这项工作得到了加拿大卫生研究与自然科学研究所的支持。

所有程序均按照麦吉尔大学动物保护委员会和加拿大动物保护委员会批准的议定书和准则进行。 急性海马体外制备注意:分离完整的海马制剂涉及三个主要步骤:（1）溶液和设备的制备，（2）海马的解剖和（3）建立产生内在θ振荡所需的快速灌注速率系统。在该方案中，及时执行手术 - 从解剖到记录 - 特别重要，因为分离的海马构成如此密集但精细的制剂，其保持体外结构的功能连接性需要非常小心。准备事先确保尽可能早地提供足够的灌注水平以最小化细胞d保持生理功能。 <ol><li> 基于蔗糖的人造脑脊液（aCSF）溶液 <ol><li>准备1X的蔗糖溶液用于解剖和解剖后孵育。将表1中列出的所有组分（CaCl 2除外）合并成1升去离子水，并在4℃下储存长达2周。 </li></ol></li><li> 标准aCSF解决方案 <ol><li>在电生理记录期间准备标准aCSF用于高流量灌注分离的海马。为了使浓缩（5X）的库存aCSF中，溶解在表2中列出的所有部件，除了氯化钙2，在2升的去离子水，并储存于4℃。 </li></ol></li><li> 准备设备 <ol><li>在开始解剖之前，用冰块塑料托盘（30 x 20 x 5 cm），碳链连接管线和three玻璃培养皿（10 x 2厘米）（ 见图1 ）。 </li><li>添加300mL的蔗糖溶液（1X股票）至500mL烧杯中，气泡将其与碳合（95％O 2，5％CO 2），并添加的Ca 2+ [1.2毫]。 </li><li>将60 mL该蔗糖溶液转移到玻璃培养皿中，并在室温下放置。将其余部分置于4°C冷冻箱中10 - 15分钟。 </li><li>在整个解剖过程中用碳水泡沫冰冷的蔗糖溶液。 </li><li>用蔗糖溶液（4℃）填充第二培养皿（冷藏室）并保持在冰上。 </li><li>将30mL冰冷的蔗糖溶液加入到50mL烧杯中。 </li></ol></li></ol> 2.全海马解剖注意:解剖分离的海马的方法基本上与原来22描述的方法相同，但是关于pe的附加细节和变化灌注速率和记录技术。 <ol><li> 脑解剖 <ol><li>麻醉小鼠（P20 - 35）在通风橱下用浸有1 mL加入到笼子中的异氟烷（100％）浸泡的小纸巾。 </li><li>斩断麻醉的小鼠，并迅速将头浸入冰冷的蔗糖溶液（50mL烧瓶中，约5秒）。转移到纸巾上。 </li><li>使用手术刀做一个内侧皮肤切口（尾部到流行），并推动两侧的皮肤暴露头颅。 </li><li>用小剪刀剪开颅骨，用铲子迅速提取大脑，并将其放入含冰冷蔗糖溶液的培养皿中。让大脑冷却1-2分钟。 </li><li>当大脑正在恢复时，将2-3毫升蔗糖溶液加到覆盖在冰上的Petri（夹层）培养皿上的透镜纸上。 </li></ol></li><li> 隔离海马 <ol><li>将大脑放置在解剖盘中，直立位置ñ。 </li><li>用剃刀刀片去除小脑和额叶皮质。将大脑沿中心面切成两半，并将两个隔离的半球返回到保持室。再允许1 - 2分钟恢复。 </li><li>将单个半透明的脑垂直放置在解剖盘上。 </li><li>旋转盘，直到中间结构面向观察者，并且隔膜复合体的嵌入轮廓可见为丘脑前面的薄的梨形层组织。 </li><li>将半透明的大脑与微球保持稳定，并将聚四氟乙烯（PTFE）涂抹的小铲的小端立即放置在隔膜区（尾部）的后面。 </li><li>将涂抹的刮刀插入隔膜下方，并向下移动尖端，直到达到解剖盘。切断头部连接隔膜区域的纤维。沿着隔膜的前边缘重复相同的操作，并切割连接到大脑前部的纤维。 </li><li>用铲子轻轻地将刚好在海马上方的皮质的内部保持在直立的位置，使用微球来小心地拉下丘脑，下丘脑和剩余的脑干核。使用刮刀切断并取出拉出的组织。 </li><li>将隔离的半球置于其侧面，并确定海马的轮廓。 </li><li>将涂抹的刮刀插入侧脑室，背侧海马的末端。 </li><li>握住刮刀与半透明大脑的中间平面水平对齐，并将其滑过心室壁的平滑轮廓，直到尖端尾端出现。 </li><li>将刮铲放在海马下面，轻轻按压到连接的纤维上，沿着海马与上覆皮层连接的内层。将该微球涂在该层的外侧，并与涂抹的刮刀滑动，穿过连接纤维别尔斯。 </li><li>要完成提取，旋转解剖盘，并将涂抹的刮刀插入腹侧海马下。用刮刀轻轻握住皮质褶皱的内侧，并使用针对微球的切片运动切断内嗅连接。 注意:通过将涂覆的刮刀放置在整个海马下，并从下面拉出剩余的脑组织，可以去除整个septooppocampal复合物。 </li><li>隔离完成后，将海马放在盘子上，加入一滴冰冷的蔗糖溶液以保持凉爽。仔细地剪除任何剩余的皮层和纤维，并通过轻轻地将剃刀刀片穿过穹窿，将海马与隔膜分开。 注意:如果从锥体细胞进行膜片钳记录（见第4.6节光代谢对照），则可以以45°角横向切割分离的海马，以暴露CA1的锥体层（"angl切割"准备），而不损害海马剩余部分中的本地网络电路。 </li><li>将制剂转移到室温蔗糖溶液中，使其恢复15-30分钟，然后转移到记录室。 </li></ol></li></ol> 3.设置快速灌注记录孤立的海马<ol><li>设置重力灌注系统，使得氧化aCSF以20-25mL / min的连续高速流入。 <ol><li>提前准备aCSF（1X）烧瓶，并在开始实验前至少15分钟将其浸入温热浴（〜30℃）。打开在线解决方案加热器系统（30°C）。 </li><li>在整个实验中使用与碳水化合物连接的水族泡沫器和管线对aCSF进行氧化，并在灌注开始之前加入CaCl 2 [2mM]。 </li><li>停止aCSF流动，并使用广泛的海马转移到记录室玻璃移液器的末端。让蔗糖饱和的制剂沉淀在底部。 </li><li>将准备物放在记录室的中心（与入口 - 出口轴线一致），CA1和SUB的光滑表面在顶部。在中隔和颞叶处稳定小重量的海马，并重新启动aCSF流。 </li></ol></li></ol> 4.孤立性海马中的电生理学<ol><li> 细胞外记录体外海马theta振荡 <ol><li>对于体外分离的海马的局部场电位（LFP）或单一单位活性的细胞外监测，使用填充有aCSF的玻璃微量吸管（1-3MΩ）。使用增益x1000的片钳钳位放大器记录低噪声交流耦合场电位信号，在线带通滤波0.1 - 500 Hz，采样率为5-20 kHz。 注意:本协议中使用的定制显微镜配备了低（2.5X）和高倍率（40X）目标，用于海马低放大视图，以及用于单细胞识别的红外视频显微镜和落射荧光。该系统的组件包括立式荧光显微镜，荧光过滤器立方体，模拟摄像机和具有成像软件的数字帧采集器，一个定位于其上的定制灌注腔室（ 图2A ，插图i）和用于神经元记录的高精度显微操作器光纤放置。 </li><li>使用安装在显微镜上的相机连接到计算机显示屏，以低放大倍数（2.5X）检查海马制剂，并快速检查是否没有底切或损坏外表面。沿着CA1的光滑表面，沿着对角线排列的alveus纤维应该是可见的（ 图2A ，插图ii）当透过光照射通过海马脓。 </li><li>使用低倍率的宽场物镜来观察孤立的海马和LFP电极在特定记录位置的位置。 注意: 图2A中示出了放置在不同记录位置的多个电极的分离的海马制剂的布局。 </li><li>将LFP电极放入浴中，直到其接触CA1区域的表面（alveus）。 注意:这通常在交流耦合记录中产生快速瞬变，类似于当电极与记录介质接触时会发生什么。在此步骤之后，音频监视器可用于收听LFP通道信号。 注意:早期的活动迹象通常只会在10 - 15分钟之后出现，因此重要的是不要进入准备阶段。如果在这段时间之后，LFP电极的表面仍然没有起作用，通过层次上的进一步向下延伸并定期暂停，看看在接近主要细胞层时是否有任何形式的活动出现。如果在进一步的5 - 10分钟后没有检测到任何东西，请小心地收回电极，并将其放置在同一区域的其他位置。 </li><li>通过金字塔层推进LFP电极。观察到细胞外刺激活性最初增加，因为检测到来自个体神经元（大多是金字塔形和抑制性筐细胞）的单次排出。进一步放下电极，注意当尖端穿过辐射线时，尖峰再次褪色。观察到，随着记录位置通过辐射降低，在θ频率范围（4 - 12 Hz）中清晰可见的网络振荡变得明显，并达到最大幅度（〜100 - 300μV）。 </li></ol></li><li> Theta振荡在一个单一的地区 <ol><li>为了测试穿过CA1区域的自发性θ振荡的空间特性，将尖端ofa参考LFP电极在中间CA1部位（沿着纵向的颞轴位于中间位置），并将其降低到如上所述的辐射状态。 </li><li>将第二个LFP电极放入CA1位点（距离第一个LFP为200 - 800μm间隔或时间），并观察CA1 theta振荡可以在相对较大的距离上同步。 </li></ol></li><li> Theta振荡跨层 <ol><li>为了测试跨越海马层的theta振荡的性质，在CA1辐射位点留下参考LFP电极。从地层上方开始，将第二个电极放入锥体细胞层，并通过辐射体。观察跨越锥体层的LFP信号（相对相反）的逐渐反转。 