Name: examining monosynaptic connections in drosophila using tetrodotoxin resistant sodium channels
Uploaded: 2018-02-14T14:00:00
Description: Watch this Scientific Journal Video about Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels at JoVE.com

我们要感谢约书亚歌手乔纳森申克, 以及周到的评论家对手稿的评论。我们还要感谢本怀特和哈罗德 Zakon 关于这项技术的讨论。乔纳森申克提供了图 3A的数据。这项工作得到了白厅基金会赠款和 NIH R21 QG 的支持。

1. 准备飞行并识别 GAL4 启动子线
<ol><li>选择一个 GAL4 启动子线和交叉与携带的苍蝇 NaChBac 转基因和 optogenetic 转基因的活化。 注意: CsChrimson11之所以被选中, 是因为它的电导率很高, 并且它可以轻松地激活 NaChBac 介导的动作电位。NaChBac 和无人 CsChrimson 都可从布卢明顿库存库中获得。</li>
	<li>在乙醇 (35 毫米) 中制备全反式视网膜, 并将溶液存放在冰箱中-20 摄氏度。</li>
	<li>将这?...

<ol>
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TERPS 分析称赞目前的技术用于电路裂解通过使检测的突触之间的识别神经元。具体来说, 该方法揭示了 monosynaptic 连接的广泛沉默的行动潜力与 TTX, 同时恢复兴奋性的选择人群的 TTX 不敏感的钠通道 NaChBac。突触释放是由 optogenetic 刺激诱发的, 而突触的事件被监测与全细胞记录。TERPS 区别于其他方法, 如掌握敏感的散装或体积传输在更长的距离引起, 并有能力进行药理操作。该技术相对简单, 只需要?...

神经科学的一个主要目标是映射神经元之间的连接, 以了解信息如何流经电路。已有许多方法和资源可用于测试一系列模型系统中的神经元之间的功能连接1,2。要使用电生理学生成最精确的接线图, 必须解决两个细胞之间的观察连接是直接的, monosynaptic 的, 还是间接的和 polysynaptic 的。在哺乳动物神经元中进行这种区分的金标准是测量突触前细胞中单个动作电位与第二神经元兴奋性突触电流 (EPSCs) 发生之间的潜伏期。Monosynaptic 连接应具有少量毫秒和低可变性3的短延迟。这种方法可能是复杂的无脊椎动物神经元, 因为突触可能发生在其树突远离躯体记录站点, 导致发现延迟, 由于长 electrotonic 距离突触后电导和记录电极.这种延迟可能会对 monosynaptic 的贡献产生歧义。此外, 小突触事件可能会腐烂之前到达体细胞记录网站, 并推动更强的突触前活动, 可能会招募 polysynaptic 事件。
各种技术已经开发, 以测试 monosynaptic 连接在无脊椎动物。一种方法使用高价阳离子溶液 (高 Di), 含有过量的 Mg++和 Ca++。此解决方案通过减少释放概率和增加动作电位阈值来阻止 polysynaptic 连接, 以支持检测 monosynaptic 输入4、5。但是, 确定 Mg++与 Ca++所需的精确比率并不微不足道, polysynaptic 的贡献可能会持续, 即使是适度的刺激6也是如此。另一种称为 GFP 重建跨突触伙伴 (掌握) 利用的前突和突触的膜之间的接近性发现, 以推断 monosynaptic 连接7。在这里, 绿色荧光蛋白 (GFP) 的一个组成部分, 表达在一个神经元, 和互补片段的分子表达在一个假定的突触的合作伙伴。荧光的存在表明, 两个神经元在足够接近, 允许重组 GFP 分子, 并暗示存在的突触。如果两个细胞有紧密的并列膜, 如在神经或束簇中, 抓地能力会错误地报告突触。通过将 GFP 的突触前片段与 synaptobrevin 直接绑在一起, 可以消除这种误报, 从而只允许在主动突触8 上进行重构。虽然掌握及其变体在确定果蝇中枢神经系统的功能连通性方面发挥了重要作用, 但如果前突和后发性伙伴之间的距离相对较大, 某些连接可能无法通过掌握而可见。这在评估与调节9或 GABAergic 抑制10相关的卷传输时尤其恰当。
这里演示了一种互补和新颖的技术, 用于测试果蝇CNS 中的直接 monosynaptic 连接。这种方法, 称为河豚毒素工程抵抗探测突触 (TERPS), 工作的共同表达的河豚毒素 (TTX) 耐药性钠通道, NaChBac 和 optogenetic 激活剂在突触前细胞, 而记录从假定突触的伙伴9。在 TTX 的存在下, 所有动作电位介导的活动都被抑制在那些表达 NaChBac 的细胞之外。NaChBac 通道有选择地恢复兴奋性的靶向突触前细胞, 允许光诱发突触传播。这种方法允许突触前神经元的强活化, 这样可以通过体细胞记录 postsynaptically 来解决连接问题, 同时减少了 polysynaptic 电路的招募几率。重要的是, 这种技术允许对体积传输进行研究, 并揭示了由单个神经元在整个电路中释放的发射机的传播。此外, TERPS 可以通过常规药理学来揭示连接的化学性质。TERPS 适用于任何模型系统, 允许转基因表达 TTX 不敏感的钠通道。

