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>

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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, 只需要一个记录电极和一个 optogenetic 刺激光源, 这比在突触前和突触后的部分获得双全细胞记录更可行。TERPS 的一个基本要求是, 在所研究的系统中, TTX 可以很容易地阻止动作电位。但是, 由于 TTX 对各种分类学门的神经元作用, TERPS 可能广泛应用于哺乳动物和无脊椎动物模型系统。
TERPS 可以很容易地修改在一些方式, 以促进刺激的假定突触前人口或记录从突触的目标。在这里, 一个 optogenetic 工具被用来刺激目标人群, 虽然通过多种机制激活是可能的。例如, 神经刺激的感觉轴突, 是例行的研究传输从触角神经在果蝇16和适用于其他感官系统, 包括周围神经包含 mechanosensory 神经元。在果蝇中测试功能连接性的类似方法通过离子型 purinoceptor P2X2 和 ATP 应用程序 2, 在一个神经元群中表达 GCaMP 钙指示器, 同时驱动另一人群中的活动.这种方法应该与 TERPS 兼容, 简单地将 NaChBac 转基因与 P2X2 受体在一个神经元和 GCaMP 在另一个群体中, 以完全无补丁的方法。但是, 应谨慎使用导致慢 depolarizations 的方法, 例如由设计器药物 (DREADDs)17、18专门激活的基于毒蕈的设计器受体。慢退极化可能导致 NaChBac 失活在行动潜力世代之前。
哺乳动物系统也采用了类似的 TERPS 方法。这些技术结合 TTX 和 channelrhodopsin 来映射神经元之间的连通性。Channelrhodopsin 激活一般不去极化神经元充分, 因此钾通道阻滞剂 4 AP 被应用于 TTX 引出释放 19, 20, 21.虽然这在许多突触中起作用, 但如果受体的下游靶点是 4 AP 敏感的钾通道, 它可能无法在某些代谢型突触中起作用。例如, 血清素调制的嗅觉反应在飞蛾直接依赖于调制这样一个 4 AP 敏感 K + 电流 (I a) 22. 这种方法在 CSDn 的突触中也不起作用 (没有显示数据)。因此, 与其他常用方法相比, TERPS 有更少的药理干预的好处, 并且可以在更广泛的范围内或突触中工作。
TERPS 有几个限制, 应该考虑。首先, 在整个发展过程中, NaChBac 通道的慢性表达可能会产生潜在的副作用。通过比较缺乏 NaChBac 结构的控制蝇神经元的活性, 并在没有 TTX 的情况下与结构飞行, 可以确定正确的电路组装和功能。为避免任何此类问题, NaChBac 转基因的表达可以通过温度敏感 GAL4 阻遏的表达, GAL8023来进行世俗控制。另外, 如果突触仅以状态依赖性的方式观察, 可以想象, 用 TTX 抑制网络活动可能会隐藏这种连接。重要的是要强调, 与 TERPS 观察到的积极联系可能代表真正的单突触连接, 而消极的结果并不证明没有连接。
虽然 TERPS 能清楚地揭示一个神经元的发射机释放能力对突触后伙伴的影响, 但 NaChBac 的慢通道动力学和与其活化相关的高原电位使其不太适用于经典神经传递.改变 TERPS 以更快、更自然的发射机释放的一种方法是, 表达一个修改后的 TTX 不敏感版本的内生果蝇钠通道第, 而不是 NaChBac. 这种改变将导致突触前细胞的自然峰值活动, 并允许更常规的分析, 在缺乏网络活动的突触传递。这将有很大的潜力, 研究速率编码突触, 在这种情况下, 吸引广泛的突触前活动, 而不招募 polysynaptic 网络将是可取的。