注意:有关这些类型活动的代表性示例，请参见图2B 。层流/深度剖面和电流源密度（CSD）分析的例子被示出在分离海马相位反转的位点先前已经23，24，25出版。 </li></ol></li><li> 完整海马中的伽玛振荡和θ-γ耦合 <ol><li>将场电极放置在CA1 / SUB边界处，并将其降低，直到其位于金字塔形和分子层之间的界面处。在这个水平上，可以记录显示具有相位锁定到局部θ节奏的振幅变化的清晰伽马振荡的场电位24 。调整缩放以观察theta-gamma耦合的慢时间尺度。注意，伽马脉冲串发生在两个不同的频带中，这可以通过对在慢（25-50Hz）和快速伽马（150-250Hz）范围内的正在进行的LFP信号的带通滤波来显示（参见图2C代表性滤波痕迹）。 </li></ol></li><li> 体外海马θ振荡期间的全细胞膜片钳记录 注意:在本协议的这一部分，目标是获得视觉上指导的全细胞膜片钳记录从分离的海马识别的中间神经元从表达荧光蛋白的转基因PV-TOM小鼠tdTomato，在PV启动子的控制下（对于更多细节见材料和参考文献26 ）。 <ol><li>使用低（2.5X）和高功率（40X）放大倍数的荧光视频显微镜来显示位于来自PV-TOM小鼠的海马制剂表面附近的tdTomato阳性中间神经元（ 图3 ）。注意，虽然PV-TOM神经元可以被成功地可视化并且通过阿尔卑斯（达到100μm）的斑块靶向，但是当用角切割技术制备海马时，也可以相对容易地达到非荧光锥体细胞（参见第2.2.14节注）。</li><li>在海马低放大视图下，在CA1 / SUB中放置LFP电极以监测θ振荡，同时准备膜片钳实验。 </li><li>切换到40倍放大倍数并将目标物体沉入目标区域;降低它直到顶层变得可见。操纵显微镜的位置，以确保从相对侧开启补片移液器的充分开放通道。 </li><li>在荧光显微镜下，选择荧光PV-TOM细胞，并用填充有标准细胞内溶液的补片移液管（2-3MΩ）接近。 </li><li>使用口器将轻度正压施加到移液管上，并在电极下降到电池上时监测电阻。当尖端遇到目标细胞时，移液管阻力增加，并且当正压力推在膜上时出现明显的凹坑。释放压力并检查当前的脉冲是否平整，因为密封开始形成。马上放大至超极化电位（-70 mV），一旦实现GΩ密封，就会通过移液管支架施加少量负压使细胞膜破裂。 </li><li>一旦进入全细胞配置，检查在自发性海马振荡期间鉴定的PV细胞的生理特性（ 图3B ）。 </li><li>观察到来自PV细胞的细胞内膜电位记录的特征在于快速刺激行为和与正在进行的CA1 /SUBθ节律同步的动作电位突发。 </li><li>切换到电压钳位并将细胞保持在不同的膜电位，以表征由内在节奏活动产生的兴奋性与抑制性突触电流之比。 图3A中显示了来自锥体细胞的膜片钳记录的实例用于比较。 </li></ol></li><li> 孤立性海马中的光合作用控制 </stroNG&gt; 注:对于海马网络活动的光遗传控制，涉及含PV-GABA能细胞的节奏活化的细胞类型特异性的策略在这里被用作这些细胞显示在所述分离海马25同步主要细胞群中发挥重要的作用。通过将PV-Cre小鼠系与组成型表达ChR2 Cre-dependent（Ai32）的小鼠杂交，从而产生PV-ChY小鼠，实现兴奋性通道视紫红质-2（ChR2）在PV细胞中的选择性表达。或者，可以将病毒载体构建体（ 例如 ，AAVdj-ChETA-eYFP）注射到PV-Cre或PV-TOM小鼠的海马中（P15-16）以驱动兴奋性视蛋白在CA1 / SUB中间神经元中的表达（ 图4A ）。 注意:此策略已在前面描述25 。 <ol><li>将LFP电极放置在CA1 / SUB区域，并在隔离的海马中贴附附近的锥体细胞在PV中间神经元中表达蓝光敏感兴奋性视蛋白ChR2的小鼠的安瓿。 </li><li>在海马制剂上方放置一个光纤导轨（直径0.2-1毫米），并将其置于记录区域上。使用来自LED光源的蓝光（473 nm）进行光生刺激，由光脉冲（10 - 20 ms，由晶体管晶体管逻辑信号触发）或在θ频率（4 - 12 Hz）传送的正弦波电压指令组成。 注意:定制的基于LED的光刺激组件已在其他地方描述25 。 </li><li>切换到电流钳位并表征自发θ振荡期间金字塔形的活动。 </li><li>启动刺激方案并记录光响应。观察记录的神经元中的场振荡和突触活动在光遗传刺激期间变得越来越同步，并且PV细胞的节奏起搏导致对频率的鲁棒控制θ振荡的强度和强度（ 图4 ）。 </li></ol></li></ol>

