Name: assessment of long-term depression induction in adult cerebellar slices
Uploaded: 2019-10-16T13:00:00
Description: Watch this Scientific Journal Video about Assessment of Long-term Depression Induction in Adult Cerebellar Slices at JoVE.com

我们感谢奥巴的技术援助。这项研究得到了对K.Y的科研援助资助（C）17K01982的部分支持。

所有实验程序都通过了RIKEN在实验中照顾和使用动物委员会。老鼠被关在RIKEN脑科学中心的动物设施中，温度（23-25°C）和湿度（45%~65%）良好控制。条件。使用雄性小鼠和雌性WT小鼠（C57BL/6，3~6个月）。
1. 实验中使用的解决方案的制备
注：所有溶液均应在不含金属（电阻率 > 18.2 MΩ）和其他杂质（总有机碳 （TOC） < 5.0 ppb）的超纯水中制成。工?...

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四个协议之间的差异
在LTD诱导协议1和2中，Cjs在1Hz时300次足以诱导小脑LTD. CF的刺激频率似乎在生理范围内，因为警报成年小鼠（P60）的复杂尖峰发射速率报告为1.25赫兹36。然而，单单CF刺激并没有导致PF-CF突触的长期可塑性，如协议1和2（图4，图5）中使用的，虽然仅CF刺激在较高频率诱导LTD24。协?...

小脑皮层的精心设计的神经元网络的突触组织，由PC、分子层内神经元（篮子和硬质细胞）、Golgi细胞、颗粒细胞的PF、青苔纤维和攀爬纤维（CFs）组成，已经得到阐明。在激发/抑制和发散/收敛方面，组织良好的电路图表明小脑是一个"神经机"1，尽管以前不知道这个"机器"的目的。后来Marr提出，PC的PC输入构成一个三层关联学习网络2。他还建议，每个CF传达一个大脑指令的元素运动2。他假设同时激活 PF 和 CF 将增强 PF-PC 突触活动，并导致 PF-PC 突触的长期强权 （LTP）。另一方面，Albus 假设 PF 和 CF 的同步激活导致 PF-PC 突触3的 LTD。上述两项研究均将小脑解释为一种独特的记忆装置，将小脑并入小脑皮质网络导致Marr_Albus模型学习机模型的形成。
根据这些理论预测，两条证据线表明小脑中存在突触可塑性。第一线的证据是由絮凝器的解剖组织建议的;这里前庭器官起源的MF通路和视网膜起源的CF通路收敛在PC4上。这种独特的收敛模式表明，在絮凝中发生的突触可塑性导致前视反射的显著适应性。第二，记录花团的PC反应和絮状物的病变也支持了上述假设5，6，7。此外，在猴子手部运动8的适应过程中，PC放电模式支持了突触可塑性假说，尤其是阿不思的LTD-假说3。
为了直接确定突触可塑性的性质，一束PF的重复结合刺激（Cjs）和CF，专门在体内对PC进行内联作用，被证明诱导LTD对PF_PC突触的传输功效9， 10，11.在随后使用小脑切片12和培养 PC 的体外探索中，共培养的颗粒细胞刺激和橄榄细胞刺激13或电磷基质和体细胞结合去极化14，15引起 LTD.使用体外制剂16、17，对LTD诱导背后的信号转导机制也进行了深入的研究。
VOR 和 OKR 的适应通常用于定量评估基因操作对小脑运动学习的影响，因为前庭-小脑皮层被证明是 VOR18适应性学习的基本来源，19、20和 OKR19、21 LTD 诱导失败与行为运动学习障碍之间的相关性已被视为 LTD 在电机中起着重要作用的证据学习机制22.这些观点统称为运动学习的LTD假说，或马尔-阿布斯-伊托假说23，24，25，26。
使用类似的方案测量了眼动的自适应学习，同时利用各种实验条件诱导在切片制备27、28、29、30、31中诱导LTD.最近，Schonewille等人26日报告说，一些基因操纵的小鼠表现出正常的运动学习，但小脑切片没有表现出LTD，因此得出结论，LTD对运动学习来说并非必要。然而，在室温下只尝试使用一种类型的协议来诱导LTD。因此，我们在30°C左右的记录条件下使用几种LTD诱导协议，并证实在接近生理温度32的温度下使用这些协议在基因操纵的小鼠中可靠地诱导了LTD。
然而，关于结合刺激的基本特性，仍然存在一些问题。第一个是复杂尖峰的形状和LTD的振幅之间的关系。其次，结合PF刺激和躯体去极化，是否有必要使用刺激的数量是难以捉摸的。在本研究中，用野生型（WT）小鼠调查了这些问题。

