作者谨感谢布卢明顿股票中心，利群罗，查尔斯·朱克和莉莉和裕侬扬慷慨分享飞股票及以下资金:肉体-URM（以JD和SW），本科福特学院暑期奖学金（申银万国），BD公司暑期研究奖学金（以CL和DL），该蕾妮和Anthony M.马龙，MD &#39;63暑期研究奖学金（以DL），詹姆斯·&#39;75和简Colihan暑期研究奖学金（TO）通过校友/圣十字学院的家长暑期研究基金和Stransky基金会暑期研究奖学金（为TO）。特别感谢生物系和教务处在学院的圣十字对实验室全部工作的支持。

1.十字到生成MARCM苍蝇<ol><li>保持苍蝇玉米面糖蜜培养基（6.5克/升琼脂; 23.5克/升酵母; 60 g / L的玉米面; 60毫升/升糖蜜; 4毫升/升酸混合物; 0.13％Tegosept）或标准果蝇介质在22摄氏度在12小时的光照/黑暗循环。 注:比标准温度下是用来保持MARCM-股健康。这延伸到14天蛋和成年之间的发育时间。 </li><li>从一个MARCM就绪库存 - （8日龄约1）含有必要的遗传元件（GFP的上游活化序列（UAS-GFP）的GAL80阻遏蛋白，该Gal4的转录因子，的控制下使用处女雌性热休克驱动的重组，和FRT重组位点）。 注:我们MARCM现成的个股包括以下基因型:elavGal4，UAS-CD8-GFP，HS-flipase; FRT40A，tubGal80 / CYO; tubGal4 / TM3Sb。 注意:特定FRT位点，将取决于突变的位置在基因组中。例如，FRT40A用于左臂2号染色体，如NOMPC（见10的更多详细信息MARCM协议）测试突变。 </li><li>交叉MARCM就绪处女雌性到男性包含感兴趣的突变和其相应的FRT位点（例如，W-; NOMPC3，FRT40A / CYO + / +）。为了控制有效MARCM感应，设置使用雄性苍蝇含有相同FRT位点的第二交叉，但没有突变。使用5位女性的大致比例:1男在十字架。 </li></ol> 2.定时产卵<ol><li>使苍蝇交配用于至少一个晚上后，移动大人到新的小瓶中，用新鲜培养基。允许苍蝇之前再次转移杂交到新的小瓶产卵在黑暗环境中进行大约为4小时。注意产蛋期的开始和结束时间。 注意:当似乎有高数量的媒体卵经4小时interva结束之前更短的时间间隔是可以接受的湖<ol><li>保持小瓶鸡蛋足够数量（大约大于20），并存储在一个12小时的光/暗周期在22摄氏度至晚热冲击。 注意:小瓶用鸡蛋的介质上一个数低（小于大约20）是不可能产生足够数量的成蝇对于行为测试和效率起见，建议他们不被保留用于进一步的测试。 注:我们的苍蝇就提出用灯光从早上8点到晚上8点。我们已经找到了傍晚的时候分的领先优势，以更高的蛋密度，因此通常执行4之间定时十字 - 晚上8点到晚上8点 - 午夜。苍蝇奠定在室温下的鸡蛋放在一个黑暗的橱柜，然后返回到孵化第二天，以免干扰常规明暗周期在孵化器。 </li></ol></li></ol> 3.执行热休克来诱导有丝分裂重组<ol><li>大约85 - 产蛋后100小时，地点vIALS进入水浴在37摄氏度下1小时。确保水位到达媒体在小瓶的高度以上，但不完全淹没小瓶以防止幼虫溺水。 </li><li>从水浴中取出药瓶，并允许在室温下1小时的恢复期。 </li><li>地方小瓶放回37摄氏度的水浴中1小时。 </li><li>取下水浴和存储小瓶在孵化器12小时光照/黑暗周期在22摄氏度左右，直至羽化。 </li></ol> 4.准备行为测试<ol><li>羽化之后，麻醉苍蝇在冰上。选择苍蝇与包含所有为MARCM遗传元件和表示GFP表达标记的克隆很可能会根据在父个股中使用的可观察到的表型的标记物观察到的基因型。斩首苍蝇与虹膜切除术剪刀。 注:例如，我们选择了苍蝇不遗传标记CYO（大翅膀）和Sb（茬毛）来鉴定FY苍蝇，将包含所有必要创造马赛克区域的遗传元件标有绿色荧光蛋白。 注:斩首是既要防止成年人，当刺激飞走。 </li><li>放置无头苍蝇在封闭，潮湿的环境，并允许约10 - 20小时的恢复。只有用苍蝇这种权利本身扰动时进行进一步的测试。 30小时断头内的测试飞行。 </li></ol> 5.行为测试<ol><li>通过荧光解剖显微镜下观察断头苍蝇，发现标有绿色荧光蛋白的猪鬃外部感觉器官纯合子克隆。记录刷毛名称和左，右两侧。以消除任何潜在的偏见，该实验室构件进行刷毛刺激应盲目刷毛，野生型或突变体的基因型。 </li><li>通过偏转GFP引发疏导反射标塞得满满的向飞机身采用了硬毛或细镊子，观察腿再sponse。给苍蝇解除他们的腿在应对猪鬃刺激和苍蝇不动自己的腿得分为零分之一。 注:以前的研究6,7有特点的是通过刺激特定的刷毛引起腿部的反应。这包括，响应各刷毛的腿和苍蝇该特定刷毛刺激响应的比例。我们只打进了我们预期的基础上Vandervorst的分类6响应腿的动作。 </li><li>开展间隔2分钟间隔五位审判。 </li></ol>

