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>

<ol>
	<li>Williams, S. R., Mitchell, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+measurement+of+somatic+voltage+clamp+errors+in+central+neurons.">Direct measurement of somatic voltage clamp errors in central neurons.</a> Nat Neurosci. 7, 790-798 (2008).</li><li>Denk, W., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photon+upmanship:+why+multiphoton+imaging+is+more+than+a+gimmick.">Photon upmanship: why multiphoton imaging is more than a gimmick.</a> Neuron. 18, 351-357 (1997).</li><li>Yuste, R., Denk, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+spines+as+basic+functional+units+of+neuronal+integration.">Dendritic spines as basic functional units of neuronal integration.</a> Nature. 375, 682-684 (1995).</li><li>Nimchinsky, E. A., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Function+of+Dendritic+Spines.">Structure and Function of Dendritic Spines.</a> Annu Rev Physiol. 64, 313-353 (2002).</li><li>Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+calcium+channels+in+single+spines+using+optical+fluctuation+analysis.">Analysis of calcium channels in single spines using optical fluctuation analysis.</a> Nature. 408, 589-593 (2000).</li><li>Mainen, Z. F., Malinow, R., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+calcium+transients+in+single+spines+indicate+that+NMDA+receptors+are+not+saturated.">Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated.</a> Nature. 399, 151-155 (1999).</li><li>Yasuda, R., Sabatini, B. L., Svoboda, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+calcium+channels+in+dendritic+spines.">Plasticity of calcium channels in dendritic spines.</a> Nat Neurosci. 6, 948-955 (2003).</li><li>Carter, A. G., Sabatini, B. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=State-dependent+calcium+signaling+in+dendritic+spines+of+striatal+medium+spiny+neurons.">State-dependent calcium signaling in dendritic spines of striatal medium spiny neurons.</a> Neuron. 44, 483-493 (2004).</li><li>Topolnik, L., Congar, P., Lacaille, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+metabotropicglutamate+receptor-+and+AMPA+receptor-mediated+dendritic+Ca2++signals+by+presynaptic+and+postsynaptic+activity+in+hippocampal+interneurons.">Differential regulation of metabotropicglutamate receptor- and AMPA receptor-mediated dendritic Ca2+ signals by presynaptic and postsynaptic activity in hippocampal interneurons.</a> J Neurosci. 25, 990-1001 (2005).</li><li>Topolnik, L., Chamberland, S., Pelletier, J. G., Ran, I., Lacaille, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activity-dependent+compartmentalized+regulation+of+dendritic+Ca2++signaling+in+hippocampal+interneurons.">Activity-dependent compartmentalized regulation of dendritic Ca2+ signaling in hippocampal interneurons.</a> J Neurosci. 29, 4658-4663 (2009).</li><li>Camir&#233;, O., Topolnik, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+calcium+nonlinearities+switch+the+direction+of+synaptic+plasticity+in+fast-spiking+interneurons.">Dendritic calcium nonlinearities switch the direction of synaptic plasticity in fast-spiking interneurons.</a> J Neurosci. 34, 3864-3877 (2014).</li><li>Jaffe, D. B., Johnston, D., Lasser-Ross, N., Lisman, J. E., Miyakawa, H., Ross, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thespread+of+Na++spikes+determines+the+pattern+of+dendritic+Ca2++entry+into+hippocampal+neurons.">Thespread of Na+ spikes determines the pattern of dendritic Ca2+ entry into hippocampal neurons.</a> Nature. 357, 244-246 (1992).</li><li>Nakamura, T., Barbara, J. G., Nakamura, K., Ross, W. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+release+of+Ca2++from+IP3-sensitive+stores+evoked+by+synaptic+activation+of+mGluRs+paired+with+backpropagating+action+potentials.">Synergistic release of Ca2+ from IP3-sensitive stores evoked by synaptic activation of mGluRs paired with backpropagating action potentials.</a> Neuron. 24, 727-737 (1999).</li><li>Kovalchuk, Y., Eilers, J., Lisman, J., Konnerth, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA+receptor-mediated+subthreshold+Ca(2+)+signals+in+spines+of+hippocampal+neurons.">NMDA receptor-mediated subthreshold Ca(2+) signals in spines of hippocampal neurons.</a> J Neurosci. 20, 1791-1799 (2000).</li><li>Goldberg, J. H., Yuste, R., Tamas, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ca2++imaging+of+mouse+neocortical+interneurone+dendrites:+contribution+of+Ca2+-permeable+AMPA+and+NMDA+receptors+to+subthreshold+Ca2++dynamics.">Ca2+ imaging of mouse neocortical interneurone dendrites: contribution of Ca2+-permeable AMPA and NMDA receptors to subthreshold Ca2+ dynamics.