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>将这股28µL 混合成大约5毫升的水化薯片食品, 并将这种混合物添加到传统的果蝇培养基的顶部。</li>
	<li>在含有0.2 毫米全反式视网膜的食物中, 培养成年苍蝇, 表达 csChrimson 1–3天11。</li>
</ol>2. 准备 TTX 库存
<ol><li>在蒸馏或去离子水中创建1毫米 TTX 的库存溶液。将此解决方案存储在实验室冰箱中几个月。检查机构关于 TTX 储存的政策, 因为许多机构将 TTX 归类为危险的或受控制的物质。</li>
</ol>3. 准备飞行电生理学
<ol><li>收集1–3天的老成年苍蝇, 是在食物上提出的全跨视网膜。 注: 请参阅 Murthy 和特纳在2013年详细描述补丁夹紧果蝇神经元12,13。</li>
	<li>把苍蝇放在玻璃闪烁瓶里。把瓶子放在冰上1分钟以固定苍蝇。</li>
	<li>使用粗钳将苍蝇插入一个特制的录音室。房间由3½ "圆激光切开从 1/16" 丙烯酸。切割¾ "孔进入中心, 其中一个自定义箔附有2部分环氧树脂。箔包含三角形形状的希望 , 苍蝇入。</li>
	<li>在动物周围涂抹蜡或 UV 固化环氧树脂, 在记录室内牢固地保护它。</li>
	<li>用鹅颈光纤在显微镜下进行可视化照明。将鹅颈纤维设置为斜光照, 调整它们以便直接从侧面照亮苍蝇。</li>
	<li>将光照水平调整为尽可能低的设置, 仍然允许清晰地显示苍蝇。这种光强度在个别实验中会有所不同。 注意: 此协议可通过使用原位脑外植体方法14来进行修改。</li>
	<li>检索外部记录生理盐水。生理盐水含有毫米: 103 氯化钠, 3 氯化钾, 5 n-三 (羟甲基) 甲基-2-aminoethane 磺酸, 8 海藻糖, 10 葡萄糖, 26 NaHCO3, 1 NaH2PO4, 1.5 CaCl2, 4 氯化镁2 (调整为 270–275 mΩ)。盐水冒泡与 95% O2/5% CO2 (卡波金) 和有 pH 值调整到 7.315。</li>
	<li>将4-6 滴胞外记录生理盐水放在苍蝇的制剂上。在这一点上, 增加光水平在解剖显微镜, 以更好的能见度。</li>
	<li>用电解法锐化钨丝。要制作电线, 请准备一个3度的饱和溶液, 并在20伏的交流电流的同时, 将导线的尖端浸在溶液中20倍于100毫秒, 直到尖端被削尖。使用削尖的钨线去除头上的角质层, 从而暴露大脑。</li>
	<li>通过去除周围的气管和脂肪体进一步揭露大脑。如果访问触角裂片, 使用弯曲的钨线或类似的工具, 轻轻地把触角放在箔录音室下面, 以增加能见度。使用锋利的镊子轻轻地撕掉覆盖的范围内, 将作出录音的胶质鞘。</li>
	<li>将录音室传送到电生理学钻机。立即开始灌注与外部记录盐水冒泡与卡波金保存大脑。盐水溶液储层放置在显微镜阶段以上, 并被重力喂入制备。使用真空线和2升锥形瓶收集盐水。</li>
</ol>4. 获得全细胞记录
<ol><li>把补丁吸管在一个商业吸管拉拔器上。参考手册的设置, 以达到适当的吸管与尖端电阻近 7 mΩ。</li>
	<li>将4µL 的内部记录解决方案添加到补丁吸管中。内部生理盐水包括140钾天门冬氨酸, 10 HEPES, 4 MgATP, 0.5 Na3GTP, 1 EGTA 和1氯化钾在毫米。</li>
	<li>为全细胞记录设置膜片钳放大器。将放大器的偏移量设为零, 并设置放大器以提供测试脉冲。在这里, 一个 AM 系统补丁钳放大器2400使用。</li>