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作者有没有竞争经济利益。

这个协议利用一个成人行为分析以筛选影响机械感觉在果蝇的突变。因为突变体的集合包含致死对元件的突变，将排除筛选作为成年人，该协议使得使用第一Lee和罗述的复杂的遗传技术的，（1999年）和详细的协议吴，罗，（2006年），以规避成人杀伤力。 MARCM诱导同源染色体之间有丝分裂重组以产生纯合细胞的克隆的区域。因为阻遏蛋白，浴盆-GAL80中有丝分裂重组发生的损失的这些?...

Humans rely on the ability to convert mechanical stimuli from their environment, such as touch, pressure, vibration, or sound waves, into sensory information that can be processed by the nervous system, in a process termed mechanotransduction. Many of the overall mechanistic features of mechanotransduction between humans and invertebrates are the same1, making Drosophila a useful model to study the molecular mechanisms of mechanotransduction. Drosophila melanogaster contain two sets of specialized sensory organs (Type I and II) that are capable of converting mechanical stimuli into action potentials. Type I mechanoreceptors have a neuron with a single dendrite or sensory process, surrounded by three support cells2. The Type I mechanoreceptors include bristle mechanoreceptors, hearing sensitive chordotonal organs (the Johnston's organ), and campaniform sensilla that convey information about wing beats3. The bristles that cover the dorsal side of the fly are the most abundant and easily accessible of the Type I organs. Similar to the extracellular environment of hair cells associated with hearing and balance in vertebrates, the support cells surrounding fly mechanosensitive neurons secrete a high potassium endolymph that creates an unusual concentration gradient for potassium1. Mechanosensitive neurons, of both the mammalian and fly systems, utilize this high extracellular potassium to depolarize the cell. In response to mechanical stimulation of the macrochaete bristles towards the body wall, the sensory neurons respond with a burst of action potentials driven by this potassium depolarization of the cell membrane4. The Drosophila sensory neurons that innervate bristles resemble the mechanosensitive cells of other organisms, including vertebrate hair cells, in both structure and function1,5. The accessibility of Drosophila external sense organs to experimental manipulation and the abundance of genetic techniques available to researchers make Drosophila an excellent model system to investigate the molecular underpinnings of mechanosensation. 
In the fly, stimulation of a single sensory neuron that innervates a bristle leads to an observable behavioral response. Stimulation of different bristles evokes specific, reproducible behavioral responses, depending on the bristle that is stimulated. Upon tactile stimulation, decapitated wild-type flies exhibit a complex grooming reflex wherein they clean the area near the stimulated bristle with a patterned set of leg movements5-7. When homozygous for known single-gene mutations that interfere with learning7 or coordination and locomotor activity5, flies respond abnormally to mechanical stimulation. This grooming reflex is therefore a useful tool to study the effects of single-gene mutations on a specific, replicable behavior.
The robust behavioral response to stimulation of a single macrochaete bristle holds the potential to assist in identifying new genes involved in mechanotransduction. This protocol being used to test a collection of mutant flies for the absence of a behavioral response to indicate that the mutation interferes with mechanosensation. In the mutant collection selected for screening, the mutations cause lethality before adulthood, and therefore would be impossible to test using traditional adult behavior screens. Originally, this collection of lethal p-elements was combined with FRT recombination sites to test cell growth defects in clones. The clones were made