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II. The differential influence of drugs upon both the septal cell firing pattern and the hippocampus theta activity.</a> Electroencephalogr Clin Neurophysiol. 14, 212-219 (1962).</li><li>Mitchell, S. J., Ranck, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+theta+rhythm+in+medial+entorhinal+cortex+of+freely+moving+rats.">Generation of theta rhythm in medial entorhinal cortex of freely moving rats.</a> Brain Res. 189 (1), 49-66 (1980).</li><li>Alonso, A., Garcia-Austt, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+sources+of+theta+rhythm+in+the+entorhinal+cortex+of+the+rat.+I.+Laminar+distribution+of+theta+field+potentials.">Neuronal sources of theta rhythm in the entorhinal cortex of the rat. I. Laminar distribution of theta field potentials.</a> Exp Brain Res. 67 (3), 493-501 (1987).</li><li>Vertes, R. P., Kocsis, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Brainstem-diencephalo-septohippocampal+systems+controlling+the+theta+rhythm+of+the+hippocampus.">Brainstem-diencephalo-septohippocampal systems controlling the theta rhythm of the hippocampus.</a> Neuroscience. 81 (4), 893-926 (1997).</li><li>Bland, B. H., Colom, L. V., Konopacki, J., Roth, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+records+of+carbachol-induced+theta+rhythm+in+hippocampal+slices.">Intracellular records of carbachol-induced theta rhythm in hippocampal slices.</a> Brain Res. 447 (2), 364-368 (1988).</li><li>Cobb, S. R., Buhl, E. H., Halasy, K., Paulsen, O., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synchronization+of+neuronal+activity+in+hippocampus+by+individual+GABAergic+interneurons.">Synchronization of neuronal activity in hippocampus by individual GABAergic interneurons.</a> Nature. 378 (6552), 75-78 (1995).</li><li>Williams, J. H., Kauer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+carbachol-induced+oscillatory+activity+in+rat+hippocampus.">Properties of carbachol-induced oscillatory activity in rat hippocampus.</a> J Neurophysiol. 78 (5), 2631-2640 (1997).</li><li>Chapman, C. A., 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=Cholinergic+induction+of+theta-frequency+oscillations+in+hippocampal+inhibitory+interneurons+and+pacing+of+pyramidal+cell+firing.">Cholinergic induction of theta-frequency oscillations in hippocampal inhibitory interneurons and pacing of pyramidal cell firing.</a> J Neurosci. 19 (19), 8637-8645 (1999).</li><li>Strata, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrinsic+oscillations+in+CA3+hippocampal+pyramids:+physiological+relevance+to+theta+rhythm+generation.">Intrinsic oscillations in CA3 hippocampal pyramids: physiological relevance to theta rhythm generation.</a> Hippocampus. 8 (6), 666-679 (1998).</li><li>Kocsis, B., Bragin, A., Buzsaki, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interdependence+of+multiple+theta+generators+in+the+hippocampus:+a+partial+coherence+analysis.">Interdependence of multiple theta generators in the hippocampus: a partial coherence analysis.</a> J Neurosci. 19 (14), 6200-6212 (1999).</li><li>Fellous, J. M., Sejnowski, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+induction+of+oscillations+in+the+hippocampal+slice+in+the+slow+(0.5-2+Hz),+theta+(5-12+Hz),+and+gamma+(35-70+Hz)+bands.">Cholinergic induction of oscillations in the hippocampal slice in the slow (0.5-2 Hz), theta (5-12 Hz), and gamma (35-70 Hz) bands.</a> Hippocampus. 10 (2), 187-197 (2000).</li><li>Gillies, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+of+atropine-resistant+theta+oscillations+in+rat+hippocampal+area+CA1.">A model of atropine-resistant theta oscillations in rat hippocampal area CA1.</a> J Physiol. 543 (Pt 3), 779-793 (2002).</li><li>Gloveli, T., 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=Orthogonal+arrangement+of+rhythm-generating+microcircuits+in+the+hippocampus.">Orthogonal arrangement of rhythm-generating microcircuits in the hippocampus.</a> Proc Natl Acad Sci U S A. 102 (37), 13295-13300 (2005).</li><li>Konopacki, J., Eckersdorf, B., Kowalczyk, T., Golebiewski, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Firing+cell+repertoire+during+carbachol-induced+theta+rhythm+in+rat+hippocampal+formation+slices.">Firing cell repertoire during carbachol-induced theta rhythm in rat hippocampal formation slices.</a> Eur J Neurosci. 23 (7), 1811-1818 (2006).</li><li>Hajos, N., 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=Maintaining+network+activity+in+submerged+hippocampal+slices:+importance+of+oxygen+supply.">Maintaining network activity in submerged hippocampal slices: importance of oxygen supply.</a> Eur J Neurosci. 29 (2), 319-327 (2009).</li><li>Manseau, F., Goutagny, R., Danik, M., Williams, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+hippocamposeptal+pathway+generates+rhythmic+firing+of+GABAergic+neurons+in+the+medial+septum+and+diagonal+bands:+an+investigation+using+a+complete+septohippocampal+preparation+in+vitro.">The hippocamposeptal pathway generates rhythmic firing of GABAergic neurons in the medial septum and diagonal bands: an investigation using a complete septohippocampal preparation in vitro.</a> J Neurosci. 28 (15), 4096-4107 (2008).</li><li>Goutagny, R., Jackson, J., Williams, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Self-generated+theta+oscillations+in+the+hippocampus.">Self-generated theta oscillations in the hippocampus.</a> Nat Neurosci. 12 (12), 1491-1493 (2009).</li><li>Jackson, J., Goutagny, R., Williams, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fast+and+slow+gamma+rhythms+are+intrinsically+and+independently+generated+in+the+subiculum.">Fast and slow gamma rhythms are intrinsically and independently generated in the subiculum.</a> J Neurosci. 31 (34), 12104-12117 (2011).</li><li>Amilhon, B., 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=Parvalbumin+Interneurons+of+Hippocampus+Tune+Population+Activity+at+Theta+Frequency.">Parvalbumin Interneurons of Hippocampus Tune Population Activity at Theta Frequency.</a> Neuron. 86 (5), 1277-1289 (2015).</li><li>Huh, C. 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作者声明没有竞争的商业或经济利益。