	<li>使用正压通过吸管和接近细胞。使用机器人引导吸管并与细胞接触。释放正压。将膜的保持电位设置为-60 mV。吸管应形成一个 gigaohm 密封与细胞的膜, 证明了低次 picoamp 保持电流。 注: 如果使用 GFP 引导细胞靶向, 尽量减少使用 epifluorescent 光, 以防止 ChR2 激活和潜在的突触抑郁症。在应用步骤5之前, 还要等待几分钟。</li>
	<li>设置放大器提供测试脉冲从-50 mv 到-60 mv。此设置是膜片钳放大器的标准。测试脉冲将显示一个大的 capacitative 瞬态, 代表当前需要充电的补丁吸管。通过调整 AM 2400 或类似放大器上的容量补偿旋钮, 消除 capacitative 瞬态。</li>
	<li>应用负压短脉冲, 破裂细胞膜, 获得全细胞构型。 注意: 当 capacitative 瞬时显示神经元的电容时, 会观察到整个细胞的构型。保持电流 (基线) 的小幅增加也可能会被观察到。将放大器设置为当前的钳位模式, 并将电池的电位调整到-50 到-60 mV。</li>
</ol>5. 测试突触连通性
<ol><li>配置数据采集 (数据采集) 系统以触发 LED 驱动程序以生成光脉冲。在这里, 一个国家仪器数据采集系统使用 Matlab, 提供一个模拟信号的 LED 驱动器。LED 强度通过模拟信号调整到驱动程序。led 是一个 620-630 nm 波长 led。</li>
	<li>将高功率红色 LED 直接放置在准备下方。调整亮度, 使0.238 兆瓦/毫米2达到飞行。</li>
	<li>激活突触前神经元, 同时记录从突触后细胞的兴趣。实现细胞活化 optogenetically, pharmacogentically, 或通过神经刺激。在这里, csChrimson 和短暂的红光脉冲被应用。 注: 突触连接可以被监视在当前夹子作为 EPSPs 或电压钳作为 EPSCs。在应用 TTX 响应40毫秒光脉冲之前, 突触连接应该是可见的。如果没有观察到连接, 那么神经元就不太可能 synaptically 连接单声道或 polysynaptically。保持电位可以调整, 以强调兴奋或抑制连接相应。</li>
	<li>如果在生理盐水中观察到连接, 则在灌注系统中加入 TTX, 以达到最终浓度为1µM. 使用循环泵来捕获和重复使用盐水来保存 TTX。蠕动泵将从浴缸中提取盐水溶液, 并将其返回到盐水水库。</li>
	<li>调整泵的速度, 提供一个强大的恒定真空, 不允许盐水平在浴缸上升和下降。 注意: TTX 的应用应导致神经元中的峰值活动停止, 在整个细胞中被监测。这证实了足够的 TTX 已经添加到盐水。</li>
	<li>再次应用红光 (每脉冲40毫秒) 的短脉冲。在这一点上, 所有的突触连接可能 monosynaptic。添加药理拮抗剂到循环盐水中, 以测试残留在 TTX 的突触连接的化学性质。 注: 当将光遗传学和荧光靶向神经元结合时, 在尝试获取记录时, 不要持续激活神经元的突触前, 这可能是很重要的。这可能导致突触抑郁症, 并使它很难看到连接。由于 csChrimson 有一个长的励磁带, 广泛延伸到绿色荧光蛋白的范围, 一个红色的荧光可以用来可视化神经元和 channel2rhodopsin (Ch2R) 可以用来刺激细胞的数量。这样做需要特定的遗传学和自定义筛选器多维数据集。为了生成合适的苍蝇, 在莱克萨或 Q 系统二进制表达系统下, 制作出表示红色荧光 (RFP 或 mCherry) 的苍蝇。这些将需要与苍蝇表达 Gal4 启动子和 UAS-Ch2R 和无人 NaChBac 效应基因。荧光的最佳滤波立方体将有一个狭窄的励磁范围来激发红色荧光, 但不会激发 Ch2R. 此外, 该过滤器集应包括一个长通量发射过滤器, 以收集尽可能多的发射光。我们使用了自定义过滤器集的色度, 包括部件号 et580/25x 和 t600lpxr (从49306集), 但与 et610lp 屏障/发射过滤器。</li>
</ol>

