由眼视光健康科学学院西部大学DJC提供资金。特别感谢IRISA谭图形设计。

所有的畜牧业和实验应该得到批准，并按照规定由相应的机构或其他法律规定的机构动物护理和使用委员会的准则进行。 1。斑马鱼护理<ol><li>保持在标准条件下的斑马鱼在28.5°C下10小时黑暗14小时光照周期16。 </li><li>保持成年斑马鱼在大约3鱼/ L的油箱密度</li></ol> 2。视动反应（OKR） <ol><li>用14.5厘米直径的旋转鼓轮，立体显微镜可调节光强度设置（300-8,000勒克斯）和计算机（ 图1a）构建自定义OKR录音设备。 <ol><li>安装一个摄像头的显微镜，将提供一个实时输入到附近的监视器，并允许图像捕捉和记录。 </li><li>使用链接到一台计算机，将住宿，一个微控制器控制旋转鼓日期不同速度和方向的选择。 </li></ol></li><li>在0.016％三卡因麻醉的鱼2-3分钟，然后将鱼放在一个小平台，用眼睛和鳃悬在边缘。 </li><li>放置一薄海绵/毛巾在鱼的身体和脚2-3枚泡沫，成形以容纳鱼，在鱼上，以保持它固定。限制了鱼的尾部的运动被认为是固相化的重要方面，而不会造成伤害的鱼，并被最好使用平片的泡沫来实现的。 （ 图1b） </li><li>圆筒形充水水箱内的鱼的位置，即适合的OKR记录装置的旋转滚筒的内部。使用该平台上的磁铁对鱼直立位置时，用眼睛从鼓的边缘约7.3厘米该鱼应该从麻醉恢复在几分钟之内 - 正常的呼吸恢复和随机眼球运动应得到遵守。 </li><li>放置一个BASË光栅每度（CPD）0.07周期进入转鼓，搞计算机的控制，开始旋转和视频捕捉（12 rpm的速度被用于本文，但速度8-16转应该得到相似的结果）。 </li><li>一旦初始OKR由基引起的光栅，暂停旋转简要地，并以较小的光栅（高空间频率）代替光栅。 </li><li>重复这个过程直到一个OKR不再能引出。复试与导致OKR以下修改楼梯的方式，然后重复使用没有引起反应来验证OKR的损失光栅最小的光栅。这个过程可以重复进行，以验证对检测到的最小光栅真实响应如果最终改变光栅相对于第一灭绝事件。 </li><li>通过放置一个黑色的塑料封堵器在相邻的另外一只眼睛的条纹取得单眼视力的测量。重复上述过程（步骤2.6-2.7）。 </li><li>通过重新定位阻塞器，并再次重复该步骤，得到2.6-2.7的相对眼睛的视力。 </li><li>注意每个眼睛距离的条纹通过使用参考测量带材的鱼箱的下部，以便准确敏锐的措施可以在下面的步骤来计算在测试过程中的距离。 </li></ol> 3。计算视力<ol><li>通过计算CPD获得视力: <img alt="式（1）" fo:content-width="0.7in" src="/files/ftp_upload/50832/50832eq1.jpg" width="70px" /> 其中，a是从透镜中心到光栅的距离，h是最小的光栅在这OKR观察到（ 图1c）中的一个周期的长度。 </li><li>对合并的视力测量值，平均同时从左眼和右眼的一个值被使用。 </li></ol>