虽然来自急性海马切片的电生理记录构成了体外标准技术，但是本文提出的方法与传统方法有很大不同。不同于其中特定细胞层在表面可见并且可以直接检查的薄片制剂，完整的海马制剂更类似于体内构型，其中电极在穿过单个层时下降到目标脑区域中。海马的完整性与局部神经元群体的功能连通性和性质一起保存。这提供了一个复杂而强大的工具，用于调查海马中的小型和大型网?...

海马THETA振荡（4 - 12赫兹）之间是在哺乳动物脑节律活动的最主要的形式，并且据信在认知功能中发挥关键作用，如中的情节记忆1，2，3时空信息和形成处理。虽然一些体内研究，突出THETA调制地方细胞具有空间导航和病变的研究，以及临床证据的关系，支持海马THETA振荡参与记忆形成4，5，6，相关联的机制视图伴随着海马theta振荡的产生尚未完全了解。早期体内研究表明，θ活性主要依赖于外在振荡器，特别是节奏输入从传入大脑结构，例如隔膜和内嗅皮层7，8，9，10。与海马神经元的属性一起海马神经网络的内部连通- -一种内在因素作用也假定的基于体外观察11，12，13，14，15，16，17，18。然而，除了少数标志性开发方法研究19，20，21，困难可以复制的简单体外切片准备生理现实群体活动长期以来，延迟了对海马和相关领域的内在能力进行更为详细的实验检查来自我产生θ振荡。 标准体外薄片实验设置的一个重要缺点是脑结构的3D细胞和突触组织通常受到损害。这意味着不能支持基于空间分布的细胞组件的许多形式的协调网络活动，范围从局部组（半径≤1mm）到遍及一个或多个脑区域（&gt; 1mm）的神经元群体。考虑到这些考虑，需要一种不同类型的方法来研究θ振荡如何在海马中出现并传播到相关的皮层和皮质下输出结构。 近年来，初步开展了"完整的海马"准备，以检查双向intera两个结构22和"分离的海马"制剂的随后演变ctions，已经揭示，在缺少外部节奏输入23海马自发发生固有THETA振荡。这些方法的价值在于初步的认识，即这些区域的整个功能结构必须被保留以便在体外作为θ节律发生器22 。