<ol>
	<li>Petreanu, L., Huber, D., Sobczyk, A., 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=Channelrhodopsin-2-assisted+circuit+mapping+of+long-range+callosal+projections.">Channelrhodopsin-2-assisted circuit mapping of long-range callosal projections.</a> Nat Neurosci. 10, 663-668 (2007).</li><li>Yao, Z., Macara, A. M., Lelito, K. R., Minosyan, T. Y., Shafer, O. T. <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+functional+neuronal+connectivity+in+the+Drosophila+brain.">Analysis of functional neuronal connectivity in the Drosophila brain.</a> J Neurophysiol. , 684-696 (2012).</li><li>Doyle, M. W., Andresen, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reliability+of+monosynaptic+sensory+transmission+in+brain+stem+neurons+in+vitro.">Reliability of monosynaptic sensory transmission in brain stem neurons in vitro.</a> J Neurophysiol. 85, 2213-2223 (2001).</li><li>Nicholls, J. G., Purves, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monosynaptic+chemical+and+electrical+connexions+between+sensory+and+motor+cells+in+the+central+nervous+system+of+the+leech.">Monosynaptic chemical and electrical connexions between sensory and motor cells in the central nervous system of the leech.</a> J Physiol-London. 209, 647-667 (1970).</li><li>Byrne, J. H., Castellucci, V. F., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+individual+mechanoreceptor+sensory+neurons+to+defensive+gill-withdrawal+reflex+in+Aplysia.">Contribution of individual mechanoreceptor sensory neurons to defensive gill-withdrawal reflex in Aplysia.</a> J Neurophysiol. 41, 418-431 (1978).</li><li>Liao, X., Walters, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+elevated+divalent+cation+solutions+to+isolate+monosynaptic+components+of+sensorimotor+connections+in+Aplysia.">The use of elevated divalent cation solutions to isolate monosynaptic components of sensorimotor connections in Aplysia.</a> J Neurosci Meth. 120, 45-54 (2002).</li><li>Feinberg, E. H., 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=GFP+Reconstitution+Across+Synaptic+Partners+(GRASP)+Defines+Cell+Contacts+and+Synapses+in+Living+Nervous+Systems.">GFP Reconstitution Across Synaptic Partners (GRASP) Defines Cell Contacts and Synapses in Living Nervous Systems.</a> Neuron. 57, 353-363 (2008).</li><li>Macpherson, L. 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=Dynamic+labelling+of+neural+connections+in+multiple+colours+by+trans-synaptic+fluorescence+complementation.">Dynamic labelling of neural connections in multiple colours by trans-synaptic fluorescence complementation.</a> Nat Commun. 6, 10024-10029 (2015).</li><li>Zhang, X., Gaudry, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+integration+of+a+serotonergic+neuron+in+the+Drosophila+antennal+lobe.">Functional integration of a serotonergic neuron in the Drosophila antennal lobe.</a> Elife. 5, (2016).</li><li>Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+olfactory+processing+in+Drosophila:+mechanisms+and+principles.">Early olfactory processing in Drosophila: mechanisms and principles.</a> Annu Rev Neurosci. 36, 217-241 (2013).</li><li>Klapoetke, N. C., 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=Independent+optical+excitation+of+distinct+neural+populations.">Independent optical excitation of distinct neural populations.</a> Nat Methods. 11, 338-346 (2014).</li><li>Murthy, M., Turner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-cell+in+vivo+patch-clamp+recordings+in+the+Drosophila+brain.">Whole-cell in vivo patch-clamp recordings in the Drosophila brain.