</a> J Physiol. 551, 67-78 (2003).</li><li>Goldberg, J. H., Lacefield, C. O., Yuste, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+dendritic+calcium+spikes+in+mouselayer+5+low+threshold+spiking+interneurones:+implications+for+control+of+pyramidal+cell+bursting.">Global dendritic calcium spikes in mouselayer 5 low threshold spiking interneurones: implications for control of pyramidal cell bursting.</a> J Physiol. 558, 465-478 (2004).</li><li>Oertner, T. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+imaging+of+single+synapses+in+brain+slices.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+calcium+concentration+dynamics+in+small+neuronal+compartments.">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="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Estimating+intracellular+calcium+concentrations+and+buffering+without+wavelength+rationing.">Estimating intracellular calcium concentrations and buffering without wavelength rationing.</a> Biophys J. 78, 2655-2667 (2000).</li><li>Camir&#233;, O., Topolnik, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+compartmentalization+and+regulation+of+postsynaptic+CaTs+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., Camir&#233;, O., Topolnik, L., Skinner, F. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+a+semi-automated+strategy+to+develop+multi-compartment+models+that+predict+biophysical+properties+of+interneuron-specific+3+(IS3)+cells+in+hippocampus.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+and+activity-dependent+dynamics+of+action+potential-evoked+Ca2++signals+in+dendrites+of+hippocampal+inhibitory+interneurons.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+calcium+mechanisms+and+long-term+potentiation+in+cortical+inhibitory+interneurons.">Dendritic calcium mechanisms and long-term potentiation in cortical inhibitory interneurons.</a> Eur J Neurosci. 35, 496-506 (2012).</li><li>Camir&#233;, O., Lacaille, J. C., Topolnik, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dendritic+Signaling+in+Inhibitory+Interneurons:+Local+Tuning+via+Group+I+Metabotropic+Glutamate+Receptors.">Dendritic Signaling in Inhibitory Interneurons: Local Tuning via Group I Metabotropic Glutamate Receptors.</a> Front Physiol. 3, 259 (2012).</li><li>Bourque, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Central+mechanisms+of+osmosensation+and+systemic+osmoregulation.">Central mechanisms of osmosensation and systemic osmoregulation.</a> Nat RevNeurosci. 9, 519-531 (2008).</li><li>Aghajanian, G. K., Rasmussen, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+studies+in+the+facial+nucleus+illustrating+a+simple+new+method+for+obtaining+viable+motoneurons+in+adult+rat+brain+slices.">Intracellular studies in the facial nucleus illustrating a simple new method for obtaining viable motoneurons in adult rat brain slices.</a> Synapse. 3, 331-338 (1989).</li><li>Pettit, D. L., Wang, S. S., Gee, K. R., Augustine, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+two-photon+uncaging:+anovel+approach+to+mapping+glutamate+receptors.">Chemical two-photon uncaging: anovel approach to mapping glutamate receptors.</a> Neuron. 19, 465-471 (1997).</li><li>Salom&#233;, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+random-access+scanning+in+two-photon+microscopy+usingacousto-optic+deflectors.">Ultrafast random-access scanning in two-photon microscopy usingacousto-optic deflectors.</a> J Neurosci Methods. 154, 161-174 (2006).</li><li>Lechleiter, J. D., Lin, D. T., Sieneart, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-photon+laser+scanning+microscopy+using+an+acoustic+optical+deflector.">Multi-photon laser scanning microscopy using an acoustic optical deflector.</a> Biophys J. 83, 2292-2299 (2002).</li></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 border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;">Animal Strain: Mouse CD1&#160;</td>
			<td style="width:148px;">Charles River</td>
			<td style="width:159px;">022</td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;">Isoflurane</td>
			<td style="width:148px;">AbbVie Corporation</td>
			<td>0B506-099</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">CGP 55845 hydrochloride&#160;</td>
			<td>Abcam</td>
			<td>ab120337</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Calcium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">D-(+)-Glucose&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>G8270</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td>H3375</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnesium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>M8266</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnesium sulfate heptahydrate</td>