<table border="0" cellpadding="0" cellspacing="0" width="946">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">UAS-csChrimson</td>
			<td style="width:219px;">Bloomington Drosophila Stock Center</td>
			<td style="width:344px;">55135</td>
			<td style="width:167px;">Used as a neural activator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">UAS-NaChBac</td>
			<td>Bloomington Drosophila Stock Center</td>
			<td>9466</td>
			<td style="width:167px;">Resotores excitibility in cells in TTX</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tetrodotoxin</td>
			<td>Tochris</td>
			<td>1078</td>
			<td style="width:167px;">Special permission may be needed to purchase TTX as it is a controlled substance</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">all trans-Retinal</td>
			<td>Sigma-Aldrichall trans-Retinal</td>
			<td>R2500</td>
			<td style="width:167px;">Require co-factor for channelrhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:216px;">Weldable 321 Stainless Steel&#160;Sheet,&#160;0.002&#34;&#160;Thick,&#160;10&#34;&#160;Wide</td>
			<td>McMaster Carr</td>
			<td>3254K7</td>
			<td style="width:167px;">Used to make custom fly holder. Custom foil can be laser cut at pololu.com from the provided PDF file</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissection Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000-C</td>
			<td style="width:167px;">Used for dissection of preparation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Waxer</td>
			<td>Almore</td>
			<td>Eectra Waxer 66000</td>
			<td style="width:167px;">Used during dissection to secure fly in foil</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Paraffin Wax</td>
			<td>Joann</td>
			<td>4917217</td>
			<td style="width:167px;">Used with waxer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Number 5 forceps</td>
			<td>Fine Science Tools</td>
			<td>#5CO</td>
			<td style="width:167px;">Used for dissection and desheathing</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dissection Scissors</td>
			<td>Fine Science Tools</td>
			<td>15001-08</td>
			<td style="width:167px;">Used to remove parts of the cuticle during dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Tungsten wire</td>
			<td>A-M Systems</td>
			<td>797500</td>
			<td style="width:167px;">Use with electrolysis to make sharpened needles for dissection</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Reciculating Peristaltic Pump</td>
			<td>Simply Pumps (Amazon)</td>
			<td>PM200S</td>
			<td style="width:167px;">for recirculating TTX</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Speed Controller for peristaltic pump</td>
			<td>Zitrades (Amazon)</td>
			<td style="width:344px;">N/A</td>
			<td style="width:167px;">PWM Dimming Controller For LED Lights or Ribbon, 12 Volt 8 Amp,Adjustable Brightness Light Switch Dimmer Controller DC12V 8A 96W for Led Strip Light B</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Versa-Mount&#160;Precision Compressed Air Regulator</td>
			<td>McMaster Carr</td>
			<td style="width:344px;">1804T1</td>
			<td style="width:167px;">For applying positive pressure during patching</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Glass capilaries</td>
			<td>World Precision Instruments</td>
			<td style="width:344px;">TW150F-3</td>
			<td style="width:167px;">For patch pipettes</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Multipurpose Gauge</td>
			<td>McMaster Carr</td>
			<td>3846K431</td>
			<td style="width:167px;">Gauge for pressure regulator</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Electrophysiology Camera</td>
			<td>Dage MTI&#160;</td>
			<td>IR-1000</td>
			<td style="width:167px;">Any camera that works in the IR range (850 nm) will work. You do not want to use red illumination as this can activate csChrimson</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IR LED</td>