<table border="0" cellpadding="0" cellspacing="0" width="820">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Reagents</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td style="width:247px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>S9625</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sucrose</td>
			<td>Sigma Aldrich</td>
			<td>S9378</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NaH2PO4 - sodium phosphate monobasic</td>
			<td>Sigma Aldrich</td>
			<td>S8282</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Magnesium sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P3911</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">D-(+)-Glucose</td>
			<td>Sigma Aldrich</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Calcium chloride dihydrate</td>
			<td>Sigma Aldrich</td>
			<td>C5080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium Ascorbate</td>
			<td>Sigma Aldrich</td>
			<td>A7631-25G</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:319px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Standard Dissecting Scissors</td>
			<td>Fisher Scientific</td>
			<td>08-951-25</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel Handle #4, 14cm</td>
			<td>WPI</td>
			<td>500237</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Filter forceps, flat jaws, straight (11cm)</td>
			<td>WPI</td>
			<td>500456</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paragon Stainless Steel Scalpel Blades #20</td>
			<td>Ultident</td>
			<td>02-90010-20</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fine Point Curved Dissecting Scissors</td>
			<td>Thermo Fisher Scientific</td>
			<td>711999</td>
			<td>brain extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Teflon (PTFE) -coated thin spatula</td>
			<td>VWR</td>
			<td>82027-534</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hayman Style Microspatula</td>
			<td>Fisher Scientific</td>
			<td>21-401-25A</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab spoon</td>
			<td>Fisher Scientific</td>
			<td>14-375-20</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Borosilicate Glass Pasteur Pipets</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Droper</td>
			<td>Fisher Scientific</td>
			<td></td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Razor blades Single edged</td>
			<td>VWR</td>
			<td>55411-055</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lens paper (4X6 inch)</td>
			<td>VWR</td>
			<td>52846-001</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass petri dishes (100 x 20 mm)</td>
			<td>VWR</td>
			<td>25354-080</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic tray for ice; size 30 x 20 x 5 cm</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>hippocampal preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Single Inline Solution Heater</td>
			<td>Warner Instruments</td>
			<td>SH-27B</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aquarium air stones for bubbling</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tygon E-3603 tubing (ID 1/16 OD 1/8)</td>
			<td>Fisherbrand</td>
			<td>14-171-129</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Electric Skillet</td>
			<td>Black &#38; Decker</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">95% O2/5% CO2 gas mixture (carbogen)&#160;</td>
			<td>Vitalaire</td>
			<td>SG466204A</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass bottles/flasks (4 x 1 L)</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>perfusion system</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Submerged recording Chamber</td>
			<td>custom design (FM)</td>
			<td>n.a.</td>
			<td style="width:247px;">Commercial alternative may be used</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass pipettes (1.5 / 0.84 OD/ID (mm) )</td>
			<td>WPI</td>
			<td>1B150F-4</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hum Bug 50/60 Hz Noise Eliminator</td>
			<td>Quest Scientific</td>
			<td>Q-Humbug</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B patch-clamp amplifier</td>
			<td>Molecular devices</td>
			<td>MULTICLAMP</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiclamp 700B Commander Program</td>
			<td>Molecular devices</td>
			<td>MULTICLAMP</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital/Analogue converter</td>
			<td>Molecular devices</td>
			<td>DDI440</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PCLAMP10</td>
			<td>Molecular devices</td>
			<td>PCLAMP10</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibration isolation table&#160;</td>
			<td>Newport</td>
			<td>n.a.</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulators (manually operated )</td>
			<td>Siskiyou&#160;</td>
			<td>MX130</td>
			<td>electrophysiology (LFP)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micromanipulators (automated)</td>
			<td>Siskiyou&#160;</td>
			<td>MC1000e</td>
			<td>electrophysiology (patch)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Audio monitor&#160;</td>
			<td>A-M Systems</td>
			<td>Model 3300</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Micropipette/Patch pipette puller</td>
			<td>Sutter</td>
			<td>P-97</td>
			<td>electrophysiology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Custom-built upright fluorescence microscope</td>
			<td>Siskiyou</td>
			<td>n.a.</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Analogue video camera</td>
			<td>COHU</td>
			<td>4912-2000/0000</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Digital frame grabber with imaging software</td>
			<td>EPIX, Inc</td>
			<td>PIXCI-SV7</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus 2.5x objective</td>
			<td>Olympus</td>
			<td>MPLFLN</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Olympus 40x water immersion objective</td>
			<td>Olympus</td>
			<td>UIS2 LUMPLFLN</td>
			<td>Imaging</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Custom-made light-emitting diode (LED) system&#160;</td>
			<td>custom</td>
			<td>n.a.</td>
			<td>optogenetic stimulation (Amhilon et al., 2015)</td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;width:319px;">Name</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Animals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">PV::Cre (KI) mice</td>
			<td style="width:135px;">Jackson Laboratory</td>
			<td style="width:119px;">stock number 008069</td>
			<td style="width:247px;">Allow&#160; Cre-directed gene expression in PV interneurons</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Constitutive-conditional Ai9 mice (R26-lox-stop-lox-tdTomato (KI))</td>
			<td style="width:135px;">Jackson Laboratory</td>
			<td style="width:119px;">stock number 007905</td>
			<td style="width:247px;">Express TdTomato following Cre-mediated recombination</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:319px;">Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP</td>
			<td style="width:135px;">Jackson Laboratory</td>
			<td style="width:119px;">stock 
			number 012569</td>
			<td style="width:247px;">Express the improved channelrhodopsin-2/EYFP fusion protein following exposure to Cre recombinase</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PVChY mice</td>
			<td>In house breeding</td>
			<td>n.a.</td>
			<td style="width:247px;">Offspring obtained from cross-breeding the PV-Cre line with Ai32 mice (R26-lox-stop-lox-ChR2(H134R)-EYFP</td>
		</tr>
	</tbody>
</table>

tuning in the hippocampal theta band in vitro: methodologies for recording from the isolated rodent septohippocampal circuit

该方案概述了从孤立的整个海马，WT和转基因小鼠制备和记录的程序，以及近来对θ振荡研究的方法和应用的改进。提出了分离的海马制剂的简单表征，其中检查内部海马θ振荡器之间的关系以及玉米螟-1（CA1）和亚细胞（SUB）区域的锥体细胞和GABA能中间神经元的活性。总体而言，我们显示分离的海马能够在体外产生内在的θ振荡，海马内产生的节律性可以通过对小白蛋白阳性（PV）中间神经元的光诱导刺激进行精确的操纵。 体外分离的海马制剂提供了独特的机会，使用来自视觉识别的neu的同时场和细胞内膜片钳记录更好地了解θ节奏生成的机制。

<html>本节说明通过在体外研究小鼠分离的海马制剂中的 θ振荡可以获得的结果的实例。提取分离的海马的解剖程序如图1所示 。使用这种制备，可以在放置多个场电极期间检查内在θ振荡，记录整体活动和将突触输入同步到分离的海马的不同区域和层中的神经元群体（ 图2 ）。提出了同时进行的全细胞膜片钳和细胞...

Watch this Scientific Journal Video about Tuning in the Hippocampal Theta Band In Vitro: Methodologies for Recording from the Isolated Rodent Septohippocampal Circuit at JoVE.com

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

在这里，我们提出了从孤立的整个海马制剂记录节律神经元网络theta和γ振荡的协议。我们描述从海马提取到场，单元和全细胞膜片钳记录的细节以及θ节律的光起搏的实验步骤。

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

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

tuning-hippocampal-theta-band-vitro-methodologies-for-recording-from

Research

JoVE Journal

Neuroscience

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

9.7K 次观看.  McGill University. 在这里，我们提出了从孤立的整个海马制剂记录节律神经元网络theta和γ振荡的协议。我们描述从海马提取到场，单元和全细胞膜片钳记录的细节以及θ节律的光起搏的实验步骤。 

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

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

逆行荧光染料标记允许有针对性的外单细胞记录技术鉴定神经元<em&gt;在体内</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