specifically in the eye because adult flies can survive in the lab setting without functional vision8. However, removal of all mechanosensation can cause adults to be severely uncoordinated or die before eclosion5. This protocol uses a mosaic approach to circumvent the lethality of the mutations and allow for adult stage testing. A genetic technique called Mosaic Analysis with a Repressible Cell Marker (MARCM)9 is used to generate homozygous mutant cells in a limited number of adult fly sensory organs, while the rest of the organism remains heterozygous. These MARCM flies readily survive until adulthood, yet the bristles are homozygous for the lethal genes of interest.
MARCM allows for regions of homozygous mutant cells to be generated and marked by the expression of green fluorescent protein (GFP), while the rest of the organism remains heterozygous at that particular locus and unmarked9. MARCM combines individual p-element mutations with five common genetic elements: GFP under the control of an upstream-activating sequence (UAS-GFP), Gal80 repressor protein under control of a general promoter (tub-Gal80), Gal4 transcription factor under control of a general promoter (tub-Gal4), the FRT recombinase enzyme expressed through a heat-shock controlled promoter, and a FRT recombination site10. By driving mitotic recombination through a heat-shock activated recombinase, a limited number of cells are made homozygous for the mutation and marked with GFP. GFP expression is repressed in heterozygous cells by the presence of Gal80 on the wild-type copy of the chromosome. 
A heat-shock protocol for MARCM was optimized to induce recombination in the fly bristle external sense organs, while much of the organism remains heterozygous, and thus unmarked with GFP. Mosaic flies generated using this protocol contained homozygous mutations most frequently in the post alar or dorsal central bristles on the surface of the notum. 
We have tested the utility of this combination of MARCM and the grooming behavior screen with a known mechanosensitive mutant, NOMPC. The ion channel, no mechanoreceptor potential C (NOMPC), is an essential component of the mechanotransduction pathway in Drosophila5,11-13. NOMPC belongs to the transient receptor potential (TRP) superfamily of cation channels5 and satisfies all of the criteria to qualify as a mechanosensitive channel in Drosophila14,15: 1) NOMPC is expressed in the ciliate tips of type 1 sensory neurons of Drosophila 13,16-18, 2) NOMPC null larvae do not have an electrical response to tactile stimulation13, 3) Ectopic expression of NOMPC in touch insensitive cells can induce sensitivity to mechanical stimulation13, 4) heterologous expression of NOMPC in Schneider 2 cells yields a mechanosensitive channel13, and 5) NOMPC adult mutants display defects in their response to mechanical stimulation5. Given this evidence, we predicted that NOMPC mutant clones would show an altered or inhibited grooming response in response to mechanical stimulation of bristles.
A MARCM-stock containing the NOMPC mutation was developed for use in a proof of principle experiment of the grooming assay. Mosaic flies were stimulated at macrochaete bristles containing homozygous NOMPC mutant cells. We expected an inhibition of the grooming response following stimulation of the macrochaete bristle. We found that only 2 of 14 mutant bristle flies tested gave a single response to repeated stimulation; most did not respond to stimulation of the homozygous mutant bristle. Having confirmed that this MARCM-based behavioral assay produces an abnormal grooming reflex in a known mechanosensitive mosaic mutant, this technique can be used in a screen for additional mechanosensitive mutations.

