通过计算机跟踪检测大鼠嗅觉行为<em>果蝇</em>在四象限嗅
Summary
We describe here a behavioral setup and data analysis method for assaying olfactory responses of up to 100 vinegar flies (Drosophila melanogaster). This system may be used with single or multiple olfactory stimuli, and adaptable for optogenetic activation or silencing of neuronal subsets.
Abstract
神经生物学的一个主要挑战是要了解如何电路的神经，由于其复杂的行为，强大的基因技术，紧凑的神经系统功能，引导适当的动物行为， 果蝇是这种调查的一个极好的模型系统。实验室行为分析已经长期用于与果蝇模拟自然环境性质和研究下面的相应的行为的神经机制（ 例如趋光性，趋化性，感官学习和记忆）1-3。随着最近的那个标签特定的神经元亚群的转基因果蝇线大集合的可用性，行为检测已采取了突出的作用与行为4-11链接神经元。多功能和可重复的模式，以数据分析的基本计算程序一起，是各种genotyp候选飞线的快速检测不可缺少的上课。特别有用的是设置了在试验动物，实验持续时间和呈现刺激性质的数目灵活。选择的测定法也应生成可再生的数据，很容易获取和分析。在这里，我们提出了测定果蝇的行为反应的系统和协议的详细说明，在一个大的四场舞台上飞起来。设置在这里用来测定苍蝇单个嗅觉刺激的反应;然而，相同的设置可以被修改以测试多个嗅觉，视觉或光遗传学刺激，或这些的组合。嗅觉设置记录蝇种群响应气味的活性，并计算分析方法被应用到量化苍蝇的行为。所收集的数据进行分析，以得到一个快速读出一个实验运行，这是有效的数据收集和实验条件的优化必不可少的。
Introduction
适应并响应外部环境的能力，对于所有动物的生存至关重要。动物需要避免危险，寻找食物和寻找配偶，并从以前的经验中学习。感觉系统功能，以接收各种刺激，如视觉，化学和mechanosensory的，和发送这些信号到中枢神经系统来解释和解码。大脑然后指示基于感知环境适宜的运动行为，如觅食或捕食逃逸。了解感觉系统如何检测外部世界，大脑如何解码并指导决策，是神经生物学的一个重大挑战。
果蝇是研究如何神经回路引导行为的强大模型系统。除了 是简单和廉价的维护， 果蝇表现出许多不同的和复杂的刻板行为，但用COMPAC这样做ŧ约10万神经元的神经系统。强大遗传技术用于操纵果蝇基因组的存在，以及数以千计的转基因品系已生成的选择性和可重复性地标记的神经元10-13的相同的子集。这些转基因品系可以用来选择性地操纵标记神经元（激活或抑制）的活性，并且这些操作可以被用来研究如何神经功能导向的行为。
多种行为检测已制定了各种研究果蝇的行为。 果蝇 ，像许多动物，用他们的指导许多行为选择，比如寻找食物，寻找配偶，避免危险的嗅觉。因此嗅觉是研究外部刺激是如何检测和动物的神经系统解释，引导适当的选择一个良好的感觉系统。这样，一些试验中已经开发了用于investiga婷幼虫和成虫嗅觉行为。传统上，在果蝇嗅觉行为是由两选择T-迷宫范例，它可用于测定先天和了解到嗅觉行为3测定。在该试验中，约50苍蝇给出了两种管之间：一个管包含有问题的气味和其它含有一个控制气味（通常是气味溶剂）。苍蝇给出的设定时间做出选择，然后是在不同的腔室蝇的数目进行计数。尽管T-迷宫是许多实验的简单测定中，也有一些限制。例如，嗅觉行为仅在一个时间点测量的，并且该时间点之前进行不同的选择将被丢弃。同样，人口内苍蝇的个体行为被忽略。另外，T型迷宫要求苍蝇人工计数，这可能会引入错误。最后，因为只有两种测量的选择，这减少经常需要察觉的细微的行为变化的统计力量。到两选择T-迷宫一个替代方案是一个四象限（四场）嗅觉14-18。在该试验中，动物探究在其中每个竞技场的四个角的填充有加臭空气的潜在来源的舞台。竞技场有皱褶星形最大化四个实验确定的气味象限的形成。如果气味在随后的一个角供给它包含只在那一个象限。动物的行为可以相比，其在三个控制象限行为，因为他们进入和离开气味象限被跟踪，和容易。因此四象限嗅觉试验记录到刺激气味在大的试验舞台空间和时间的行为反应。
四象限嗅觉首先由彼得森等人的 15和兽医等人开发的。17，调查醇个别寄生蜂的工厂行为反应。福彻等 18 Semmelhack和王16改编设置监视单个果蝇的嗅觉反应。四象限嗅觉是吸引和排斥反应同样敏感，允许广泛的测试增味剂和条件。自定义编写飞跟踪软件，由Alex Katsov 19开发，目前由儒略布朗（在材料详述）保持，引入额外的优点，以更近的四象限嗅觉14,20-23的实现。现在有可能以测定到在高空间同时100蝇（27.5像素/厘米）和分辨率，它允许提取的各种参数，如在任何时间点的位置，速度和苍蝇的加速度随时间（每秒30帧）。这使得调查果蝇的行为反应的动力学气味20 </s了>。然而，应该指出的是，在人口中的各个蝇在整个跟踪期间的身份不能保持。相反，每个飞行轨道被记录为只要两个飞轨道不相交。在这一点，苍蝇发散后新的轨道进行分配。通过将其它视频俘获软件（在材料表详述），相同的配置允许灵活的跟踪时段，并且可以使用通过以较低的帧速率拍摄图像来跟踪苍蝇长达24小时。此选项被用来研究果蝇产卵行为和喜好产卵14比较他们的身体姿势。四字段嗅觉也可用于研究的反应在多峰（ 例如嗅觉和视觉）刺激，或以光遗传学9或thermogenetic 21刺激感官刺激的演示相结合。此外，高时间分辨率允许轨迹的FO提取R IN乐团数据集的每一个人飞。因此，该方法允许调查嗅觉引导人口行为和也从个人的社交互动。通过该测定所产生的数据是健壮和高度可再现的，允许使用的四场嗅觉用于行为屏幕。
在这里，我们描述了一个四象限嗅觉安装组件。我们进一步展示其在应对苹果醋和排斥响应高度集中丙酸乙酯测定嗅觉吸引力的使用。最后，我们描述和用于记录飞跟踪数据的分析提供了示例代码。
Protocol