<table>
	<tbody>
		<tr>
			<td>Amplifier</td>
			<td>Molecular Devices-Axon</td>
			<td>Multiclamp 700B</td>
			<td></td>
		</tr>
		<tr>
			<td>Borosilicate glass capillary</td>
			<td>Sutter</td>
			<td>BF150-110-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Digitizer</td>
			<td>Molecular Devices-Axon</td>
			<td>Digidata1322A</td>
			<td></td>
		</tr>
		<tr>
			<td>Electrode puller</td>
			<td>Sutter</td>
			<td>Model P-97</td>
			<td></td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>FUJIFILM Wako Pure Chemical</td>
			<td>26675-46-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Isolator</td>
			<td>A.M.P.I.</td>
			<td>ISOflex</td>
			<td></td>
		</tr>
		<tr>
			<td>Linear slicer</td>
			<td>Dosaka EM</td>
			<td>PRO7N</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>NIKON</td>
			<td>Eclipse E600FN</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>MP1 Single Channel Pump</td>
			<td></td>
		</tr>
		<tr>
			<td>Picrotoxin</td>
			<td>Sigma-Aldrich</td>
			<td>P1675</td>
			<td></td>
		</tr>
		<tr>
			<td>Pure water maker</td>
			<td>Merck-Millipore</td>
			<td>MilliQ 7000</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for experiment</td>
			<td>Molecular probe-Axon</td>
			<td>pClamp 10</td>
			<td></td>
		</tr>
		<tr>
			<td>Software for statistics</td>
			<td>KyensLab</td>
			<td>KyPlot 5.0</td>
			<td></td>
		</tr>
		<tr>
			<td>Stimulator</td>
			<td>WPI</td>
			<td>DS8000</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature controller</td>
			<td>Warner</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetrodotoxin</td>
			<td>Tocris</td>
			<td>1078</td>
			<td></td>
		</tr>
	</tbody>
</table>

assessment of long-term depression induction in adult cerebellar slices

突触可塑性为学习和记忆提供了一种机制。对于小脑运动学习，从平行纤维（PF）到Purkinje细胞（PC）的突触透射的长期凹陷（LTD）被认为是运动学习的基础，并且观察到各种基因操纵的动物。常用的运动学习集，如光动反射（OKR）、前庭-眼反射（VOR）和轮状测试等，用于评估运动学习能力。然而，从GluA2-Carboxy终点体改进的撞鼠获得的结果显示，尽管缺乏PF-LTD，但VOR和OKR的正常适应。在该报告中，LTD的诱导只尝试在室温下使用一种刺激方案。因此，在接近生理温度下使用各种方案在同一敲锥突变体中探索了诱导小脑LTD的条件。最后，我们发现了刺激方案，通过这种刺激方案，可以在这些基因操纵的小鼠中诱导LTD。在这项研究中，提出了一套对LTD归纳进行评估的协议，这将更准确地用于检查LTD与电机学习之间的因果关系。总之，在评价基因操纵小鼠的LTD时，实验条件至关重要。

本研究使用了四种方案来诱导小脑LTD。在前两个协议（协议1和2）中，在电流夹紧条件下应用了PF刺激和CF刺激的结合。在其他两个协议（协议3和4）中，体体去极化被取代在电压夹条件下的CF刺激。比较了结合刺激过程中的电压轨迹或电流轨迹（图2）。
在电流夹合条件下（协议-1）中，1个PF刺激和1个CF刺激的结合通常用于切片制备26，27。</s...

Watch this Scientific Journal Video about Assessment of Long-term Depression Induction in Adult Cerebellar Slices at JoVE.com

Assessment of Long-term Depression Induction in Adult Cerebellar Slices

在一些基因操纵的动物中，使用单一协议可能无法诱导小脑Purkinje细胞中的LTD，并且LTD和运动学习之间可能存在差异。在基因操纵的动物中，需要多种协议来评估LTD诱导。显示了标准协议。

成人脑切片长期抑郁症诱导评估

Synaptic plasticity provides a mechanism for learning and memory. For cerebellar motor learning, long-term depression (LTD) of synaptic transmissions from ...

当一个特定的蛋白质功能被基因操纵改变时，刺激性失效和运动运行损伤的协调被认为是LTS和运动学习之间因果关系的必要条件。在这里，我们演示了使用多种协议来评估DLT，这可以通过基因操纵动物的补偿机制诱导。在收获大脑之前，在冰上冷却和氧化两个50毫升的ACSF烧杯。当溶液温度达到4摄氏度以下时，在一个冰冷烧杯中加入50微升的一毫摩尔四毒素。要收获大脑，请握住头部，用眼科剪刀沿着中线切割肤浅的皮肤。手动缩回皮肤以广泛暴露头骨表面，并使用剪刀将头骨水平地从主要的脊柱孔切割到眼睛和耳朵的上方，然后沿着两只眼睛上方的一条线切割头骨。使用手术刀切割大脑中间的大脑，从头骨中分离出大脑的骨细胞部分，包括小脑。将样品浸入ACSF的冰冷烧杯中，并调整气泡管，使它不会搅动烧杯中的大脑块。至少7分钟后，用铲子拾起脑块，用一张滤纸吸收多余的ACSF。用适当的医用粘合剂将组织腹侧向下安装到两厘米或两厘米的阿加片上。使用刀片尽可能切出脑组织的右半球，以与Purkinje细胞的树突状平面平行。切切和移除半球的另一边，在上优和劣等的科利库利之间切割大脑。切断脊髓，将修剪过的小脑右侧与阿加块粘在预冷的试样托盘上。然后，倾斜试样托盘，将 ACSF 倒入样品上以固定组织并洗去多余的胶水。要切入大脑样本，请调整标本，使小脑的侧侧位于前部，并倒入足够的冰冷切片ACSF，辅以四分多毒素，以完全浸入小脑。将气管放入切割溶液中，然后使用氧气-二氧化碳气体混合物开始冒泡。使用精细的钳子和放大镜，取出气管母体，切除小脑。去除脑干和阿加块后，旋转托盘 180 度，使小脑的后面朝向振动器的剃须刀刀片，并调整第一个切割位置。将振动器振幅设置为 5.5，频率设置为 85 赫兹，速度设置为 3 到 4，切片厚度设置为 300 微米。获得小脑切片后，使用尼龙网将部分转移到26摄氏度水浴中的丙烯酸培养箱中，将样品完全浸入新鲜含氧ACSF中至少一小时。对于全细胞贴片夹报告，通过超定溶解ACSF中的一微摩尔皮罗毒素三分钟，然后以每分钟2毫升的流量将30摄氏度的记录室与毒素溶液一起使用。几分钟后，将小脑切片转移到录音室，用铂金重量和尼龙线固定组织。然后用新鲜的ACSF填充刺激电极。为了刺激平行纤维，将刺激电极放在从Purkinje细胞层约50微米的分子层表面。为了刺激攀爬纤维，将刺激电极放在Purkinje细胞层的底部，并使用微加载器将8微升0.45微升过滤钾或基于镉的内部溶液填充。在将电极浸入ACSF之前，对记录电极施加微弱的正压。电极电阻应为2至4兆欧姆，应修正液体交汇电位。使用记录电极接近Purkinje细胞的健康明亮的细胞体，并稍微推动Purkinje细胞的表面。然后停止施加正压并施加负压，直到形成千兆孔密封。然后使用负压建立整个单元配置，将膜电位保持在负70毫伏，并在0.1赫兹下应用负2毫伏100毫秒脉冲，连续监测输入电阻、系列电阻和输入电容。对于长期凹陷诱导，用0.1毫秒脉冲刺激分子层，并应用双脉冲刺激来识别平行纤维兴奋性后突触电流。应观察到配对脉冲促进和相对于刺激强度增加的振幅逐渐增加。要记录测试响应，请应用单个 0.1 赫兹脉冲并调整刺激的强度，使唤起的振幅在 200 皮安附近。刺激Purkinje细胞层底部的攀爬纤维，并确定攀爬纤维激活引出的平行纤维兴奋后电流，应用双脉冲刺激。应以全或无方式观察配对脉冲抑郁，与刺激强度的增加相关。在这个具有代表性的实验中，在目前夹紧条件下，使用一个平行纤维刺激和一个爬升纤维刺激的结合在切片制备中。结膜刺激引出的复杂尖峰的形状与攀爬纤维刺激所引出的复杂尖峰的形状相似，第一个陡峭的尖峰后跟两到三个尖峰。当一个平行纤维刺激在50毫秒后由连结二次平行和爬升纤维刺激跟随时，观察到一个类似形状的复杂尖峰。在使用基于氦的内部溶液的电压夹条件下进行的此测试中，在 50 毫秒后，同时应用第二次并联光纤刺激和体电去极化，随后进行了平行光纤刺激。体向极化从负70到零毫伏时引出一个向内电流，尾电流在极化后也引起。最后，在电压夹条件下，在100赫兹的5个平行纤维刺激与体电去极化同时给予。同样，在去极化过程中又产生反复的内流，尾电流在极化后被引出。为了评估小脑DLT与基因操纵动物的运动学习之间的关系，应采用多种方案在舒适的生理条件下诱导LTD。如果小脑在运动学习后在基因操纵的动物中可视化，它们之间的因果关系可以更直接地被检查。