<table border="0" cellpadding="0" cellspacing="0" width="648">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Brewers Yeast (25 lb)</td>
			<td style="width:163px;">MP Biomedicals&#160;</td>
			<td style="width:163px;">ICN90331225&#160;</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Corn (25lb)</td>
			<td style="width:163px;">MP Biomedicals&#160;</td>
			<td style="width:163px;">ICN90141125&#160;</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Agar (1lb)</td>
			<td style="width:163px;">MoorAgar Inc.</td>
			<td style="width:163px;">41004</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Tegosept (5kg)</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">20-259</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:163px;">Molasses (1Gallon)</td>
			<td style="width:163px;">Sugarmill Brand - Thomsen Food Service</td>
			<td style="width:163px;">0 2625</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Propionic Acid</td>
			<td style="width:163px;">Fisher</td>
			<td style="width:163px;">A258-500</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Phosphoric acid</td>
			<td style="width:163px;">Fisher</td>
			<td style="width:163px;">A260-500</td>
			<td style="width:163px;">Fly Food</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">Drosophila Vials, Narrow (PS)</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">32-109</td>
			<td style="width:163px;">Fly Cultures</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:163px;">6oz Square Bottom Bottle (PP)</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">32-130</td>
			<td style="width:163px;">Fly Cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Flugs - Plastic Fly Bottles</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">49-100</td>
			<td style="width:163px;">Fly Cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Rayon Balls, Large</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">51-100</td>
			<td style="width:163px;">Fly Cultures</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Droso-Filler, Narrow</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">59-168</td>
			<td style="width:163px;">Fly Food Preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Droso-Filler, Bottles</td>
			<td style="width:163px;">Genesee</td>
			<td style="width:163px;">59-170</td>
			<td style="width:163px;">Fly Food Preparation</td>
		</tr>
		<tr height="95">
			<td height="95" style="height:95px;width:163px;">8A-C / gear driven lab stirrer with c-clamp mount&#160; 1/15HP, 700RPM variable speed, 115V, 50/60Hz</td>
			<td style="width:163px;">Cleveland Mixer</td>
			<td style="width:163px;">8A-C</td>
			<td style="width:163px;">Fly Food Preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Water jacketed Kettle</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
			<td style="width:163px;">Fly Food Preparation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">Diurnal Growth Chamber</td>
			<td style="width:163px;">Forma Scientific</td>
			<td style="width:163px;"></td>
			<td style="width:163px;">Temperature and light/dark cycle controlled</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Water bath</td>
			<td style="width:163px;">VWR</td>
			<td style="width:163px;"></td>
			<td style="width:163px;">For heat shock</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">MicroScissors&#160;</td>
			<td style="width:163px;">Fine Science Tools</td>
			<td style="width:163px;">&#160;15000-08</td>
			<td style="width:163px;">For removing heads</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Fluroscence Dissecting Microscope</td>
			<td style="width:163px;">Zeiss</td>
			<td style="width:163px;">SteREO Discovery V8&#160;</td>
			<td style="width:163px;">With GFP cube (KSC295-814D) band pass filter</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">Fluroscence Light Source</td>
			<td style="width:163px;">Zeiss</td>
			<td style="width:163px;">X-Cite 120</td>
			<td style="width:163px;">Fiber optic light pipe makes this easy to configure&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Camera for Scope</td>
			<td style="width:163px;">Zeiss</td>
			<td style="width:163px;">AxioCam ICc1</td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Image acquistion software</td>
			<td style="width:163px;">Zeiss</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:163px;">Ice bucket</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
			<td style="width:163px;">for cold anthesia</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:163px;">Homemade cold anthesia tray</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
			<td style="width:163px;">for cold anthesia decapitation</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:163px;">Plastic boxes</td>
			<td style="width:163px;"></td>
			<td style="width:163px;"></td>
			<td style="width:163px;">for recovery of decaptitated flies in humid environment</td>
		</tr>
	</tbody>
</table>

a behavioral assay for mechanosensation of marcm-based clones in drosophila melanogaster