</a> Cold Spring Harb Protoc. , 140-148 (2013).</li><li>Murthy, M., Turner, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+the+head+cuticle+and+sheath+of+living+flies+for+whole-cell+patch-clamp+recordings+in+the+brain.">Dissection of the head cuticle and sheath of living flies for whole-cell patch-clamp recordings in the brain.</a> Cold Spring Harb Protoc. 2013, 134-139 (2013).</li><li>Gu, H., O'Dowd, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+Cell+Recordings+from+Brain+of+Adult+Drosophila.">Whole Cell Recordings from Brain of Adult Drosophila.</a> J Vis Exp. , (2007).</li><li>Wilson, R. I., Turner, G. C., Laurent, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+olfactory+representations+in+the+Drosophila+antennal+lobe.">Transformation of olfactory representations in the Drosophila antennal lobe.</a> Science. 303, 366-370 (2004).</li><li>Kazama, H., Wilson, R. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homeostatic+Matching+and+Nonlinear+Amplification+at+Identified+Central+Synapses.">Homeostatic Matching and Nonlinear Amplification at Identified Central Synapses.</a> Neuron. 58, 401-413 (2008).</li><li>Becnel, 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=DREADDs+in+Drosophila:+a+pharmacogenetic+approach+for+controlling+behavior,+neuronal+signaling,+and+physiology+in+the+fly.">DREADDs in Drosophila: a pharmacogenetic approach for controlling behavior, neuronal signaling, and physiology in the fly.</a> Cell Reports. 4, 1049-1059 (2013).</li><li>Armbruster, B. N., Li, X., Pausch, M. H., Herlitze, S., Roth, 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=Evolving+the+lock+to+fit+the+key+to+create+a+family+of+G+protein-coupled+receptors+potently+activated+by+an+inert+ligand.">Evolving the lock to fit the key to create a family of G protein-coupled receptors potently activated by an inert ligand.</a> P Natl Acad Sci USA. 104, 5163-5168 (2007).</li><li>Sun, Q. Q., Wang, X., Yang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laserspritzer:+A+Simple+Method+for+Optogenetic+Investigation+with+Subcellular+Resolutions.">Laserspritzer: A Simple Method for Optogenetic Investigation with Subcellular Resolutions.</a> PLoS One. 9, e101600-e101608 (2014).</li><li>Holloway, B. 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=Monosynaptic+Glutamatergic+Activation+of+Locus+Coeruleus+and+Other+Lower+Brainstem+Noradrenergic+Neurons+by+the+C1+Cells+in+Mice.">Monosynaptic Glutamatergic Activation of Locus Coeruleus and Other Lower Brainstem Noradrenergic Neurons by the C1 Cells in Mice.</a> J Neurosci. 33, 18792-18805 (2013).</li><li>Petreanu, L., Mao, T., Sternson, S. M., 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=The+subcellular+organization+of+neocortical+excitatory+connections.">The subcellular organization of neocortical excitatory connections.</a> Nature. 457, 1142-1145 (2009).</li><li>Kloppenburg, P., Ferns, D., Mercer, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotonin+enhances+central+olfactory+neuron+responses+to+female+sex+pheromone+in+the+male+sphinx+moth+manduca+sexta.">Serotonin enhances central olfactory neuron responses to female sex pheromone in the male sphinx moth manduca sexta.</a> J Neurosci. 19, 8172-8181 (1999).</li><li>Suster, M. L., Seugnet, L., Bate, M., Sokolowski, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refining+GAL4-driven+transgene+expression+in+Drosophila+with+a+GAL80+enhancer-trap.">Refining GAL4-driven transgene expression in Drosophila with a GAL80 enhancer-trap.</a> Genesis. 39, 240-245 (2004).</li></ol>

作者没有什么可透露的。

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><tbody><tr><td>UAS-csChrimson</td><td>Bloomington Drosophila Stock Center</td><td>55135</td><td>Used as a neural activator</td></tr><tr><td>UAS-NaChBac</td><td>Bloomington Drosophila Stock Center</td><td>9466</td><td>Resotores excitibility in cells in TTX</td></tr><tr><td>Tetrodotoxin</td><td>Tochris</td><td>1078</td><td>Special permission may be needed to purchase TTX as it is a controlled substance</td></tr><tr><td>all trans-Retinal</td><td>Sigma-Aldrichall trans-Retinal</td><td>R2500</td><td>Require co-factor for channelrhodopsin</td></tr><tr><td>Weldable 321 Stainless Steel&amp;nbsp;Sheet,&amp;nbsp;0.002&quot;&amp;nbsp;Thick,&amp;nbsp;10&quot;&amp;nbsp;Wide</td><td>McMaster Carr</td><td>3254K7</td><td>Used to make custom fly holder. Custom foil can be laser cut at pololu.com from the provided PDF file</td></tr><tr><td>Dissection Microscope</td><td>Zeiss</td><td>Stemi 2000-C</td><td>Used for dissection of preparation</td></tr><tr><td>Waxer</td><td>Almore</td><td>Eectra Waxer 66000</td><td>Used during dissection to secure fly in foil</td></tr><tr><td>Paraffin Wax</td><td>Joann</td><td>4917217</td><td>Used with waxer</td></tr><tr><td>Number 5 forceps</td><td>Fine Science Tools</td><td>#5CO</td><td>Used for dissection and desheathing</td></tr><tr><td>Dissection Scissors</td><td>Fine Science Tools</td><td>15001-08</td><td>Used to remove parts of the cuticle during dissection</td></tr><tr><td>Tungsten wire</td><td>A-M Systems</td><td>797500</td><td>Use with electrolysis to make sharpened needles for dissection</td></tr><tr><td>Reciculating Peristaltic Pump</td><td>Simply Pumps (Amazon)</td><td>PM200S</td><td>for recirculating TTX</td></tr><tr><td>Speed Controller for peristaltic pump</td><td>Zitrades (Amazon)</td><td>N/A</td><td>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><td>Versa-Mount&amp;nbsp;Precision Compressed Air Regulator</td><td>McMaster Carr</td><td>1804T1</td><td>For applying positive pressure during patching</td></tr><tr><td>Glass capilaries</td><td>World Precision Instruments</td><td>TW150F-3</td><td>For patch pipettes</td></tr><tr><td>Multipurpose Gauge</td><td>McMaster Carr</td><td>3846K431</td><td>Gauge for pressure regulator</td></tr><tr><td>Electrophysiology Camera</td><td>Dage MTI&amp;nbsp;</td><td>IR-1000</td><td>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><td>IR LED</td><td>Thorlabs&amp;nbsp;</td><td>M850F2</td><td>For oblique illumination</td></tr><tr><td>fiber optic for IR LED</td><td>Thorlabs</td><td>M89L01</td><td>Couples to IR LED</td></tr><tr><td>Objective lens&amp;nbsp;</td><td>Olympus 40X</td><td>LUMPlanFLN</td><td>This can be used on most microscipes and works well for visualizing fly neurons.</td></tr><tr><td>Amber LED</td><td>Thorlabs</td><td>M590L3d</td><td>For visualizing RFP and mCherry</td></tr><tr><td>Blue LED</td><td>Thorlabs</td><td>&lt;strong&gt;M470L3&lt;/strong&gt;&lt;sup&gt;d&lt;/sup&gt;</td><td>For visualizing GFP</td></tr><tr><td>GFP filter set</td><td>Chroma</td><td>&lt;strong&gt;49011&lt;/strong&gt;</td><td>For visualizing GFP or stimulating channel2rhodopsin</td></tr><tr><td>Custom mCherry Filter Set</td><td>Chroma</td><td>&amp;nbsp;et580/25x and t600lpxr (from the 49306 set) but with an et610lp barrier/emission optic</td><td>Use only if you wish to patch identified neurons with channel2rhodopsin</td></tr><tr><td>Dichroic to combine Amber and blue LED</td><td>Thorlabs</td><td>DMLP550R</td><td>Use only patch under mCherry and excite channel2rhodopsin with blue light.</td></tr><tr><td>Red LED</td><td>LEDSupply</td><td>Cree XPE 620 - 630 nm</td><td>Used to drive csChrimson</td></tr><tr><td>LED Driver</td><td>LEDSupply</td><td>3021-D-E-1000</td><td>Used to drive LEDs for optogenetic stimulation</td></tr><tr><td>Manipulator</td><td>Sutter Instruments</td><td>MP-225</td><td>Used to position pipette during recordings</td></tr><tr><td>Patchclamp Amplifier</td><td>A-M Systems</td><td>Model 2400</td><td>An equivalent amplifier is suitable</td></tr><tr><td>Bessel Filter</td><td>Warner Instruments&amp;nbsp;</td><td>LPF 202A</td><td>Auxillary filter used to filter current trace to oscilloscope during patching.</td></tr><tr><td>Data Acquisition System&amp;nbsp;</td><td>National Instruments</td><td>NI PCIe-6321&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; 781044-01</td><td>Used to record data from amplifier to computer</td></tr><tr><td>Connector Block - BNC Terminal BNC-2090A</td><td>National Instruments</td><td>779556-01</td><td>Used to connect amplifier to DAQ card.</td></tr><tr><td>Steel Foil&amp;nbsp;</td><td>McMaster Carr</td><td>3254K7</td><td>Steel foil for custom recording chamber</td></tr><tr><td>Magnets</td><td>K&amp;amp;J Magnetics</td><td>D42</td><td>To secure recording chamber to ring stand</td></tr><tr><td>1/16&quot; Cell Cast Acrylic Clear</td><td>Pololu</td><td></td><td>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 ...

examining-monosynaptic-connections-drosophila-using-tetrodotoxin

Research

JoVE Journal

Neuroscience

Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

9.9K 次观看.  University of Maryland. 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。

视频: Examining Monosynaptic Connections in Drosophila Using Tetrodotoxin Resistant Sodium Channels

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The Drosophila larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila is well known for the ease of powerful genetic manipulations and the larval nervous system has proven particularly useful in studying not only normal function but also perturbations that accompany some neurological disease (Lloyd and Taylor, 2010). Many key synaptic molecules found in Drosophila are also found in mammals and like most CNS excitatory synapses in mammals, the Drosophila NMJ is glutamatergic and demonstrates activity-dependent remodeling (Kohet al. , 2000). Additionally, Drosophila neurons can be individually identified because their innervation patterns are stereotyped and repetitive making it possible to study identified synaptic terminals, such as those between motor neurons and the body-wall muscle fibers that they innervate (Keshishian and Kim, 2004). The existence of evolutionarily conserved synapse components along with the ease of genetic and physical manipulation make the Drosophila model ideal for investigating the mechanisms underlying synaptic function (Budnik, 1996).
The active zones at synaptic terminals are of particular interest because these are the sites of neurotransmitter release. NC82 is a monoclonal antibody that recognizes the Drosophila protein Bruchpilot (Brp), a CAST1/ERC family member that is an important component of the active zone (Waghet al. , 2006). Brp was shown to directly shape the active zone T-bar and is responsible for effectively clustering Ca2+ channels beneath the T-bar density (Fouquetet al. , 2009). Mutants of Brp have reduced Ca2+ channel density, depressed evoked vesicle release, and altered short-term plasticity (Kittelet al. , 2006). Alterations to active zones have been observed in Drosophila disease models. For example, immunofluorescence using the NC82 antibody showed that the active zone density was decreased in models of amyotrophic lateral sclerosis and Pitt-Hopkins syndrome (Ratnaparkhiet al. , 2008; Zweieret al. , 2009). Thus, evaluation of active zones, or other synaptic proteins, in Drosophila larvae models of disease may provide a valuable initial clue to the presence of a synaptic defect.
Preparing whole-mount dissected Drosophila larvae for immunofluorescence analysis of the NMJ requires some skill, but can be accomplished by most scientists with a little practice. Presented is a method that provides for multiple larvae to be dissected and immunostained in the same dissection dish, limiting environmental differences between each genotype and providing sufficient animals for confidence in reproducibility and statistical analysis.

Dissection and Imaging of Active Zones in the Drosophila Neuromuscular Junction

The neuromuscular junction (NMJ) of Drosophila melanogaster is an important model system for studying normal synaptic function as well as perturbations to synaptic function found in certain neurological diseases. We present a protocol for dissection of the Drosophila larval motor system and immunostaining for active zone proteins within the NMJ.

在活动区域​​的解剖和影像果蝇神经肌肉交界处

The Drosophila  larvae neuromuscular junction (NMJ) is an excellent model for the study of synaptic structure and function. Drosophila  is well known for ...

科研

神经科学

Electrophysiological Methods for Recording Synaptic Potentials from the NMJ of Drosophila Larvae

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. The larval neuromuscular system is a model synapse for the study of synaptic physiology and neurotransmission, and is a valuable research tool that has defined genetics and is accessible to experimental manipulation. Larvae can be dissected to expose the body wall musculature, central nervous system, and peripheral nerves. The muscles of Drosophila and their innervation pattern are well characterized and muscles are easy to access for intracellular recording. Individual muscles can be identified by their location and orientation within the 8 abdominal segments, each with 30 muscles arranged in a pattern that is repeated in segments A2 - A7. Dissected drosophila larvae are thin and individual muscles and bundles of motor neuron axons can be visualized by transillumination1. Transgenic constructs can be used to label target cells for visual identification or for manipulating gene products in specific tissues. In larvae, excitatory junction potentials (EJP&#8217;s) are generated in response to vesicular release of glutamate from the motoneurons at the synapse. In dissected larvae, the EJP can be recorded in the muscle with an intracellular electrode. Action potentials can be artificially evoked in motor neurons that have been cut posterior to the ventral ganglion, drawn into a glass pipette by gentle suction and stimulated with an electrode. These motor neurons have distinct firing thresholds when stimulated, and when they fire simultaneously, they generate a response in the muscle. Signals transmitted across the NMJ synapse can be recorded in the muscles that the motor neurons innervate. The EJP&#8217;s and minature excitatory junction potentials (mEJP&#8217;s) are seen as changes in membrane potential. Electrophysiological responses are recorded at room temperature in modified minimal hemolymph-like solution2 (HL3) that contains 5 mM Mg2+ and 1.5 mM Ca2+. Changes in the amplitude of evoked EJP&#8217;s can indicate differences in synaptic function and structure. Digitized recordings are analyzed for EJP amplitude, mEJP frequency and amplitude, and quantal content.