科研

神经科学

Investigations on Alterations of Hippocampal Circuit Function Following Mild Traumatic Brain Injury

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological impairments 1. Two pervasive and disabling symptoms experienced by TBI survivors, memory deficits and a reduction in seizure threshold, are thought to be mediated by TBI-induced hippocampal dysfunction 2,3. In order to demonstrate how altered hippocampal circuit function adversely affects behavior after TBI in mice, we employ lateral fluid percussion injury, a commonly used animal model of TBI that recreates many features of human TBI including neuronal cell loss, gliosis, and ionic perturbation 4-6.
	Here we demonstrate a combinatorial method for investigating TBI-induced hippocampal dysfunction. Our approach incorporates multiple ex vivo physiological techniques together with animal behavior and biochemical analysis, in order to analyze post-TBI changes in the hippocampus. We begin with the experimental injury paradigm along with behavioral analysis to assess cognitive disability following TBI. Next, we feature three distinct ex vivo recording techniques: extracellular field potential recording, visualized whole-cell patch-clamping, and voltage sensitive dye recording. Finally, we demonstrate a method for regionally dissecting subregions of the hippocampus that can be useful for detailed analysis of neurochemical and metabolic alterations post-TBI.
	These methods have been used to examine the alterations in hippocampal circuitry following TBI and to probe the opposing changes in network circuit function that occur in the dentate gyrus and CA1 subregions of the hippocampus (see Figure 1). The ability to analyze the post-TBI changes in each subregion is essential to understanding the underlying mechanisms contributing to TBI-induced behavioral and cognitive deficits. 
	The multi-faceted system outlined here allows investigators to push past characterization of phenomenology induced by a disease state (in this case TBI) and determine the mechanisms responsible for the observed pathology associated with TBI.

A multi-faceted approach to investigating functional changes to hippocampal circuitry is explained. Electrophysiological techniques are described along with the injury protocol, behavioral testing and regional dissection method. The combination of these techniques can be applied in similar fashion for other brain regions and scientific questions.

调查轻度创伤性脑损伤海马电路功能的改变

Traumatic Brain Injury (TBI) afflicts more than 1.7 million people in the United States each year and even mild TBI can lead to persistent neurological ...

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 ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

多单位记录方法来描述蝗虫的神经活动（<em&gt; Schistocerca美洲</em&gt;）嗅觉电路

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Direct Intraventricular Delivery of Drugs to the Rodent Central Nervous System

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses specifically on antisense oligonucleotides (ASOs), though the techniques shown in the video here can also be used to deliver a plethora of other drugs to the CNS. Antisense oligonucleotides (ASOs) have the capability to knockdown sequence-specific targets 1 as well as shift isoform ratios of specific genes 2. To achieve widespread gene knockdown or splicing in the CNS of mice, the ASOs can be delivered into the brain using two separate routes of administration, both of which we demonstrate in the video.
The first uses Alzet osmotic pumps, connected to a catheter that is surgically implanted into the lateral ventricle. This allows the ASOs to be continuously infused into the CNS for a designated period of time. The second involves a single bolus injection of a high concentration of ASO into the right lateral ventricle. Both methods use the mouse cerebral ventricular system to deliver the ASO to the entire brain and spinal cord, though depending on the needs of the study, one method may be preferred over the other.

We describe a method to target drugs to the central nervous system by either implanting a catheter or performing a bolus injection into the right lateral ventricle in mice. We focus specifically on the delivery of antisense oligonucleotides. This technique is readily adaptable to other drugs and to rats.

啮齿动物的中枢神经系统药物直接脑室交货

Due to an inability to cross the blood brain barrier, certain drugs need to be directly delivered into the central nervous system (CNS). Our lab focuses ...

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls gastrointestinal motility. A procedure for the isolation and culture of a mixed population of neurons and glia from the myenteric plexus is described. The primary cultures can be maintained for over 7 days, with connections developing among the neurons and glia. The longitudinal muscle strip with the attached myenteric plexus is stripped from the underlying circular muscle of the mouse ileum or colon and subjected to enzymatic digestion. In sterile conditions, the isolated neuronal and glia population are preserved within the pellet following centrifugation and plated on coverslips. Within 24-48 hr, neurite outgrowth occurs and neurons can be identified by pan-neuronal markers. After two days in culture, isolated neurons fire action potentials as observed by patch clamp studies. Furthermore, enteric glia can also be identified by GFAP staining. A network of neurons and glia in close apposition forms within 5 - 7 days. Enteric neurons can be individually and directly studied using methods such as immunohistochemistry, electrophysiology, calcium imaging, and single-cell PCR. Furthermore, this procedure can be performed in genetically modified animals. This methodology is simple to perform and inexpensive. Overall, this protocol exposes the components of the enteric nervous system in an easily manipulated manner so that we may better discover the functionality of the ENS in normal and disease states.

An In-vitro Preparation of Isolated Enteric Neurons and Glia from the Myenteric Plexus of the Adult Mouse

We demonstrate a cell culture protocol for the direct study of neuronal and glial components of the enteric nervous system. A neuron/glia mixed culture on coverslips is prepared from the myenteric plexus of adult mouse providing the ability to examine individual neuron and glia function by electrophysiology, immunohistochemical, etc.