Representative Results
Discussion
这里所描述的四场嗅觉是研究大量人口的野生型和突变体果 蝇苍蝇的嗅觉反应一个通用行为系统。每个实验以〜1小时（包括设置，实验运行和清洁），并且4-6实验每天可常规进行。用40-50苍蝇5分钟的典型试验产生大约45万跟踪的数据点进行分析。所描述的结构也可使用，以小的修改，并监控其他昆虫或昆虫幼虫的运动响应于嗅觉或其它感觉刺激在一段时间内，从最小到几天。四象限检…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We thank Terry Shelley for manufacturing the fly arena and the light-tight enclosure, Liz Marr for help with fly stock maintenance, and Xiaojing Gao and Junjie Luo for help with the Matlab code used for data analysis. We thank Johan Lundström at the Monell Chemical Senses Center for demonstrating his odor delivery setup. This work was supported by grants from the Whitehall Foundation (CJP) and NIH NIDCD (R01DC013070, CJP).
Materials
| Air delivery system | (Quantity needed) | ||
| Tubing and connectors | |||
| Thermoplastic NPT(F) Manifolds | Cole-Parmer, IL, USA | R-31522-31 | 1 |
| Hex reducing nipple (1/4MNPT->1/8MNPT) | McMaster-Carr, IL, USA | 5232T314 | 1 |
| Tubing (ID:1/8) | McMaster-Carr, IL, USA | 5108K43 | 50Ft |
| Tubing (ID:1/16) | McMaster-Carr, IL, USA | 52355K41 | 100Ft |
| Barbed tube fittings | McMaster-Carr, IL, USA | 5117K71 | 1pack |
| Push-to-connect tube fittings | McMaster-Carr, IL, USA | 5779K102 | 4 |
| Barbed Tube Fittings (1/4MNPT->1/8BF) | McMaster-Carr, IL, USA | 5463K439 | 1 pack (10) |
| Barbed Tube Fittings (1/8MNPT->1/8BF) | McMaster-Carr, IL, USA | 5463K438 | 2 pack (10) |
| Barbed Tube Fittings (1/8MNPT->1/16BF) | McMaster-Carr, IL, USA | 5463K4 | 2 pack (10) |
| Barbed Tube Fittings (1/4MNPT->1/4BF) | McMaster-Carr, IL, USA | 5670K84 | 1 |
| Hex head plug | McMaster-Carr, IL, USA | 48335K152 | 1 |
| Air pressure regulator, air filter and flowmeters | (Quantity needed) | ||
| Labatory gas drying unit | W A HAMMOND DRIERITE CO LTD, OH, USA | Model: L68-NP-303; stock #26840 | 1 |
| Multitube frames for 150-mm flowtubes | Cole-Parmer, IL, USA | R03215-30 | 1 |
| Multitube frames for 150-mm flowtubes | Cole-Parmer, IL, USA | R03215-76 | 1 |
| 150-mm flowtubes | Cole-Parmer, IL, USA | R-03217-15 | 9 |
| Valve Cartridge | Cole-Parmer, IL, USA | R-03218-72 | 9 |
| Precision Air regulator | McMaster-Carr, IL, USA | 6162K13 | 1 |
| Soleniod valves | Automate Scientific, Berkeley, CA | 02-10i | 4 |
| Solenoid valve controller | ValveLink 8.2, Automate Scientific, Berkeley, CA | 01-18 | 1 |
| Electronic flow meter | Honeywell | AWM3100V | 1 |
| DAQ (NI USB-6009, National Instruments) and a | National Instruments | NI USB-6009 | 1 |
| Power supply | Extech Instruments | 382200 | 1 |
| Odor chambers | |||
