Name: in vivo optical calcium imaging of learning-induced synaptic plasticity in drosophila melanogaster
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Description: Watch this Scientific Journal Video about In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster at JoVE.com

这项工作得到了德国研究委员会的支持，通过合作研究中心SFB 889"感官处理机制"和2705年"脑回路解剖：结构，可塑性和行为功能" 果蝇蘑菇体"。

1. 转基因果蝇，果蝇黑色素
<ol><li>交叉雌性处女和雄蝇（在12小时光/暗周期中，在60%的相对湿度下提高25°C）分别携带所需的Gal4和UAS构造25，以产生苍蝇，其中感兴趣的特定神经元表示基因编码钙指示器。</li>
	<li>年龄的雌性后代的上述十字架，直到他们在3-6天后，环状。雌性苍蝇更可取，因为它们的体型稍大。</li>
</ol>2. 果蝇在体内培养钙成像的制剂...

<ol>
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神经回路的解剖是神经科学领域的一个突出目标。果蝇的遗传可及性以及行为测试的广度和易用性使其成为研究此类现象的理想工具。在这里，提出了一种方法，可以在单个苍蝇中可视化由于嗅觉调节而在亚细胞水平上发生的调制。通过进行预训练和训练后对气味引起的钙动力学的可视化（由我们第17组首先建立），可以在天真和学到的气味反应之间画出直接的、动物内?...

通过学习在大脑中获取信息的位置和方式，然后存储为记忆，是神经科学中最具挑战性的任务之一。神经科学研究已经导致突触传播的概念改变，作为神经元基质的基础，学习和记忆形成2，3。据推测，在学习期间，在刺激感知期间活跃的神经元集合之间的突触连接会被修改，从而在记忆记忆记忆期间可以检索其组合活动模式，从而指导未来的行为行动4。这些"基因细胞"及其突触通常分布在大脑区域和处理水平，这使得很难将突触传播的观察到的变化分配给学习任务或刺激。要本地化和可视化那些与特定学习任务有因果关系的突触变化，需要一个适当的模型系统，以便精确限制这些突触。
对于这样的努力，果蝇黑色素是特别适合的，因为它结合了相对的大脑简单性，行为丰富，和实验可访问性。在成熟的模型生物中，果蝇位于线虫和基因可处理的哺乳动物（如小鼠）之间的神经元复杂性。在C.elegans中观察到神经元的立体数量（+300）和有限的行为表谱。另一方面，哺乳动物拥有数百万个神经元和惊人的行为复杂性。果蝇的大脑有10万欧元，神经元比大多数脊椎动物的大脑小得多，许多神经元是单独可识别的5。然而，果蝇表现出广泛的复杂行为，包括表现出强大的关联嗅觉学习和记忆形成的能力，第一次描述超过40年前6。在这个经典的调理过程中，成群的苍蝇受到一种气味作为条件刺激（CS+），而他们接受惩罚电击作为无条件的刺激（美国）。然后，第二种气味 （ CS-） 被呈现，没有任何惩罚.因此，动物学会避免与惩罚相关的气味，这可以在两种气味，CS+和CS-之间的后续选择情况进行测试。在果蝇中解剖这种行为背后的神经元基质的工作已经确定蘑菇体（MB）是"engram"7、8、9、10的主要部位。因此，这个大脑区域的电路是，也是密集研究的主题，以揭示记忆密码被获取和存储的逻辑（最近回顾在11，12）。
果蝇MB由每半球2，000个内在神经元（Kenyon细胞）组成，以平行的斧子投影13组织。嗅觉投影神经元的斧子扩展到侧原体和MB calyces，MB的主要树突输入位点，并从天线叶接收嗅觉输入。长，平行的斧子束的肯扬细胞构成脚轮和叶。大多数Kenyon细胞通过将一个抵押品伸向大脑的中线，形成水平β/β-叶，并通过向背向前方向延伸第二个抵押品，形成水平β/β-叶。另一组肯扬细胞形成MB的水平β-叶13，其中学习过程和随后的短期记忆形成可以本地化10。MB 叶接收一个正位输入并提供有效输出，这两个输出通常都限制在肯扬细胞斧14、15、16上不同的隔间子区域。特别是，一个多巴胺能MB输入神经元已被证明调解基于价值，例如，惩罚性，增强作用在关联嗅觉学习15，17。来自蘑菇体叶的立体和单独可识别的可识别的 MB 输出神经元集成了大量 Kenyon 细胞中的信息，针对不同的大脑区域，并承担行为指导性或厌恶性信息15.这种神经元结构导致了关联形图的组织概念。气味由稀疏激活的Kenyon细胞集合相对精确编码。这些Kenyon细胞组合的重合活性和多巴胺的释放 - 通过惩罚刺激引起的 - 调节从Kenyon细胞前突子到MB输出神经元的传输，使动物随后避免这种特殊的气味10 ， 12.我们使用这个相当精确定义和本地化的语法作为范例案例来说明如何确定和监测突触活动中的这些与学习相关的变化。
果蝇作为模型系统的价值强烈依赖于无与伦比的基因工具箱，它允许人们表达转基因，以识别，监测和控制复杂电路18中的单个神经元。神经元活动监测技术的出现（如钙成像，这里讨论）使得能够确定神经元活动模式以响应特定的刺激。通过将基因编码的钙指标（GECIs）的特定Gal4驱动表达与嗅觉刺激相结合，可以可视化感兴趣的神经元的异味引起的钙动力学19。在此协议中，通过进一步将此技术与经典调理范式耦合，可以在学习环境中检查这些嗅觉响应。学习诱导的可塑性可以使用 GECIs 进一步解剖，这些 GECIs 不仅被本地化为单个特定神经元，而且还可分解到神经元的特定子隔间。Pech等人20建立了一系列工具，正是允许这一点。通过将GCaMP321定位到前或后发孔-通过与脊椎动物突触素或d荷马，分别20个链接- 这些位点的差分调制可以区分。在此上下文中，这种本地化比大多数在细胞醇中普遍存在的 GECA 具有优势，例如，GCaMP 22、GCaMP321或 GCaMP623 ，因为这意味着前和后瞬变可以区别于神经元激活后发生的整体综合钙流入。这可以提供有关由于或导致学习和记忆形成而发生的可塑性的位置和类型的线索。例如，此处提供的协议显示了此工具在嗅觉关联学习期间破译 MB 输出神经元调制的价值，将钙传感器的表达定向到仅对贴子的表达。通过监测，在单个苍蝇内，嗅觉调理前后的异味活动可以直接比较天真的气味反应和所学的气味反应。虽然固定在同一成像室，苍蝇暴露于多种气味。然后，他们收到一个厌恶的关联调理协议，其中一种气味与电击（成为CS+）配对，而另一种气味在没有强化的情况下呈现（成为CS-）。最后，苍蝇再次暴露在与第一步相同的气味中。使用双光子显微镜观察钙动力学。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>1-Octen-3-ol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>O5284</td>
			<td>Chemical used as odorant</td>
		</tr>
		<tr>
			<td>3-Octanol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>218405</td>
			<td>Chemical used as odorant</td>
		</tr>
		<tr>
			<td>4-Methylcyclohexanol</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>153095</td>
			<td>Chemical used as odorant</td>
		</tr>
		<tr>
			<td>Bandpass filter for EGFP (525/50 nm)</td>
			<td>Carl Zeiss Microscopy GmbH, Jena, Germany</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Clear adhesive tape</td>
			<td>Tesa SE, Norderstedt, Germany</td>
			<td></td>
			<td>Standard claer adhesive tape</td>
		</tr>
		<tr>
			<td>Concave-convex jaws</td>
			<td>Fine Science Tools, North Vancouver, Canada</td>
			<td>10053-09</td>
			<td>Blade Holders with concave-convex jaws</td>
		</tr>
		<tr>
			<td>Fine forceps</td>
			<td>Fine Science Tools, North Vancouver, Canada</td>
			<td>11412-11</td>
			<td>Forceps with tip 0.1 x 0.06mm</td>
		</tr>
		<tr>
			<td>Hypodermic needle</td>
			<td>Sterican - B. Braun, Melsungenk, Germany</td>
			<td>4665120</td>
			<td>1.20x40mm</td>
		</tr>
		<tr>
			<td>Insect Minutien pins</td>
			<td>Fine Science Tools, North Vancouver, Canada</td>
			<td>26002-10</td>
			<td>Diameter 0.1mm, tip 0.0125mm</td>
		</tr>
		<tr>
			<td>Kentoflow</td>
			<td>Kent Express Dental Supplies, Gillingham, UK</td>
			<td>953683</td>
			<td>Blue light-curing glue</td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Carl Roth GmbH &#38; Co. KG, Karlsruhe, Germany</td>
			<td>0656.1</td>
			<td>Standard objective slide 76 x 26 mm</td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma-Aldrich, St. Louis, MO, USA</td>
			<td>M8410</td>
			<td>Used as diluent for odorants</td>
		</tr>
		<tr>
			<td>Mode-locked Ti-Sapphire laser Chameleon Vision 2</td>
			<td>Coherent Inc., Santa Clara, CA, USA</td>
			<td></td>
			<td>Tunable infrared femtosecond laser</td>
		</tr>
		<tr>
			<td>Multiphoton Microscope LSM 7MP equipped with BiG detectors</td>
			<td>Carl Zeiss Microscopy GmbH, Jena, Germany</td>
			<td></td>
			<td>Multiphoton microscope, multiple companies provide similar devices.</td>
		</tr>
		<tr>
			<td>Plan-Apochromat 20x (NA = 1.0) water immersion objective</td>
			<td>Carl Zeiss Microscopy GmbH, Jena, Germany</td>
			<td>421452-9900-000</td>
			<td>Objective W &#34;Plan-Apochromat&#34; 20x/1.0 DIC M27 70mm</td>
		</tr>
		<tr>
			<td>Ringer&#39;s solution</td>
			<td>n.a.</td>
			<td>n.a.</td>
			<td>5mM KCl, 130mM NaCl, 2mM MgCl2, 2mM CaCl2, 5mM Hepes-NaOH, 36mM sucrose, pH = 7.4</td>
		</tr>
		<tr>
			<td>Stab knife</td>
			<td>Sharpoint, Surgical Specialties Corporation, Reading, PA, USA</td>
			<td>72-1551</td>
			<td>5.0mm Straight restricted blade depth</td>
		</tr>
		<tr>
			<td>Surgical scalpel blade</td>
			<td>Swann-Morton, Sheffield, UK</td>
			<td>0303</td>
			<td>Product No. 11</td>
		</tr>
		<tr>
			<td>Surgical scalpel handle</td>
			<td>Swann-Morton, Sheffield, UK</td>
			<td>0907</td>
			<td>Product No. 7S/S</td>
		</tr>
		<tr>
			<td>Visual Basics of Applicatons (VBA) software to receive a trigger 
			from the odor-delivery device and the electric shock 
			application device (power supply) to interact with the 
			ZEN software from Zeiss that controls the microscope.</td>
			<td>Custom-written and available upon request</td>
			<td>n.a.</td>
			<td>n.a.</td>
		</tr>
	</tbody>
</table>

in vivo optical calcium imaging of learning-induced synaptic plasticity in drosophila melanogaster

几十年来对许多模型生物体的研究导致了目前作为学习和记忆形成基础的突触可塑性的概念。突触传播中由学习引起的变化通常分布在大脑的许多神经元和处理水平上。因此，需要一些方法来可视化神经元间学习依赖性突触可塑性。果蝇果蝇黑色素体是研究学习基础神经元回路的一个特别有利的模型有机体。这里提出的协议演示了一种方式，其中可以监测形成关联嗅觉记忆的过程，即突触活动及其变化。利用果蝇中现有的广泛遗传工具，可以具体表达确定细胞群甚至单个细胞中的遗传编码钙指标。通过将苍蝇固定到位，并打开头部胶囊，可以在提供嗅觉刺激的同时，可视化这些细胞中的钙动力学。此外，我们演示了一种设置，即苍蝇可以同时受到对身体的电击。这提供了一个系统，苍蝇可以经历经典的嗅觉调节 - 其中以前天真的气味被学习与电击惩罚 - 同时，这种气味（和其他未经训练的气味）的表示是通过双光子显微镜在大脑中观察到。我们的实验室以前曾报告过合成局部钙传感器的产生，这使得人们可以将荧光钙信号限制在午睡前或午合室。双光子显微镜提供了一种在空间上解决精细结构的方法。我们通过关注神经元来整合来自蘑菇体内的信息，这是昆虫大脑的高阶中心，从而说明了这一点。总体而言，该协议提供了一个检查神经元之间的突触连接的方法，这些神经元的活动由于嗅觉学习而被调节。

图2中可以看到使用上述协议获取的图像示例。d荷马-GCaMP3在MB输出神经元中表示，其树突内压在MB+-lobe的隔间1（神经元称为MVP228，29），并使用分裂Gal4线MB112C16进行基因定位。此外，演示的还有细胞细胞的亚细胞定位和合成后局部钙指标的差异。在比较图 2a和图 2f...

Watch this Scientific Journal Video about In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster at JoVE.com

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

在这里，我们提出了一个协议，其中前和/或午睡钙可以在果蝇学习和记忆的上下文中可视化。体内钙成像使用显着局部钙传感器与经典的嗅觉调节范式相结合，因此可以确定这种类型的关联学习背后的突触可塑性。

在Vivo光学钙成像学习诱导突触可塑性在果蝇黑色素增生

Decades of research in many model organisms have led to the current concept of synaptic plasticity underlying learning and memory formation. ...