			<td>Sigma-Aldrich</td>
			<td>230391</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraformaldehyde powder, 95%</td>
			<td>Sigma-Aldrich</td>
			<td>158127</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Potassium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Potassium gluconate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>P1847</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium azide&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S2002</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium bicarbonate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S8875</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium chloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S5886</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sucrose&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Triton X-100&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T9284</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma base&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T1503</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Trizma hydrochloride&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T3253</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Sodium phosphate dibasic dihydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>71643</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;"> 
			Sodium phosphate monobasic 
			monohydrate&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>S9638</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;">Biocytin</td>
			<td>Sigma-Aldrich</td>
			<td style="width:159px;">B4261</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Alexa Fluor 594 Hydrazide</td>
			<td>ThermoFisher Scientific</td>
			<td style="width:159px;">A10438</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;">SR95531 (Gabazine)&#160;</td>
			<td style="width:148px;">Abcam</td>
			<td style="width:159px;">ab120042</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Adenosine triphosphate (ATP)-Tris</td>
			<td>Sigma-Aldrich</td>
			<td>A9062</td>
			<td></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:239px;"> 
			Guanosine (GTP)-Na+</td>
			<td>Sigma-Aldrich</td>
			<td>G8877</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Oregon Green BAPTA-1</td>
			<td>ThermoFisher Scientific</td>
			<td>O6812</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Phosphocreatine di(tris) salt</td>
			<td>Sigma-Aldrich</td>
			<td>P1937</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Streptavidin-conjugated Alexa-546</td>
			<td>ThermoFisher Scientific</td>
			<td>S11225</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;"> 
			Patch Borosilicate Glass Capillaries</td>
			<td>World Precision Instruments</td>
			<td>1B100F-4</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;"> 
			Theta Borosilicate Glass Capillaries</td>
			<td>Sutter Instrument</td>
			<td>BT-150-10</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;"> 
			P-97 Flaming/Brown Micropipette puller</td>
			<td style="width:148px;">Sutter Instrument</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;"> 
			TCS SP5 Confocal Multiphoton Microscope</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;"> 
			Chameleon Ultra II Ti:Sapphire multiphoton laser&#160;</td>
			<td>Coherent</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;">LAS AF Imaging Acquisition Software</td>
			<td>Leica Microsystems</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Temperature Controller TC-324B</td>
			<td style="width:148px;">Warner Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:239px;"> 
			MultiClamp 700B Amplifier</td>
			<td>Molecular Devices</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Digidata 1440A Digitizer</td>
			<td>Molecular Devices</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Confocal Translator</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Micromanipulator</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			pClamp Data Acquisition Software</td>
			<td>Molecular Devices</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:239px;"> 
			A365 Constant Current Stimulus Isolator</td>
			<td style="width:148px;">World Precision Instruments</td>
			<td style="width:159px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:239px;"> 
			Vibraplane Optical Table</td>
			<td style="width:148px;">Kinetic Systems</td>
			<td style="width:159px;"></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