一个<em在体外</em从成年小鼠的肌间神经丛制备隔离肠道神经元和神经胶质

The enteric nervous system is a vast network of neurons and glia running the length of the gastrointestinal tract that functionally controls ...

Utilizing Combined Methodologies to Define the Role of Plasma Membrane Delivery During Axon Branching and Neuronal Morphogenesis

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular guidance cues like netrin-1 and results in dramatic increases to the surface area of the axonal plasma membrane. Netrin-1-dependent axon branching likely involves temporal and spatial control of plasma membrane expansion, the components of which are supplied through exocytic vesicle fusion. These fusion events are preceded by formation of SNARE complexes, comprising a v-SNARE, such as VAMP2 (vesicle-associated membrane protein 2), and plasma membrane t-SNAREs, syntaxin-1 and SNAP25 (synaptosomal-associated protein 25). Detailed herein isa multi-pronged approach used to examine the role of SNARE mediated exocytosis in axon branching. The strength of the combined approach is data acquisition at a range of spatial and temporal resolutions, spanning from the dynamics of single vesicle fusion events in individual neurons to SNARE complex formation and axon branching in populations of cultured neurons. This protocol takes advantage of established biochemical approaches to assay levels of endogenous SNARE complexes and Total Internal Reflection Fluorescence (TIRF) microscopy of cortical neurons expressing VAMP2 tagged with a pH-sensitive GFP (VAMP2-pHlourin) to identify netrin-1 dependent changes in exocytic activity in individual neurons. To elucidate the timing of netrin-1-dependent branching, time-lapse differential interference contrast (DIC) microscopy of single neurons over the order of hours is utilized. Fixed cell immunofluorescence paired with botulinum neurotoxins that cleave SNARE machinery and block exocytosis demonstrates that netrin-1 dependent axon branching requires SNARE-mediated exocytic activity.

Light microscopy techniques coupled with biochemical assays elucidate the involvement of SNARE-mediated exocytosis in netrin-dependent axon branching. This combination of techniques permits identification of molecular mechanisms controlling axon branching and cell shape change.

采用组合运用各种方法，定义质膜交付在轴突分支和神经元形态发生中的作用

During neural development, growing axons extend to multiple synaptic partners by elaborating axonal branches. Axon branching is promoted by extracellular ...

Recording Gamma Band Oscillations in Pedunculopontine Nucleus Neurons

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the centrolateral/parafascicular; Cl/Pf nucleus). The activation of either the PPN or Cl/Pf nuclei in vivo has been described to induce the arousal of the animal and an increment in gamma band activity in the cortical electroencephalogram (EEG). The cellular mechanisms for the generation of gamma band oscillations in Reticular Activating System (RAS) neurons are the same as those found to generate gamma band oscillations in other brains nuclei. During current-clamp recordings of PPN neurons (from parasagittal slices from 9 - 25 day-old rats), the use of depolarizing square steps rapidly activated voltage-dependent potassium channels that prevented PPN neurons from being depolarized beyond -25 mV.
Injecting 1 - 2 sec long depolarizing current ramps gradually depolarized PPN membrane potential resting values towards 0 mV. However, injecting depolarizing square pulses generated gamma-band oscillations of membrane potential that showed to be smaller in amplitude compared to the oscillations generated by ramps. All experiments were performed in the presence of voltage-gated sodium channels and fast synaptic receptors blockers. It has been shown that the activation of high-threshold voltage-dependent calcium channels underlie gamma-band oscillatory activity in PPN neurons. Specific methodological and pharmacological interventions are described here, providing the necessary tools to induce and sustain PPN subthreshold gamma band oscillation in vitro.

The pedunculopontine nucleus (PPN) is located in the brainstem and its neurons are maximally activated during waking and rapid eye movement (REM) sleep brain states. This work describes the experimental approach to record in vitro gamma band subthreshold membrane oscillation in PPN neurons.

录音伽玛乐队在振荡脚桥核神经元

Synaptic efferents from the PPN are known to modulate the neuronal activity of several intralaminar thalamic regions (e.g., the ...

Personalized Needles for Microinjections in the Rodent Brain

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to deliver gene or cell therapy products. Unfortunately, current microinjection techniques use steel or glass needles that are suboptimal for multiple reasons: in particular, steel needles may cause tissue damage, and glass needles may bend when lowered deeply into the brain, missing the target region. In this article, we describe a protocol to prepare and use quartz needles that combine a number of useful features. These needles do not produce detectable tissue damage and, being very rigid, ensure reliable delivery in the desired brain region even when using deep coordinates. Moreover, it is possible to personalize the design of the needle by making multiple holes of the desired diameter. Multiple holes facilitate the injection of large amounts of solution within a larger area, whereas large holes facilitate the injection of cells. In addition, these quartz needles can be cleaned and re-used, such that the procedure becomes cost-effective.

We describe here a protocol for microinjection in the rodent brain that uses quartz needles. These needles do not produce detectable tissue damage and ensure reliable delivery even in deep regions. Moreover, they can be adapted to research needs by personalized designs and can be re-used.

啮齿动物脑 Microinjections 的个性化针

Microinjections have been used for a long time for the delivery of drugs or toxins within specific brain areas and, more recently, they have been used to ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

小容积回收-灌注-浸没式室系统中维持的急性海马切片的突触可塑性记录

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...