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动作电位, 使其成为 TERPS (图 2A) 的合适准备。NaChBac 钠通道的转基因表达抢救神经元的兴奋性, 并产生巨大的高原电位 (图 2B)。
触角叶中的局部中间神经元 (LN) 显示了对嗅觉刺激的强健反应 (图3A)。然而, 由于个体 EPSPs 不易在 LN 中得到解决, 目前还不清楚这种反应是直接来自嗅觉受体神经元输入 (ORN), 还是通过投影神经元 (PN) 间接输入 (图 3B)。通过使用 TERPS, ORNs 显示在这里, 确实可以与 LNs 直接突触连接 (图 3C)。
TERPS 的一个独特特点是它能够专门解决在 GABA 或 neuromodulators (如血清素) 中可能发生的额外突触体积传输。在果蝇触角叶中刺激血清素神经元 (CSDn) 会导致局部 interneuron 中的励磁和抑制混合 (图4A 和图4B)。激发是由兴奋性发射机乙酰胆碱冥想和抑制是由血清素 (图 4C和图 4D) 引起的。TERPS 可用于通过消除 polysynaptic 连接 (图 4E) 来确定连接是否 monosynaptic。在 TTX 中, 在 LN 中仍然观察到强烈的抑制, 暗示它直接从特定的血清素神经元 (图4F 和图4G) 的激活中到达。这种连接被血清素拮抗剂 methysergide 阻断。乙酰胆碱介导的兴奋性突触在 TTX 中消除, 提示它来源于 polysynaptic 源。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57052/57052fig1.jpg"> 图 1:TERPS 消除 polysynaptic 连接并隔离 monosynaptic 输入.(A). 一个示意图显示, 增加突触前活动可以招募 polysynaptic 连接。(B). 图解说明 TERPS 如何通过使用 TTX 消除神经元中的峰值活动来去除 polysynaptic 的贡献。兴奋是完全恢复在一个神经元的群体, 然后可以兴奋 optogenetically。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57052/57052fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57052/57052fig2.jpg"> 图 2:NaChBac 恢复神经元兴奋性果蝇神经元.(A) 应用1µm TTX 取消了在果蝇神经元中的峰值。TTX 中消除了自发活性和气味诱发抑制。(B) NaChBac 和 csChrimson 在 CSDn 中表达, 大脑暴露于 TTX。csChrimson 刺激 depolarizes CSDn 和阈值交叉后, CSDn 膜电压有较大的非线性增加。这种快速退极化构成了类似于高原的电位 (嵌入)。每种颜色的模拟强度对应于相同的颜色在电压嵌入。此数字已从张和 Gaudry 20169中修改。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57052/57052fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57052/57052fig3.jpg"> 图 3:TERPS 显示 ORNs 和 LNs 之间的 monosynaptic 连接.(A) 在 LN 中显示气味响应的示例记录。这种反应可能是单一的或 polysynaptic 的性质。(B) TERPS 可以在 ORN 到突触中进行测试。TTX 用于阻断所有细胞在制备过程中的动作电位和兴奋性。兴奋是完全恢复 ORNs 通过 NaChBac 钠通道。然后, ORNs 可以兴奋与 channelrhodopsin 诱发突触释放。突触事件的测量后 synaptically 使用全细胞记录。(C) TERPS 显示 ORN 突触是 monosynaptic 的。TERPS 也可以与药理学相结合, 表明突触是胆碱能和由烟碱受体拮抗剂盐酸 (200 µM) 阻断的。垂直灰色条表示 ORN 刺激的时间。此数字已在 2016年9中从张和 Gaudry 修改。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57052/57052fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57052/57052fig4.jpg"> 图 4: TERPS 对调节神经元的批量或体积传输释放敏感.(A) 对 CSDn 的刺激会导致在果蝇触角叶中记录的 LN 的动作电位。(B) 超极化搏为 CSDn 刺激导致阈活动。in 反应包括一个快速的退极化, 其次是一个缓慢的极化。灰色条表示 CSDn 刺激的时间。Methysergide (50 µM), 一个广泛的 5 HT 受体拮抗剂, 阻止缓慢的极化, 但对退极化没有影响。盐酸阻止快速去极化, 这表明它是胆碱的性质。(C) TERPS 可用于解决 CSDn 突触中的哪些化学成分是单抗 polysynaptic。(D) CSDn 在 TTX 和突触后的状态下受到刺激, 在全细胞记录中测量反应。极化坚持认为 TTX 是 monosynaptic 性质的。这极化也被 methysergide 阻止, 与血清素传输一致。TTX 中残留的短暂退极化很可能是由电隙耦合介导的, 因为它不会被烟拮抗剂阻挡。此数字已在 2016年9中从张和 Gaudry 修改。<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57052/57052fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充文件 1: 果蝇箔.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57052/DrosophilaFoil.pdf" target="_blank">请单击此处下载此文件.</a>
补充文件 2: 生理学室.<a href="//cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57052/PhysiologyChamber.pdf" target="_blank">请单击此处下载此文件.</a>

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

Nanyang Technological University

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

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

海马脑片的器官型检查神经元损伤的文化模式

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

科研

神经科学

Examining Local Network Processing using Multi-contact Laminar Electrode Recording

Cortical layers are ubiquitous structures throughout neocortex1-4 that consist of highly recurrent local networks. In recent years, significant progress has been made in our understanding of differences in response properties of neurons in different cortical layers5-8, yet there is still a great deal left to learn about whether and how neuronal populations encode information in a laminar-specific manner.
Existing multi-electrode array techniques, although informative for measuring responses across many millimeters of cortical space along the cortical surface, are unsuitable to approach the issue of laminar cortical circuits. Here, we present our method for setting up and recording individual neurons and local field potentials (LFPs) across cortical layers of primary visual cortex (V1) utilizing multi-contact laminar electrodes (Figure 1; Plextrode U-Probe, Plexon Inc).
The methods included are recording device construction, identification of cortical layers, and identification of receptive fields of individual neurons. To identify cortical layers, we measure the evoked response potentials (ERPs) of the LFP time-series using full-field flashed stimuli. We then perform current-source density (CSD) analysis to identify the polarity inversion accompanied by the sink-source configuration at the base of layer 4 (the sink is inside layer 4, subsequently referred to as granular layer9-12). Current-source density is useful because it provides an index of the location, direction, and density of transmembrane current flow, allowing us to accurately position electrodes to record from all layers in a single penetration6, 11, 12.

A fundamental issue in our understanding of cortical circuitry is how networks in different cortical layers encode sensory information. Here we describe electrophysiological techniques utilizing multi-contact laminar electrodes to record single-units and local field potentials and present analyses to identify cortical layers.

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 ...

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

The aim of this methods paper is to describe how to implement a neuroimaging technique to examine complementary brain processes engaged by two similar tasks. Participants' behavior during task performance in an fMRI scanner can then be correlated to the brain activity using the blood-oxygen-level-dependent signal. We measure behavior to be able to sort correct trials, where the subject performed the task correctly and then be able to examine the brain signals related to correct performance. Conversely, if subjects do not perform the task correctly, and these trials are included in the same analysis with the correct trials we would introduce trials that were not only for correct performance. Thus, in many cases these errors can be used themselves to then correlate brain activity to them. We describe two complementary tasks that are used in our lab to examine the brain during suppression of an automatic responses: the stroop1 and anti-saccade tasks. The emotional stroop paradigm instructs participants to either report the superimposed emotional 'word' across the affective faces or the facial 'expressions' of the face stimuli1,2. When the word and the facial expression refer to different emotions, a conflict between what must be said and what is automatically read occurs. The participant has to resolve the conflict between two simultaneously competing processes of word reading and facial expression. Our urge to read out a word leads to strong 'stimulus-response (SR)' associations; hence inhibiting these strong SR's is difficult and participants are prone to making errors. Overcoming this conflict and directing attention away from the face or the word requires the subject to inhibit bottom up processes which typically directs attention to the more salient stimulus. Similarly, in the anti-saccade task3,4,5,6, where an instruction cue is used to direct only attention to a peripheral stimulus location but then the eye movement is made to the mirror opposite position. Yet again we measure behavior by recording the eye movements of participants which allows for the sorting of the behavioral responses into correct and error trials7 which then can be correlated to brain activity. Neuroimaging now allows researchers to measure different behaviors of correct and error trials that are indicative of different cognitive processes and pinpoint the different neural networks involved.

The goal of our research is to correlate behavior to brain activity. Accurate behavioral measures and imaging techniques allow us to elucidate brain-behavior relationships.

关联从人类前额叶皮层的fMRI信号的行为反应:检查的认知过程，使用任务分析

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

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 insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

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

Multi-photon fluorescence microscopy has enabled the analysis of morphological and physiological parameters of brain cells in the intact tissue with high spatial and temporal resolution. Combined with electrophysiology, it is widely used to study activity-related calcium signals in small subcellular compartments such as dendrites and dendritic spines. In addition to calcium transients, synaptic activity also induces postsynaptic sodium signals, the properties of which are only marginally understood. Here, we describe a method for combined whole-cell patch-clamp and multi-photon sodium imaging in cellular micro domains of central neurons. Furthermore, we introduce a modified procedure for ultra-violet (UV)-light-induced uncaging of glutamate, which allows reliable and focal activation of glutamate receptors in the tissue. To this end, whole-cell recordings were performed on Cornu Ammonis subdivision 1 (CA1) pyramidal neurons in acute tissue slices of the mouse hippocampus. Neurons were filled with the sodium-sensitive fluorescent dye SBFI through the patch-pipette, and multi-photon excitation of SBFI enabled the visualization of dendrites and adjacent spines. To establish UV-induced focal uncaging, several parameters including light intensity, volume affected by the UV uncaging beam, positioning of the beam as well as concentration of the caged compound were tested and optimized. Our results show that local perfusion with caged glutamate (MNI-Glutamate) and its focal UV-uncaging result in inward currents and sodium transients in dendrites and spines. Time course and amplitude of both inward currents and sodium signals correlate with the duration of the uncaging pulse. Furthermore, our results show that intracellular sodium signals are blocked in the presence of blockers for ionotropic glutamate receptors, demonstrating that they are mediated by sodium influx though this pathway. In summary, our method provides a reliable tool for the investigation of intracellular sodium signals induced by focal receptor activation in intact brain tissue.

We describe the combination of focal UV-induced photo-activation of neuro-active compounds with whole-cell patch-clamp and multi-photon imaging of intracellular sodium transients in dendrites and spines of hippocampal neurons in acute tissue slices of the mouse brain.

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 ...

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To complicate matters even further, the odors detected by the insect nervous system usually are not single compounds but mixtures whose composition and concentration ratios vary. This leads to an almost infinite amount of different olfactory stimuli which have to be evaluated by the nervous system.
To understand which aspects of an odor stimulus determine its evaluation by the fly, it is therefore desirable to efficiently examine odor-guided behavior towards many odorants and odor mixtures. To directly correlate behavior to neuronal activity, behavior should be quantified in a comparable time frame and under identical stimulus conditions as in neurophysiological experiments. However, many currently used olfactory bioassays in Drosophila neuroethology are rather specialized either towards efficiency or towards resolution.
Flywalk, an automated odor delivery and tracking system, bridges the gap between efficiency and resolution. It allows the determination of exactly when an odor packet stimulated a freely walking fly, and to determine the animal&#180;s dynamic behavioral reaction.

High-resolution Quantification of Odor-guided Behavior in Drosophila melanogaster Using the Flywalk Paradigm

The automated tracking system Flywalk is used for high-resolution quantification of odor-guided behavior in Drosophila melanogaster.

在气味引导行为的高分辨率定量<em&gt;果蝇</em&gt;使用<em&gt; Flywalk</em&gt;范式

In their natural environment, insects such as the vinegar fly Drosophila melanogaster are bombarded with a huge amount of chemically distinct odorants. To ...

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

Drosophila larval crawling is emerging as a powerful model to study neural control of sensorimotor behavior. However, larval crawling behavior on flat open surfaces is complex,&#160;including: pausing, turning, and meandering. This complexity in the repertoire of movement hinders detailed analysis of the events occurring during a single crawl stride cycle. To overcome this obstacle, linear agarose channels were made that constrain larval behavior to straight, sustained, rhythmic crawling. In principle, because agarose channels and the Drosophila larval body are both optically clear, the movement of larval structures labeled by genetically-encoded fluorescent probes can be monitored in intact, freely-moving larvae. In the past, larvae were placed in linear channels and crawling at the level of whole organism, segment, and muscle were analyzed1. In the future, larvae crawling in channels can be used for calcium imaging to monitor neuronal activity. Moreover, these methods can be used with larvae of any genotype and with any researcher-designed channel. Thus the protocol presented below is widely applicable for studies using the Drosophila larva as a model to understand motor control.

Using Linear Agarose Channels to Study Drosophila Larval Crawling Behavior

The Drosophila larva is a powerful model system to study neural control of behavior. This publication describes the use of linear agarose channels to elicit sustained bouts of linear crawling and methods to quantify the dynamics of larval structures during repetitive 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

We describe a protocol for measuring ethanol self-administration in fruit flies (Drosophila melanogaster) as a proxy for changes in reward states. We demonstrate a simple way to tap into the fly reward system, modify experiences related to natural reward, and use voluntary ethanol consumption as a measure for changes in reward states. The approach serves as a relevant tool to study the neurons and genes that play a role in experience-mediated changes of internal state. The protocol is composed of two discrete parts: exposing the flies to rewarding and nonrewarding experiences, and assaying voluntary ethanol consumption as a measure of the motivation to obtain a drug reward. The two parts can be used independently to induce the modulation of experience as an initial step for further downstream assays or as an independent two-choice feeding assay, respectively. The protocol does not require a complicated setup and can therefore be applied in any laboratory with basic fly culture tools.

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

We describe a protocol for inducing rewarding and nonrewarding experiences in fruit flies (Drosophila melanogaster) using voluntary ethanol consumption as a measure for changes in reward states.

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

With the rising prevalence of neurodegenerative diseases, it is increasingly important to understand the underlying pathophysiology that leads to neuronal dysfunction and loss. Fluorescence-based imaging tools and technologies enable unprecedented analysis of subcellular neurobiological processes, yet there is still a need for unbiased, reproducible, and accessible approaches for extracting quantifiable data from imaging studies. We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-based imaging studies using Drosophila models of neurodegeneration. Specifically, we describe an easy-to-follow, semi-automated approach using Fiji/ImageJ to analyze two cellular processes: first, we quantify protein aggregate content and profile in the Drosophila optic lobe using fluorescent-tagged mutant huntingtin proteins; and second, we assess autophagy-lysosome flux in the Drosophila visual system with ratiometric-based quantification of a tandem fluorescent reporter of autophagy. Importantly, the protocol outlined here includes a semi-automated segmentation step to ensure all fluorescent structures are analyzed to minimize selection bias and to increase resolution of subtle comparisons. This approach can be extended for the analysis of other cell biological structures and processes implicated in neurodegeneration, such as proteinaceous puncta (stress granules and synaptic complexes), as well as membrane-bound compartments (mitochondria and membrane trafficking vesicles). This method provides a standardized, yet adaptable reference point for image analysis and quantification, and could facilitate reliability and reproducibility across the field, and ultimately enhance mechanistic understanding of neurodegeneration.

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

We have developed a simple and adaptable workflow to extract quantitative data from fluorescence-imaging-based cell biological studies of protein aggregation and autophagic flux in the central nervous system of Drosophila models of neurodegeneration.

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

Foraging and feeding behaviors allow animals to access sources of energy and nutrients essential for their development, health, and fitness. Investigating the neuronal regulation of these behaviors is essential for the understanding of the physiological and molecular mechanisms underlying nutritional homeostasis. The use of genetically tractable animal models such as worms, flies, and fish greatly facilitates these types of studies. In the last decade, the fruit fly Drosophila melanogaster has been used as a powerful animal model by neurobiologists investigating the neuronal control of feeding and foraging behaviors. While undoubtedly valuable, most studies examine adult flies. Here, we describe a protocol that takes advantage of the simpler larval nervous system to investigate neuronal substrates controlling feeding behaviors when larvae are exposed to diets differing in their protein and carbohydrates content. Our methods are based on a quantitative colorimetric no-choice feeding assay, performed in the context of a neuronal thermogenetic-activation screen. As a read-out, the amount of food eaten by larvae over a 1 h interval was used when exposed to one of the three dye-labeled diets that differ in their protein to carbohydrates (P:C) ratios. The efficacy of this protocol is demonstrated in the context of a neurogenetic screen in larval Drosophila, by identifying candidate neuronal populations regulating the amount of food eaten in diets of different macronutrient quality. We were also able to classify and group the genotypes tested into phenotypic classes. Besides a brief review of the currently available methods in the literature, the advantages and limitations of these methods are discussed and, also, some suggestions are provided about how this protocol might be adapted to other specific experiments.

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

Described here is a protocol that enables the colorimetric quantification of the amount of food eaten within a defined interval of time by Drosophila melanogaster larvae exposed to diets of different macronutrient quality. These assays are conducted in the context of a neuronal thermogenetic screen.

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 ...