| Polypropylene Wide Mouth jar 2oz; 60ml | Nalgene | 562118-0002 | At least 5 are required per experiment, but a separate chamber is required for each dillution of each odorant. Available at Container Store, part #635114) |
| Glass odor chamber, 0.25 oz | Sunburst Bottle | LB4B | At least 5 are required per experiment |
| "In" valve for odor chamber | Smart Products, Inc., CA, USA | 214224PB-0011S000-4074 | 1 of these parts is used per odor chamber but they need to be replaced frequently |
| "Out" valve for odor chamber | Smart Products, Inc., CA, USA | 224214PB-0011S000-4074 | 1 of these parts is used per odor chamber but they need to be replaced frequently |
| O ring | RT Dygert International, MN, USA | AS568-029 Buna-N O-R | 1 pack (100) |
| Fly arena, camera and behavior boxes | (Quantity needed) | ||
| Behavior and camera box material | Interstate plastics, CA, USA | ABS black extruded (https://www.interstateplastics.com/Abs-Black-Extruded-Sheet-ABSBE~~ST.php) | 1803 sq inch |
| Teflon for fly arena and odor chamber inserts, 3/8" thick, 12"x12" | McMaster-Carr, IL, USA | 8545K27 | 1 |
| Glass plates, 1/8" Thick, 9"x 9" | McMaster-Carr, IL, USA | 8476K191 | 2 |
| Dual action thermoelectric controller | WAtronix Inc, CA, USA | DA12V-K-0 | 1 |
| IR LED array | Advanced Illumination, Rochester, VT, USA | AL4554-88024, PS24-TL | 2 LED arrays and one power supply |
| Air conditioner Unit | Melcor Store | MAA280T-12 | 1 |
| Imaging system | (Quantity needed) | ||
| Cosmicar/Pentax C21211TH (12.5mm F/1.4) C-mount Lens | B AND H PHOTO AND ELECTRONICS CORP, NY, USA | PEC21211 KP | 1 |
| CCXC-12P05N Interconnect Cable | B AND H PHOTO AND ELECTRONICS CORP, NY, USA | SOCCXC12P05N | 1 |
| DC-700 Camera Adapter | B AND H PHOTO AND ELECTRONICS CORP, NY, USA | SODC700 | 1 |
| B+W 40,5 093 IR filter | B AND H PHOTO AND ELECTRONICS CORP, NY, USA | 65-072442 | 1 |
| TiFFEN 40.5mm Circular polarizer | Amazon | 1 | |
| IR Videocamera | Industrial Vision Source, FL, USA | Sony XC-EI50 (SY-XC-E150) | 1 |
| USB video converter | The Imagingsource, NC, USA | DFG/USB2-It | 1 |
| iFlySpy2 (fly tracking software) | Julian Brown, Stanford, Calfornia: julianrbrown@gmail.com | iFlySpy2 | 1 |
| IC Capture 2.2 software | The Imagingsource, NC, USA (http://www.theimagingsource.com/en_US/products/software/iccapture/) | ||
| Miscellaneous | (Quantity needed) | ||
| Dremel rotary tool | Dremel, Racine, WI, USA | Dremel 8000-03 | 1 |
| Diamond-coated drill bits for glass cutting | Available from various suppliers; MSC industrial Supply Co, Melville, NY | 90606328 | 1 |
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