因为结构和功能同源性的哺乳动物内耳的毛细胞，即支配果蝇外部感觉器官的神经元提供机械感觉的研究的优良模型系统。这个协议描述在果蝇中，可用于识别与机械感觉干扰突变简单的触摸行为。一个macrochaete的触觉刺激刷毛上的苍蝇的胸部引出一清洁反射从第一或第三站。与机械传导（如NOMPC）干扰的突变，或者与反射弧的其他方面，可以抑制疏导响应。成人行为的传统画面就会错过这一发展过程中发挥重要作用的突变体。相反，该​​协议结合镶嵌分析与阻遏细胞标记物（MARCM）触摸屏，以允许纯合突变细胞中，只有有限的区域由EXPR生成并标分裂国家绿色荧光蛋白（GFP）的。通过测试MARCM克隆为异常的行为反应，有可能筛选的致死对元件的突变的集合以寻找涉及机械感觉新基因，将已经错过由更传统的方法。

此协议的成功在很大程度上依赖于可检验的苍蝇的总产率，热休克的有效性以诱导有丝分裂重组，并获得在果蝇以2分钟的间隔刺激一个健壮的行为反应的能力。鉴于胚胎的估计12.5％包含必要MARCM元素和成年人E关闭对GFP表达的潜在的估计的16.67％，这是关键的期间定时规定以获得高数鸡蛋。一个成功的定时的一个例子笼罩，将在该协议的其余部分被用来显示在?...

Watch this Scientific Journal Video about A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster at JoVE.com

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

一个行为分析对机械感觉MARCM为基础的克隆<em&gt;果蝇</em

In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

Because of the structural and functional homology to the hair cells of the mammalian inner ear, the neurons that innervate the Drosophila external sense ...

a-behavioral-assay-for-mechanosensation-marcm-based-clones-drosophila

Research

JoVE Journal

Neuroscience

A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

10.2K 次观看.  College of the Holy Cross. In order to identify novel mutations affecting mechanosensation, we designed an assay that measures the behavioral response to tactile stimulation of fly bristles in mutant clones generated by the MARCM method. The combination of techniques allows for the identification of mechanosensitive mutations that would otherwise be lethal.

视频: A Behavioral Assay for Mechanosensation of MARCM-based Clones in Drosophila melanogaster

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

评估大鼠蔗糖渴求育成的一个通用方法

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

科研

神经科学

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We developed a novel nanoinjection/electrophysiology assay that allows the detection of bioactive modulatory effects of compounds on the function of a neuronal circuit that mediates the escape response in Drosophila melanogaster3,4. Our in vivo assay, which uses the Drosophila Giant Fiber System (GFS, Figure 1) allows screening of different types of compounds, such as small molecules or peptides, and requires only minimal quantities to elicit an effect. In addition, the Drosophila GFS offers a large variety of potential molecular targets on neurons or muscles. The Giant Fibers (GFs) synapse electrically (Gap Junctions) as well as chemically (cholinergic) onto a Peripheral Synapsing Interneuron (PSI) and the Tergo Trochanteral Muscle neuron (TTMn)5. The PSI to DLMn (Dorsal Longitudinal Muscle neuron) connection is dependent on D&alpha;7 nicotinic acetylcholine receptors (nAChRs)6. Finally, the neuromuscular junctions (NMJ) of the TTMn and the DLMn with the jump (TTM) and flight muscles (DLM) are glutamatergic7-12. Here, we demonstrate how to inject nanoliter quantities of a compound, while obtaining electrophysiological intracellular recordings from the Giant Fiber System13 and how to monitor the effects of the compound on the function of this circuit. We show specificity of the assay with methyllycaconitine citrate (MLA), a nAChR antagonist, which disrupts the PSI to DLMn connection but not the GF to TTMn connection or the function of the NMJ at the jump or flight muscles.
Before beginning this video it is critical that you carefully watch and become familiar with the JoVE video titled "Electrophysiological Recordings from the Giant Fiber Pathway of D. melanogaster " from Augustin et al7, as the video presented here is intended as an expansion to this existing technique. Here we use the electrophysiological recordings method and focus in detail only on the addition of the paired nanoinjections and monitoring technique.