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

10.6K 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

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 ...

科研

神经科学

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

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

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

双突触诱发的星形胶质细胞和神经元的反应，在急性海马脑片电生理记录

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

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

在体内双光子成像的经验依赖的分子在大脑皮质神经元的变化

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

In vivo Neuronal Calcium Imaging in C. elegans

The nematode worm C. elegans is an ideal model organism for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, well-characterized nervous system allows identification and fluorescence imaging of any neuron within the intact animal. Simple immobilization techniques with minimal impact on the animal's physiology allow extended time-lapse imaging. The development of genetically-encoded calcium sensitive fluorophores such as cameleon 1 and GCaMP 2 allow in vivo imaging of neuronal calcium relating both cell physiology and neuronal activity. Numerous transgenic strains expressing these fluorophores in specific neurons are readily available or can be constructed using well-established techniques. Here, we describe detailed procedures for measuring calcium dynamics within a single neuron in vivo using both GCaMP and cameleon. We discuss advantages and disadvantages of both as well as various methods of sample preparation (animal immobilization) and image analysis. Finally, we present results from two experiments: 1) Using GCaMP to measure the sensory response of a specific neuron to an external electrical field and 2) Using cameleon to measure the physiological calcium response of a neuron to traumatic laser damage. Calcium imaging techniques such as these are used extensively in C. elegans and have been extended to measurements in freely moving animals, multiple neurons simultaneously and comparison across genetic backgrounds. C. elegans presents a robust and flexible system for in vivo neuronal imaging with advantages over other model systems in technical simplicity and cost.

In vivo Neuronal Calcium Imaging in C. elegans

With its small transparent body, well-documented neuroanatomy and a host of amenable genetic techniques and reagents, C. elegans makes an ideal model organism for in vivo neuronal imaging using relatively simple, low-cost techniques. Here we describe single neuron imaging within intact adult animals using genetically encoded fluorescent calcium indicators.

C.在体内神经元钙成像技术线虫

The nematode worm C. elegans is an ideal model organism  for relatively simple, low cost neuronal imaging in vivo. Its small transparent body and simple, ...

Two-photon Imaging of Cellular Dynamics in the Mouse Spinal Cord

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for live-cell imaging in the murine spinal cord. This technique is uniquely suited to analyze neural precursor cell (NPC) dynamics following transplantation into spinal cords undergoing neuroinflammatory demyelinating disorders. NPCs migrate to sites of axonal damage, proliferate, differentiate into oligodendrocytes, and participate in direct remyelination. NPCs are thereby a promising therapeutic treatment to ameliorate chronic demyelinating diseases. Because transplanted NPCs migrate to the damaged areas on the ventral side of the spinal cord, traditional intravital 2P imaging is impossible, and only information on static interactions was previously available using histochemical staining approaches. Although this method was generated to image transplanted NPCs in the ventral spinal cord, it can be applied to numerous studies of transplanted and endogenous cells throughout the entire spinal cord. In this article, we demonstrate the preparation and imaging of a spinal cord with enhanced yellow fluorescent protein-expressing axons and enhanced green fluorescent protein-expressing transplanted NPCs.

A new ex vivo preparation for imaging the mouse spinal cord. This protocol allows for two-photon imaging of live cellular interactions throughout the spinal cord.

细胞动力学在小鼠脊髓双光子成像

Two-photon (2P) microscopy is utilized to reveal cellular dynamics and interactions deep within living, intact tissues. Here, we present a method for ...

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new method of Ca2+-imaging using the bioluminescent reporter GFP-aequorin (GA) has been developed. This new technique relies on the fusion of the GFP and aequorin genes, producing a molecule capable of binding calcium and&#160;&#8212; with the addition of its cofactor coelenterazine &#8212; emitting bright light that can be monitored through a photon collector. Transgenic lines carrying the GFP-aequorin gene have been generated for both mice and Drosophila. In Drosophila, the GFP-aequorin gene has been placed under the control of the GAL4/UAS binary expression system allowing for targeted expression and imaging within the brain. This method has subsequently been shown to be capable of detecting both inward Ca2+-transients and Ca2+-released from inner stores. Most importantly it allows for a greater duration in continuous recording, imaging at greater depths within the brain, and recording at high temporal resolutions (up to 8.3 msec). Here we present the basic method for using bioluminescent imaging to record and analyze Ca2+-activity within the mushroom bodies, a structure central to learning and memory in the fly brain.