该技术有助于实时观察突触钙活性，作为细胞过程的生理参数，在果蝇黑色素素中基础学习和记忆形成。通过将神经元反应与关联训练之前和之后的气味进行比较，我们可以得出突触活动与单个果蝇记忆痕迹的形成之间的直接相关性。生产转基因果蝇，其中感兴趣的特定神经元表示一个基因编码的钙指标交叉雌性处女和雄性苍蝇携带所需的GAL4和UAS构造，并年龄雌性后代，直到三到六天后，围闭。要准备苍蝇成像，请使用细钳将冰麻醉苍蝇放入定制准备的成像室。使用解剖显微镜将胸部和腿部与腔室底部的电线接触，头部平躺。用透明胶带固定苍蝇，并使用手术刀刀片切割头部周围的胶带中的窗口，使天线被覆盖，只有胸部前部裸露。使用凹凸钳口夹住的昆虫针，用蓝光固化胶小心地环绕头部的两侧和背面。并使用蓝色发光 LED 灯来设置胶水。当胶水完全固定时，清除飞头后面的任何残留未硬胶，用一滴林格溶液盖住头部的外露角质层。使用非常细刀片的刺刀，切开穿过头部后部的角质层，从奥佩利开始，将两侧中切到眼睛，形成角质层皮瓣，使用钳子很容易撕掉。去除任何可能阻塞大脑区域的多余的角质层，并使用细钳小心地清除任何气管的后表面，注意避免脑组织本身的中断。必要时拆下并刷新林格溶液，以清除组织碎屑区域。放置一个皮下气味输送针，距离苍蝇头部约一厘米，注意没有任何东西可以阻碍气味传递到天线。然后通过皮下气味输送针将成像室连接到气味输送系统。让苍蝇10分钟从麻醉和手术中恢复过来。要可视化基于 GFP 的钙指标，请将装有红外激光和安装在振动隔离台上的多光子显微镜的激光调谐为 920 纳米的激发波长，并安装 GFP 带通滤波器。使用课程 Z 调整旋钮，扫描大脑的 Z 轴以定位感兴趣的大脑区域。使用裁剪功能将扫描重点放在感兴趣的区域上，以最大限度地减少扫描时间。旋转扫描视图，使头部前部朝下。然后将帧大小调整为 512 x 512 像素，然后选择要扫描的区域。考虑每个帧的计算扫描时间，实现至少 4 赫兹的帧速率。对于气味唤起钙瞬态可视化，启动能够连接图像采集软件和气味传递程序的预编程宏封装，并在显微镜软件中开始测量 6.25 秒，以建立 F0 基线值。在气味输送系统中，通过特定气味杯阀的打开和关闭触发的 LED 照明，提供此处指示的 2.5 秒气味刺激。接着是气味偏移结束时的 12.5 秒记录。然后以相同方式重复第二和第三气味的交付。要在此设置中执行关联调节，请使用计算机控制的气味输送系统，在 12 90 伏电击旁边显示 60 秒的调节刺激加气味。休息60秒后，仅出现60秒的调节刺激-减气味，无电击。在完成训练阶段后三分钟重复训练前气味刺激方案，测量训练后气味再次引起钙的转性。然后以适当的格式保存映像文件，以进行以后的图像分析。这里具有代表性的蘑菇体输出神经元图像，获得的证明是可以观察到的。dHomer-GCaMP3 传感器的特定分节表达（在神经元的斧弓室中未表达）显示了树突隔间中的点分信号。与表达细胞酸的苍蝇GCaMP6f相比，在表达dHomer-GCaMp3的苍蝇中可以观察到低振幅气味反应。在这里，单个苍蝇的代表性钙痕迹显示了单个制剂之间可获得的噪声水平和振幅的方差。这种技术开辟了在亚细胞水平上可视化的可能性，嗅觉表示如何调制，记忆相如何通过调节形成。这些实验对于扩大我们对蘑菇体等复杂大脑结构的理解，以及描述关联记忆如何储存在分散的神经元群体中至关重要。

Research

JoVE Journal

Neuroscience

In Vivo Optical Calcium Imaging of Learning-Induced Synaptic Plasticity in Drosophila melanogaster

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

In vivo Visualization of Synaptic Vesicles Within Drosophila Larval Segmental Axons

在果蝇幼虫分部轴突在体内的突触小泡的可视化

Elucidating the mechanisms of axonal transport has shown to be very important in determining how defects in long distance transport affect different ...

科研

神经科学

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

横向扩散和培养的神经元膜蛋白的胞吐评估使用荧光恢复和荧光损失漂白

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

In vivo Neuronal Calcium Imaging in C. elegans

In vivo Neuronal Calcium Imaging in C. elegans

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, ...

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

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

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 ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

分析突触调制<em&gt;果蝇</em&gt;曝光时间延长后，光光感受器

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Slice Patch Clamp Technique for Analyzing Learning-Induced Plasticity

分层膜片钳技术在学习诱导可塑性分析中的应用

The slice patch clamp technique is a powerful tool for investigating learning-induced neural plasticity in specific brain regions. To analyze ...

Pentylenetetrazole-Induced Kindling Mouse Model

戊四氮诱导点燃小鼠模型

Pentylenetetrazole (PTZ) is a GABA-A receptor antagonist. An intraperitoneal injection of PTZ into an animal induces an acute, severe seizure at a high ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

前体钙成像用于可视化大脑对果蝇内分泌信号的反应

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

3D Modeling of Dendritic Spines with Synaptic Plasticity

具有突触可塑性树突脊柱的三维建模

Computational modeling of diffusion and reaction of chemical species in a three-dimensional (3D) geometry is a fundamental method to understand the ...