Paired Nanoinjection and Electrophysiology Assay to Screen for Bioactivity of Compounds using the Drosophila melanogaster Giant Fiber System

A rapid in vivo assay to test for neuromodulatory compounds using the Giant Fiber System (GFS) of Drosophila melanogaster is described. Nanoinjections in the head of the animal along with electrophysiological recordings of the GFS can reveal bioactivity of compounds on neurons or muscles.

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

Screening compounds for in vivo activity can be used as a first step to identify candidates that may be developed into pharmacological agents1,2. We ...

A Lightweight, Headphones-based System for Manipulating Auditory Feedback in Songbirds

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and sensorimotor plasticity1. Consistent sensory perturbations result in compensatory changes in motor output, reflecting changes in feedforward motor control that reduce the experienced feedback error. By quantifying how different sensory feedback errors affect human behavior, prior studies have explored how visual signals are used to recalibrate arm movements2,3 and auditory feedback is used to modify speech production4-7. The strength of this approach rests on the ability to mimic naturalistic errors in behavior, allowing the experimenter to observe how experienced errors in production are used to recalibrate motor output.
Songbirds provide an excellent animal model for investigating the neural basis of sensorimotor control and plasticity8,9. The songbird brain provides a well-defined circuit in which the areas necessary for song learning are spatially separated from those required for song production, and neural recording and lesion studies have made significant advances in understanding how different brain areas contribute to vocal behavior9-12. However, the lack of a naturalistic error-correction paradigm - in which a known acoustic parameter is perturbed by the experimenter and then corrected by the songbird - has made it difficult to understand the computations underlying vocal learning or how different elements of the neural circuit contribute to the correction of vocal errors13.
The technique described here gives the experimenter precise control over auditory feedback errors in singing birds, allowing the introduction of arbitrary sensory errors that can be used to drive vocal learning. Online sound-processing equipment is used to introduce a known perturbation to the acoustics of song, and a miniaturized headphones apparatus is used to replace a songbird's natural auditory feedback with the perturbed signal in real time. We have used this paradigm to perturb the fundamental frequency (pitch) of auditory feedback in adult songbirds, providing the first demonstration that adult birds maintain vocal performance using error correction14. The present protocol can be used to implement a wide range of sensory feedback perturbations (including but not limited to pitch shifts) to investigate the computational and neurophysiological basis of vocal learning.

We describe the design and assembly of miniaturized headphones suitable for replacing a songbird&#x2019;s natural auditory feedback with a manipulated acoustic signal. Online sound processing hardware is used to manipulate song output, introduce real-time errors in auditory feedback via the headphones, and drive vocal motor learning.

一个轻量级的，耳机为基础的系统操作鸣禽听觉反馈

Experimental manipulations of sensory feedback during complex behavior have provided valuable insights into the computations underlying motor control and ...

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

A growing number of genetically encoded tools are becoming available that allow non-invasive manipulation of the neural activity of specific neurons in Drosophila melanogaster1. Chief among these are optogenetic tools, which enable the activation or silencing of specific neurons in the intact and freely moving animal using bright light. Channelrhodopsin (ChR2) is a light-activated cation channel that, when activated by blue light, causes depolarization of neurons that express it. ChR2 has been effective for identifying neurons critical for specific behaviors, such as CO2 avoidance, proboscis extension and giant-fiber mediated startle response2-4. However, as the intense light sources used to stimulate ChR2 also stimulate photoreceptors, these optogenetic techniques have not previously been used in the visual system. Here, we combine an optogenetic approach with a mutation that impairs phototransduction to demonstrate that activation of a cluster of loom-sensitive neurons in the fly's optic lobe, Foma-1 neurons, can drive an escape behavior used to avoid collision. We used a null allele of a critical component of the phototransduction cascade, phospholipase C-&beta;, encoded by the norpA gene, to render the flies blind and also use the Gal4-UAS transcriptional activator system to drive expression of ChR2 in the Foma-1 neurons. Individual flies are placed on a small platform surrounded by blue LEDs. When the LEDs are illuminated, the flies quickly take-off into flight, in a manner similar to visually driven loom-escape behavior. We believe that this technique can be easily adapted to examine other behaviors in freely moving flies.