In Vivo Functional Brain Imaging Approach Based on Bioluminescent Calcium Indicator GFP-aequorin

Here we present a novel Ca2+-imaging approach using a bioluminescent reporter. This approach uses a fused construct GFP-aequorin which binds to Ca2+ and emits light, eliminating the need for light excitation. Significantly this method permits long continuous imaging, access to deep brain structures and high temporal resolution.

在基于生物发光钙指标GFP的水母体内脑功能成像方法

Functional in vivo imaging has become a powerful approach to study the function and physiology of brain cells and structures of interest. Recently a new ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging methods exist for examining the brain tissue of live newborn mammals. Herein we summarize a protocol for imaging individual cortical neurons in living neonatal mice. This protocol includes the following two methodologies: (1) the Supernova system for sparse and bright labeling of cortical neurons in the developing brain, and (2) a surgical procedure for the fragile neonatal skull. This protocol allows the observation of temporal changes of individual cortical neurites during neonatal stages with a high signal-to-noise ratio. Labeled cell-specific gene silencing and knockout can also be achieved by combining the Supernova with RNA interference and CRISPR/Cas9 gene editing systems. This protocol can, thus, be used for analyzing the developmental dynamics of cortical neurons, molecular mechanisms that control the neuronal dynamics, and changes in neuronal dynamics in disease models.

In Vivo Two-photon Imaging of Cortical Neurons in Neonatal Mice

We present an in vivo two-photon imaging protocol for imaging the cerebral cortex of neonatal mice. This method is suitable for analyzing the developmental dynamics of cortical neurons, the molecular mechanisms that control the neuronal dynamics, and the changes in neuronal dynamics in disease models.

体内新生小鼠皮层神经元的双光子成像

Two-photon imaging is a powerful tool for the in vivo analysis of neuronal circuits in the mammalian brain. However, a limited number of in vivo imaging ...

Two-photon Imaging of Microglial Processes' Attraction Toward ATP or Serotonin in Acute Brain Slices

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of homeostasis, undergo rapid morphological changes. For example, a laser lesion induces in a few minutes an oriented growth of microglial processes, also called &#34;directional motility&#34;, toward the site of injury. A similar effect can be obtained by delivering locally ATP or serotonin (5-hydroxytryptamine [5-HT]). In this article, we describe a protocol to induce a directional growth of microglial processes toward a local application of ATP or 5-HT in acute brain slices of young and adult mice and to image this attraction over time by multiphoton microscopy. A simple method of quantification with free and open-source image analysis software is proposed. A challenge that still characterizes acute brain slices is the limited time, decreasing with age, during which the cells remain in a physiological state. This protocol, thus, highlights some technical improvements (medium, air-liquid interface chamber, imaging chamber with a double perfusion) aimed at optimizing the viability of microglial cells over several hours, especially in slices from adult mice.

Microglia, the resident immune cells of the brain, respond quickly with morphological changes to modifications of their environment. This protocol describes how to use two-photon microscopy to study the attraction of microglial processes toward serotonin or ATP in acute brain slices of mice.

微胶质过程对急性脑片 atp 或血清素的双光子成像

Microglial cells are resident innate immune cells of the brain that constantly scan their environment with their long processes and, upon disruption of ...

Transpupillary Two-Photon In Vivo Imaging of the Mouse Retina

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo&#160;microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer&#39;s disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access.
This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.

In vivo imaging is a powerful tool for the study of biology in health and disease. This protocol describes transpupillary imaging of the mouse retina with a standard two-photon microscope. It also demonstrates different in vivoimaging methods to fluorescently label multiple cellular cohorts of the retina.

小鼠视网膜的经瞳双光子体内成像

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent ...