used in combination with GFP-tagged neurons1. However, the dye is excited by ultraviolet (UV) light. The cost of the laser and the limited optical penetration depth of UV light hindered its use in many laboratories. Moreover, GFP fluorescence may interfere with the fura-2 signals2. Therefore, we decided to use a red fluorescent Ca2+ indicator dye. The huge Stokes shift of fura-red permits multicolor analysis of the red fluorescence in combination with GFP using a single excitation wavelength. We had previously good results using fura-red in combination with GFP-tagged olfactory neurons3. The protocols for olfactory tissue slices seemed to work equally well in hypothalamic neurons4. Fura-red based Ca2+ imaging was also successfully combined with GFP-tagged pancreatic &beta;-cells and GFP-tagged receptors expressed in HEK cells5,6. A little quirk of fura-red is that its fluorescence intensity at 650 nm decreases once the indicator binds calcium7. Therefore, the fluorescence of resting neurons with low Ca2+ concentration has relatively high intensity. It should be noted, that other red Ca2+-indicator dyes exist or are currently being developed, that might give better or improved results in different neurons and brain areas.

In this protocol, we update recent progress in imaging Ca2+ signals of GFP-tagged neurons in brain tissue slices using a red fluorescent Ca2+ indicator dye.

在GFP标记的下丘脑神经元的小鼠脑片成像钙反应

Despite an enormous increase in our knowledge about the mechanisms underlying the encoding of information in the brain, a central question concerning the ...

Longitudinal Two-Photon Imaging of Dorsal Hippocampal CA1 in Live Mice

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from subcellular to network levels and at temporal scales from milliseconds to weeks. In addition, two-photon imaging can be combined with a variety of behavioral tasks to explore the causal relationships between brain function and behavior. However, in mammals, limited penetration and scattering of light have limited two-photon intravital imaging mostly to superficial brain regions, thus precluding longitudinal investigation of deep-brain areas such as the hippocampus. The hippocampus is involved in spatial navigation and episodic memory and is a long-standing model used to study cellular as well as cognitive processes important for learning and recall, both in health and disease. Here, a preparation that enables chronic optical access to the dorsal hippocampus in living mice is detailed. This preparation can be combined with two-photon optical imaging at cellular and subcellular resolution in head fixed, anesthetized live mice over several weeks. These techniques enable repeated imaging of neuronal structure or activity-evoked plasticity in tens to hundreds of neurons in the dorsal hippocampal CA1. Furthermore, this chronic preparation can be used in combination with other techniques such as micro-endoscopy, head-mounted wide field microscopy or three-photon microscopy, thus greatly expanding the toolbox to study cellular and network processes involved in learning and memory.

This method describes a chronic preparation that allows optical access to the hippocampus of living mice. This preparation can be used to perform longitudinal optical imaging of neuronal structural plasticity and activity-evoked cellular plasticity over a period of several weeks.

活鼠中多萨尔希波坎卡尔CA1的纵向双光成像

Two-photon microscopy is a fundamental tool for neuroscience as it permits investigation of the brain of live animals at spatial scales ranging from ...

行为学

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as addition/removal of synapses, occur in an experience-dependent manner, providing the structural foundation of neuronal plasticity. As postsynaptic components of the most excitatory synapses in the cortex, dendritic spines are considered to be a good proxy of synapses. Taking advantages of mouse genetics and fluorescent labeling techniques, individual neurons and their synaptic structures can be labeled in the intact brain. Here we introduce a transcranial imaging protocol using two-photon laser scanning microscopy to follow fluorescently labeled postsynaptic dendritic spines over time in vivo. This protocol utilizes a thinned-skull preparation, which keeps the skull intact and avoids inflammatory effects caused by exposure of the meninges and the cortex. Therefore, images can be acquired immediately after surgery is performed. The experimental procedure can be performed repetitively over various time intervals ranging from hours to years. The application of this preparation can also be expanded to investigate different cortical regions and layers, as well as other cell types, under physiological and pathological conditions.

Two-Photon in vivo Imaging of Dendritic Spines in the Mouse Cortex Using a Thinned-skull Preparation

Time-lapse imaging in the living animal provides valuable information on structural reorganization in the intact brain. Here, we introduce a thinned-skull preparation that allows transcranial imaging of fluorescently labeled synaptic structures in the living mouse cortex by two-photon microscopy.

双光子<em&gt;在体内</em&gt;树突棘中的鼠标皮质用稀疏的头颅成像准备

In the mammalian cortex, neurons form extremely complicated networks and exchange information at synapses. Changes in synaptic strength, as well as ...

Multi-photon Intracellular Sodium Imaging Combined with UV-mediated Focal Uncaging of Glutamate in CA1 Pyramidal Neurons

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high ...

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in adult mammalian animals remains elusive. In this study, we describe an experimental procedure for measuring calcium dynamics through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy. This method enables recordings and quantifications of neural activity in bilateral mouse brain regions one at a time in the same experiment for a prolonged period in vivo. Key aspects of this method, which can be completed within an hour, include minimally invasive surgery procedures for creating dual optical windows, and the use of 2P imaging. Although we only demonstrate the technique in the S1 area, the method can be applied to other regions of the living brain facilitating the elucidation of structural and functional complexities of brain neural networks.

Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice

We describe an experimental procedure for measuring neuronal activity through dual optical windows above bilateral primary somatosensory corticies (S1) in Thy1-GCaMP6s transgenic mice using 2-photon (2P) microscopy in vivo.

胸腺1-gmcmp6 转基因小鼠在原代体血源皮质中的成像神经活性

The mammalian brain exhibits marked symmetry across the sagittal plane. However, detailed description of neural dynamics in symmetric brain regions in ...

Calcium Imaging in Neurons

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and neurotransmitter release from synaptic vesicles. Within the cell, calcium concentration is highly dynamic due to the presence of pumps that selectively transport these ions in response to a variety of signals. Calcium imaging takes advantage of intracellular calcium flux to directly visualize calcium signaling in living neurons.This video begins with an overview of the key reagents used for this technique, known as calcium indicator dyes. The discussion includes an introduction to the commonly used dye Fura-2 and some basic principles behind how both ratiometric and non-ratiometric calcium indicators work. Next, a typical calcium imaging experiment is presented, from preparing the cells and dye to capturing and analyzing the fluorescent images. Finally, several experimental applications of calcium imaging are provided, such as the study of neuronal network activity and sensory processing.

Calcium Imaging in Neurons Using Fura-2

神经元钙成像

Calcium ions play an integral role in neuron function: They act as intracellular signals that can elicit responses such as altered gene expression and ...