Optogenetic Stimulation of Escape Behavior in Drosophila melanogaster

Genetically encoded optogenetic tools enable noninvasive manipulation of specific neurons in the Drosophila brain. Such tools can identify neurons whose activation is sufficient to elicit or suppress particular behaviors. Here we present a method for activating Channelrhodopsin2 that is expressed in targeted neurons in freely walking flies.

Optogenetic刺激的逃避行为<em&gt;果蝇</em

A  growing number of genetically encoded tools are becoming available that allow non-invasive  manipulation of the neural activity of specific neurons in ...

A Single-fly Assay for Foraging Behavior in Drosophila

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster. In a light-tight box illuminated by red light that is invisible to fruit flies, a camera linked to custom data acquisition software monitors the position of six flies simultaneously. Each fly is confined to walk in individual arenas containing a food odor at the center. The testing arenas rest on a porous floor that functions to prevent odor accumulation. Latency to locate the odor source, a metric that reflects olfactory sensitivity under different physiological states, is determined by software analysis. Here, we discuss the critical mechanics of running this behavioral paradigm and cover specific issues regarding fly loading, odor contamination, assay temperature, data quality, and statistical analysis.

A Single-fly Assay for Foraging Behavior in Drosophila

In this video article, we describe an automated assay to measure the effect of hunger or satiety on olfactory dependent food search behavior in the adult fruit fly Drosophila melanogaster.

单苍蝇检测方法的觅食行为的<em&gt;果蝇</em

For many animals, hunger promotes changes in the olfactory system in a manner that facilitates the search for appropriate food sources. In this video ...

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response is a vision-based, reflexive behavior that has been observed in all vertebrates tested. Tadpole eyes are small so the tail flip response was used as alternative measure, which requires a trained technician to record the subtle response. We developed an alternative behavior assay based on the fact that tadpoles prefer to swim on the white side of a tank when placed in a tank with both black and white sides. The assay presented here is an inexpensive, simple alternative that creates a response that is easily measured. The setup consists of a tripod, webcam and nested testing tanks, readily available in most Xenopus laboratories. This article includes a movie showing the behavior of tadpoles, before and after severing the optic nerve. In order to test the function of one eye, we also include representative results of a tadpole in which each eye underwent retinal axotomy on consecutive days. Future studies could develop an automated version of this assay for testing the vision of many tadpoles at once.

A Simple Behavioral Assay for Testing Visual Function in Xenopus laevis

Xenopus laevis tadpoles prefer swimming on the white side of a black/white tank. This behavior is guided by their vision. Based on this behavior, we present a simple assay to test the visual function of tadpoles.

一个简单的行为分析在检测视觉功能<em&gt;非洲爪蟾</em

Measurement of the visual function in the tadpoles of the frog, Xenopus laevis, allows screening for blindness in live animals. The optokinetic response ...

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by stressed conspecifics due to an odorant released during stress known as the Drosophila stress odorant (dSO). Through the use of the T-maze apparatus, one can quantify the avoidance of the dSO by responder flies in a very affordable and robust assay. Conditions necessary to obtain a strong performance are presented here. A stressful experience is necessary for the flies to emit dSO, as well as enough emitter flies to cause a robust avoidance response to the presence of dSO. Genetic background, but not their group size, strongly altered the avoidance of the dSO by the responder flies. Canton-S and Elwood display a higher performance in avoiding the dSO than Oregon and Samarkand strains. This behavioral assay will allow identification of mechanisms underlying this social behavior, and the assessment of the influence of genes and environmental conditions on both emission and avoidance of the dSO. Such an assay can be included in batteries of simple diagnostic tests used to identify social deficiencies of mutants or environmental conditions of interest.

Straightforward Assay for Quantification of Social Avoidance in Drosophila melanogaster

Here, we present a protocol to quantify the avoidance of stressed individuals. This paradigm is powerful yet user-friendly and can be used to assess the influence of genes and environment on one kind of social interaction in Drosophila melanogaster.