Here we describe electrophysiological methods for measuring synaptic transmission at the neuromuscular junction of Drosophila larva. Evoked release is initiated artificially by stimulating the motor neuron axons, and transmission through the NMJ can be measured by the postsynaptic response evoked in the muscle.

电生理方法记录从果蝇幼虫NMJ突触潜力

In this video, we describe the electrophysiological methods for recording synaptic transmission at the neuromuscular junction (NMJ) of Drosophila larva. ...

生物学

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available to evoke excitatory junctional potentials (EJPs) in this preparation involves the use of suction electrodes. In both research and teaching labs, students often have difficulty maneuvering and manipulating this type of stimulating electrode. In the present work, we show how to remotely stimulate synaptic potentials at the larval NMJ without the use of suction electrodes. By expressing channelrhodopsin2 (ChR2) 4,5,6 in Drosophila motor neurons using the GAL4-UAS system 7, and making minor changes to a basic electrophysiology rig, we were able to reliably evoke EJPs with pulses of blue light. This technique could be of particular use in neurophysiology teaching labs where student rig practice time and resources are limited.

Channelrhodopsin2 Mediated Stimulation of Synaptic Potentials at Drosophila Neuromuscular Junctions

This procedure uses a blue light-activated algal channel and cell-specific genetic expression tools to evoke synaptic potentials with light pulses at the neuromuscular junction (NMJ) in Drosophila larvae. This technique is an inexpensive and easy-to-use alternative to suction electrode stimulation for synaptic physiology studies in research and teaching laboratories.

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

The Drosophila larval neuromuscular preparation has proven to be a useful tool for studying synaptic physiology1,2,3. Currently, the only means available ...

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Harvesting and Preparing Drosophila Embryos for Electrophysiological Recording and Other Procedures

This technique exposes the Drosophila embryonic neuromusculature for immunohistochemistry or electrophysiological recording. It is useful for studying early events in neuromuscular development or performing electrophysiology in mutants that cannot hatch.

收获和准备果蝇胚胎的电生理记录和其他程序

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

在电生理记录果蝇胚胎

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an embryonic fillet preparation by a microinjector. Each motor neuron whose membrane is contacted by the droplet can then be rapidly labeled. Individual motor neurons are continuously labeled, enabling fine structural details to be clearly visualized. Given that lipophilic dyes come in various colors, the technique also provides a means to get adjacent neurons labeled in multicolor. This tracing technique is therefore useful for studying neuronal morphogenesis and synaptic connectivity in the motor neuron system of Drosophila.

Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes

We describe a method for retrograde tracing of the Drosophila embryonic motor neurons using lipophilic fluorescent dyes.

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

We describe a technique for retrograde labeling of motor neurons in Drosophila. We use an oil-dissolved lipophilic dye and deliver a small droplet to an ...

发育生物学

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal degeneration are remarkably similar in humans and invertebrates as are the molecular mechanisms that drive these processes. The fruit fly, Drosophila melanogaster, provides a powerful yet simple genetic model organism to study the cellular complexities of neurodegenerative diseases. In fact, approximately 70% of disease-associated human genes have a Drosophila homolog and a plethora of tools and assays have been described using flies to study human neurodegenerative diseases. More specifically the neuromuscular junction (NMJ) in Drosophila has proven to be an effective system to study neuromuscular diseases because of the ability to analyze the structural connections between the neuron and the muscle. Here, we report on an in vivo motor neuron injury assay in Drosophila, which reproducibly induces neurodegeneration at the NMJ by 24 h. Using this methodology, we have described a temporal sequence of cellular events resulting in motor neuron degeneration. The injury method has diverse applications and has also been utilized to identify specific genes required for neurodegeneration and to dissect transcriptional responses to neuronal injury.

A Simple Neuronal Mechanical Injury Methodology to Study Drosophila Motor Neuron Degeneration

Here we describe a simple and widely accessible method to injure segmental nerves in Drosophila larvae to visualize and quantify neurodegeneration of motor neurons at the neuromuscular junction (NMJ) of third instar larvae.

一个简单的神经元机械损伤方法学研究<em&gt;果蝇</em&gt;运动神经元变性

The degeneration of neurons occurs during normal development and in response to injury, stress, and disease. The cellular hallmarks of neuronal ...

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal macropatch recordings of synaptic currents from visualized boutons at the Drosophila larval NMJ. This technique requires customized fabrication of recording micropipettes, as well as a compound microscope equipped with a high magnification, long-distance water immersion objective, differential interference contrast (DIC) optics, and a fluorescent attachment. The recording electrode is positioned on the top of a selected synaptic bouton visualized with DIC optics, epi-fluorescence, or both. The advantage of this technique is that it allows monitoring the synaptic activity of a limited number of sites of release. The recording electrode has&#160;a diameter of several microns, and the release sites positioned outside of the electrode rim do&#160;not significantly affect the recorded currents. The recorded synaptic currents have fast kinetics and can be readily resolved. These advantages are especially important for the studies of mutant fly lines with enhanced spontaneous or asynchronous synaptic activity.

Focal Macropatch Recordings of Synaptic Currents from the Drosophila Larval Neuromuscular Junction

Synaptic currents can be recorded focally from visualized synaptic boutons at the Drosophila third instar larvae neuromuscular junction. This technique enables monitoring the activity of a single synaptic bouton.

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

Drosophila neuromuscular junction (NMJ) is an excellent model system to study glutamatergic synaptic transmission. We describe the technique of focal ...

观察 果蝇 神经肌肉接头和肌肉细胞中的微管网络

Using Drosophila Larval Neuromuscular Junction and Muscle Cells to Visualize Microtubule Network

The microtubule network is an essential component of the nervous system. Mutations in many microtubules regulatory proteins are associated with neurodevelopmental disorders and neurological diseases, such as microtubule-associated protein Tau to neurodegenerative diseases, microtubule severing protein Spastin and Katanin 60 cause hereditary spastic paraplegia and neurodevelopmental abnormalities, respectively. Detection of microtubule networks in neurons is advantageous for elucidating the pathogenesis of neurological disorders. However, the small size of neurons and the dense arrangement of axonal microtubule bundles make visualizing the microtubule networks challenging. In this study, we describe a method for dissection of the larval neuromuscular junction and muscle cells, as well as immunostaining of &#945;-tubulin and microtubule-associated protein Futsch to visualize microtubule networks in Drosophila melanogaster. The neuromuscular junction permits us to observe both pre-and post-synaptic microtubules, and the large size of muscle cells in Drosophila larva allows for clear visualization of the microtubule network. Here, by mutating and overexpressing Katanin 60 in Drosophila melanogaster,&#160;and then examining the microtubule networks in the neuromuscular junction and muscle cells, we&#160;accurately reveal the regulatory role of Katanin 60 in neurodevelopment. Therefore, combined with the powerful genetic tools of Drosophila&#160;melanogaster, this protocol greatly facilitates genetic screening and microtubule dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

作者聚焦:了解果蝇神经肌肉接头中的微管网络 

Here, we present a detailed protocol to visualize the microtubule networks in neuromuscular junctions and muscle cells. Combined with the powerful genetic tools of Drosophila melanogaster, this protocol greatly facilitates genetic screening and microtubule-dynamics analysis for the role of microtubule network regulatory proteins in the nervous system.

使用河豚毒素耐药性钠通道检查果蝇中的 Monosynaptic 连接

摘要

探索更多视频

此视频中的章节

在活动区域的解剖和影像果蝇神经肌肉交界处

电生理方法记录从果蝇幼虫NMJ突触潜力

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

收获和准备果蝇胚胎的电生理记录和其他程序

在电生理记录果蝇胚胎

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

一个简单的神经元机械损伤方法学研究

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用河豚毒素耐药性钠通道检查果蝇中的 Monosynaptic 连接

摘要

探索更多视频

此视频中的章节

在活动区域​​的解剖和影像果蝇神经肌肉交界处

电生理方法记录从果蝇幼虫NMJ突触潜力

Channelrhodopsin2介导的突触电位刺激果蝇神经肌肉接头

收获和准备果蝇胚胎的电生理记录和其他程序

在电生理记录果蝇胚胎

使用亲脂荧光染料的果蝇胚胎运动神经元的逆行追踪

一个简单的神经元机械损伤方法学研究

从果蝇幼虫神经肌肉交界处的突触电流的焦 Macropatch 记录

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用 果蝇 幼虫神经肌肉接头和肌肉细胞可视化微管网络

在活动区域的解剖和影像果蝇神经肌肉交界处

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络

使用果蝇幼虫神经肌肉接头和肌肉细胞可视化微管网络