Research

JoVE Journal

Neuroscience

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

A Molecular Readout of Long-term Olfactory Adaptation in C. elegans

长期的嗅觉适应性的分子读数<em&gt; C.线虫</em

During sustained stimulation most sensory neurons will adapt their response by decreasing their sensitivity to the signal. The adaptation response helps ...

科研

神经科学

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

长时程增强一个非常稳定的和可重复的记录和保存海马鼠标片制备工艺的改进

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

Long-term Behavioral Tracking of Freely Swimming Weakly Electric Fish

自由游泳弱电鱼的长期行为跟踪

Long-term behavioral tracking can capture and quantify natural animal behaviors, including those occurring infrequently. Behaviors such as exploration and ...

Simultaneous Long-term Recordings at Two Neuronal Processing Stages in Behaving Honeybees

在两个神经元处理阶段在做人蜜蜂同时长期录音

In both mammals and insects neuronal information is processed in different higher and lower order brain centers. These centers are coupled via convergent ...

Long-term Time Lapse Imaging of Mouse Cochlear Explants

老鼠耳蜗外植体的长期时间推移成像

Here we present a method for long-term time-lapse imaging of live embryonic mouse cochlear explants. The developmental program responsible for building ...

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

Agarose Microchambers for Long-term Calcium Imaging of Caenorhabditis elegans

琼脂糖微室的长期钙成像<em&gt;秀丽隐杆线虫</em

Behavior is controlled by the nervous system. Calcium imaging is a straightforward method in the transparent nematode Caenorhabditis elegans to measure ...

Preparation of Horizontal Slices of Adult Mouse Retina for Electrophysiological Studies

成年小鼠视网膜电生理研究水平切片的制备

Vertical slice preparations are well established to study circuitry and signal transmission in the adult mammalian retina. The plane of sectioning in ...

Generation and Long-term Maintenance of Nerve-free Hydra

Generation and Long-term Maintenance of Nerve-free Hydra

无神经的一代和长期维持<em&gt;水润</em

The interstitial cell lineage of Hydra includes multipotent stem cells, and their derivatives: gland cells, nematocytes, germ cells, and nerve cells. The ...

Modified Roller Tube Method for Precisely Localized and Repetitive Intermittent Imaging During Long-term Culture of Brain Slices in an Enclosed System

封闭系统中脑切片长期培养中精确定位和重复间歇成像的改进辊筒法

Cultured rodent brain slices are useful for studying the cellular and molecular behavior of neurons and glia in an environment that maintains many of ...

Long-term Sensory Conflict in Freely Behaving Mice

自由生育小鼠的长期感觉冲突

Long-term sensory conflict protocols are a valuable means of studying motor learning. The presented protocol produces a persistent sensory conflict for ...