简单的检测方法的社交回避的量化<em&gt;果蝇</em

Drosophila melanogaster is an emerging model to study different aspects of social interactions. For example, flies avoid areas previously occupied by ...

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy and cheap methods to distinguish between individuals in a trial. A standard technique for this is CO2 anaesthesia and then labelling or wing clipping each fly. However, this is invasive and has been shown to affect behavior. Other techniques have used coloration to identify flies. This article presents a simple and non-invasive method for labelling Drosophila that allows them to be individually identified within experiments, using food coloring. This method is used in trials where two males compete to mate with a female. Dyeing allowed quick and easy identification. There was, however, some difference in the strength of the coloration across the three species tested. Data is presented showing the dye has a lower impact on mating behavior than CO2 in Drosophila melanogaster. The impact of CO2 anaesthesia is shown to depend on the species of Drosophila, with D. pseudoobscura and D. subobscura showing no impact, whereas D. melanogaster males had reduced mating success. The dye method presented is applicable to a wide range of experimental designs.

Dyeing Insects for Behavioral Assays: the Mating Behavior of Anesthetized Drosophila

This protocol describes a simple, cost effective way to individually identify Drosophila or other insects. Demonstration data investigating mating success across three species of Drosophila show that this method is comparable or better than the use of CO2 anaesthesia.

染色昆虫行为检测:麻醉的交配行为<em&gt;果蝇</em

Mating experiments using Drosophila have contributed greatly to the understanding of sexual selection and behavior. Experiments often require simple, easy ...

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.

An Inexpensive, Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Straightforward assays for measuring ethanol sensitivity and rapid tolerance in Drosophila facilitate the use of this model organism for investigating these important ethanol-related behaviors. Here, a relatively simple, scalable assay for measuring ethanol sensitivity and rapid tolerance in flies is described.

一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受<em&gt;果蝇</em

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in ...

Conditions Affecting Social Space in Drosophila melanogaster

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a straightforward manner. As we previously demonstrated 1, in a two-dimensional chamber, we first force the flies to form a tight group, subsequently allowing them to take their preferred distance from each other. After the flies have settled, we measure the distance to the closest neighbor (or social space), processing a static picture with free online software (ImageJ). The analysis of the distance to the closest neighbor allows researchers to determine the effects of genetic and environmental factors on social interaction, while controlling for potential confounding factors. Diverse factors such as climbing ability, time of day, sex, and number of flies, can modify social spacing of flies. We thus propose a series of experimental controls to mitigate these confounding effects. This assay can be used for at least two purposes. First, researchers can determine how their favorite environmental shift (such as isolation, temperature, stress or toxins) will impact social spacing 1,2. Second, researchers can dissect the genetic and neural underpinnings of this basic form of social behavior 1,3. Specifically, we used it as a diagnostic tool to study the role of orthologous genes thought to be involved in social behavior in other organisms, such as candidate genes for autism in humans 4.

Conditions Affecting Social Space in Drosophila melanogaster

The effect of genes and environment on social space of Drosophila melanogaster can be quantified through a powerful but straightforward analytical paradigm. We show here different factors that can affect this social space, and thus need to be taken into consideration when designing experiments in this paradigm.

在条件影响社会空间<em&gt;果蝇</em

The social space assay described here can be used to quantify social interactions of Drosophila melanogaster &#8212; or other small insects &#8212; in a ...

一个行为分析对机械感觉MARCM为基础的克隆

摘要

探索更多视频

此视频中的章节

评估大鼠蔗糖渴求育成的一个通用方法

电检测屏幕的配对Nanoinjection和生物活性化合物的使用果蝇巨人纤维系统

一个轻量级的，耳机为基础的系统操作鸣禽听觉反馈

Optogenetic刺激的逃避行为

单苍蝇检测方法的觅食行为的

一个简单的行为分析在检测视觉功能

简单的检测方法的社交回避的量化

染色昆虫行为检测:麻醉的交配行为

一种廉价的，可扩展的行为分析测量乙醇镇静灵敏度和快速耐受

在条件影响社会空间