			<td>Thorlabs&#160;</td>
			<td>M850F2</td>
			<td style="width:167px;">For oblique illumination</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">fiber optic for IR LED</td>
			<td>Thorlabs</td>
			<td>M89L01</td>
			<td style="width:167px;">Couples to IR LED</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Objective lens&#160;</td>
			<td>Olympus 40X</td>
			<td>LUMPlanFLN</td>
			<td style="width:167px;">This can be used on most microscipes and works well for visualizing fly neurons.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Amber LED</td>
			<td>Thorlabs</td>
			<td>M590L3d</td>
			<td style="width:167px;">For visualizing RFP and mCherry</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Blue LED</td>
			<td>Thorlabs</td>
			<td>M470L3d</td>
			<td style="width:167px;">For visualizing GFP</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">GFP filter set</td>
			<td>Chroma</td>
			<td>49011</td>
			<td style="width:167px;">For visualizing GFP or stimulating channel2rhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Custom mCherry Filter Set</td>
			<td>Chroma</td>
			<td style="width:344px;">&#160;et580/25x and t600lpxr (from the 49306 set) but with an et610lp barrier/emission optic</td>
			<td style="width:167px;">Use only if you wish to patch identified neurons with channel2rhodopsin</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Dichroic to combine Amber and blue LED</td>
			<td>Thorlabs</td>
			<td style="width:344px;">DMLP550R</td>
			<td style="width:167px;">Use only patch under mCherry and excite channel2rhodopsin with blue light.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Red LED</td>
			<td>LEDSupply</td>
			<td>Cree XPE 620 - 630 nm</td>
			<td style="width:167px;">Used to drive csChrimson</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">LED Driver</td>
			<td>LEDSupply</td>
			<td>3021-D-E-1000</td>
			<td style="width:167px;">Used to drive LEDs for optogenetic stimulation</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Manipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-225</td>
			<td style="width:167px;">Used to position pipette during recordings</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Patchclamp Amplifier</td>
			<td>A-M Systems</td>
			<td>Model 2400</td>
			<td style="width:167px;">An equivalent amplifier is suitable</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Bessel Filter</td>
			<td>Warner Instruments&#160;</td>
			<td>LPF 202A</td>
			<td style="width:167px;">Auxillary filter used to filter current trace to oscilloscope during patching.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Data Acquisition System&#160;</td>
			<td>National Instruments</td>
			<td>NI PCIe-6321&#160;&#160;&#160;&#160;&#160;&#160; 781044-01</td>
			<td style="width:167px;">Used to record data from amplifier to computer</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Connector Block - BNC Terminal BNC-2090A</td>
			<td>National Instruments</td>
			<td>779556-01</td>
			<td style="width:167px;">Used to connect amplifier to DAQ card.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Steel Foil&#160;</td>
			<td>McMaster Carr</td>
			<td>3254K7</td>
			<td style="width:167px;">Steel foil for custom recording chamber</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Magnets</td>
			<td>K&#38;J Magnetics</td>
			<td>D42</td>
			<td style="width:167px;">To secure recording chamber to ring stand</td>
		</tr>
		<tr height="100">
			<td height="100" style="height:100px;">1/16&#34; Cell Cast Acrylic Clear</td>
			<td>Pololu</td>
			<td></td>
			<td style="width:167px;">Used to make custom recording chamber. Acrylic can be laser cut at pololu.com from the provided PDF file</td>
		</tr>
	</tbody>
</table>

examining monosynaptic connections in drosophila using tetrodotoxin resistant sodium channels

在这里, 一个新的技术称为 Tetrotoxin (TTX) 工程电阻探测突触 (TERPS) 是用来测试 monosynaptic 连接的目标神经元。该方法依赖于在特定突触前神经元中与河豚毒素耐药钠通道 NaChBac 的转基因活化剂的共表达。与假定后突触伙伴的连接是由 TTX 存在的全细胞记录决定的, 它阻断了没有表达 NaChBac 的神经元的电活动。这种方法可以修改, 以使用任何活化剂或钙成像作为记者的连接。TERPS 增加了可用于确定网络内连接性的不断增长的工具集。然而, TERPS 是独一无二的, 它也可靠地报告散装或体积传输和溢出传输。

TERPS 用于区分神经元之间突触连接中的单 polysynaptic 贡献。虽然弱刺激的细胞可以用来测试直接连接, 驾驶更大的突触前活动往往招募 polysynaptic 连接(图 1A)。TERPS 通过共同表达 TTX 不敏感的钠通道 NaChBac 和 optogenetic 激活器来工作, 并在 TTX 存在的情况下测试连接以消除 polysynaptic 连接 (图 1B)。TTX 有效地阻止了果蝇大脑中的?...

Watch this Scientific Journal Video about Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels at JoVE.com

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

本文的特点是通过使用河豚毒素和河豚毒素耐药钠通道, NaChBac 来检测神经元之间的 monosynaptic 连接。

使用河豚毒素耐药性钠通道检查果蝇中的 Monosynaptic 连接

Here, a new technique termed Tetrotoxin (TTX) Engineered Resistance for Probing Synapses (TERPS) is applied to test for monosynaptic connections between ...

Research

JoVE Journal

Neuroscience

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

检查本地网络处理采用多接触层电极记录

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress ...

科研

神经科学

Correlating Behavioral Responses to fMRI Signals from Human Prefrontal Cortex: Examining Cognitive Processes Using Task Analysis

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The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Visualization of Proprioceptors in Drosophila Larvae and Pupae

可视化本体感受器<em&gt;果蝇</em&gt;幼虫和蛹

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

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

多光子细胞内钠成像结合谷氨酸海马CA1锥体神经元的UV-介导的焦点解笼锁

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

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

用线性琼脂糖渠道研究<em&gt;果蝇</em&gt;幼虫爬行行为

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat ...

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

A Simple Way to Measure Alterations in Reward-seeking Behavior Using Drosophila melanogaster

一个简单的方法来衡量在改建悬赏寻求行为使用<em&gt;果蝇</em

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We ...

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

Quantitative Cell Biology of Neurodegeneration in Drosophila Through Unbiased Analysis of Fluorescently Tagged Proteins Using ImageJ

荧光标记蛋白 ImageJ 的无偏分析在果蝇变性定量细胞生物学中的应用

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal ...

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

Quantification of Macronutrients Intake in a Thermogenetic Neuronal Screen using Drosophila Larvae

使用 德罗索菲拉· 拉瓦在恒温遗传神经元屏幕上摄入的宏量营养素的量化

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating ...

Unraveling the Role of Discrete Areas of the Rat Brain in the Regulation of Ovulation through Reversible Inactivation by Tetrodotoxin Microinjections

通过特洛多毒素微注射可逆灭活，揭开鼠脑离散区在排卵调节中的作用

Many experimental approaches have been used for studying the role of the brain in the regulation of ovulation. Examples include the lesion and ...

A Standardized Pipeline for Examining Human Cerebellar Grey Matter Morphometry using Structural Magnetic Resonance Imaging

一种使用结构磁共振成像检查人小脑灰质形态测量的标准化管道

Multiple lines of research provide compelling evidence for a role of the cerebellum in a wide array of cognitive and affective functions, going far beyond ...