<ol>
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作者什么都没有透露。

有时鱼可能逃逸到水箱。它以固定的尾部是至关重要的。另外使用一个薄海绵/毛巾在鱼的身体也被发现是非常宝贵的，以保持暂停长时间的鱼。我们可以使用这个程序，而不损害鱼，以保持鱼的固定超过30分钟。这段时间是绰绰有余做的双眼和单眼两种acuities。 光栅的演讲既可以做如这里和其他地方说明使用物理转鼓或凭数字显示18-22。这两种类型有特定的优点和缺点。我们选择使用物理旋转既出于成本考虑和技术规范。不包括计算机和成像设备，这里所描述的OKR设备可以为150美元左右建成。 该OKR是确定可见的客观手段UAL视力在成年斑马鱼。而其他一些视觉动作检测视力已被用于在以前的实验，甚至视网膜电已经在成年斑马鱼的眼睛被成功地完成，尽管它并没有被大量利用，成年斑马鱼OKR打开大门，许多新的实验条件22。这项研究表明了OKR在测量视力的变化随着时间的推移实用，识别眼部疾病模型视力差，并在确定单眼视力。 有趣的是，成年斑马鱼的愿景相比，人类视觉（20/20）大约是20/1，000（时0.60 CPD转换为-0.3 logMAR视力）。虽然这可能看起来很差，斑马鱼的眼睛是非常适合它的环境。尽管期待通过6.5厘米水，聚碳酸酯几毫米，并且空气的另一厘米的视觉任务，鱼表现相当好！我们发现，成年斑马鱼的视力是ABOUT什么将被预测给定的感光间隔23对比度70％，与最佳的人力性能〜20/12，大约是80％的预测的20/10，和OKR技术用于测量在成年斑马鱼的视觉能力是令人印象深刻的24 ，25。 测量独立眼视力的技术将允许研究，其中一只眼睛可以治疗和其他用作对照。此外，鱼眼不对称发展如白内障发展的lrp2/bugeye或鱼可以更准确地监测。应当指出的是护理需要采取准确地测量从眼睛到条纹的距离，并以阻塞视力的全部字段。这种技术应该打开大门，让斑马鱼的视觉功能更准确，更具体的措施。

斑马鱼（ 斑马鱼 ）是研究视觉生理，由于其视网膜的其他脊椎动物，它们的生命周期短，和基因变异的突变体1,2的可用性相似的良好模型。在视动反射/响应/眼球震颤（OKR）是追求平稳和快速的扫视眼球运动的组合。对于超过60年的临床医生已经表明，OKR可用于客观地测量视力的患者，并且是用于确定婴儿视觉能力3-5中特别有用。在动物中首次 ​​记载使用OKR的是在20世纪50年代6使用鸽子。最近，OKR已成为评估斑马鱼幼虫的视觉功能，通常用于筛选具有视觉障碍1,2基因突变的重要工具。而OKR已被广泛使用，以确定在斑马鱼幼鱼视觉功能，但仅在最近被证实在成年斑马鱼7-10。最近的一篇论文被Tappeiner 等人使用传统上用于小鼠市售optomotor系统，OptoMotry，以证明成年斑马鱼的视力仍然相当恒定的，〜每度0.59个周期（CPD），甚至可以跨越不同的角速度7 。 虽然OKR已被证明是在斑马鱼幼虫的研究非常有用的，其他的方法进行视觉行为已被用于斑马鱼与成人不同程度的成功。逃避反应实验结果显示，与白化斑马鱼的红宝石 ，无论是色素突变体，减少了视觉反应在明亮光线条件下11,12。同样的逃避反应实验也成功地识别夜盲症Ð突变体，虽然在2年的13岁。但是，逃避反应检测也不是没有缺点。这是很难归于一个确切的视觉功能和仅为总approximation个视觉上的变化 - 这意味着它需要一个大的变化的变化确定之前。 已经开发了用于识别的成年斑马鱼有视觉障碍的另一种方法是optomotor响应（OMR）11。在该试验中，鱼被放置在一个圆形槽，在该中心不透明的列。黑白条纹绕池和鱼的任务是在条纹运动的方向游动。像逃避反应，对OMR集中在成年斑马鱼的视觉动作能力。然而，已经成功地用于有视觉障碍，如lrp2/bugeye突变体14识别的鱼。该lrp2/bugeye斑马鱼展览牛眼，高眼压，减少OMR，并进行性视网膜变性14,15。 许多研究已经使用的视觉动作的能力在成年斑马鱼，这往往是主观的，不能定量itatively分析。通过使用OKR，可以更客观地研究成鱼的视力。我们已经建立了我们自己的OKR设备，一个最初用于幼虫的研究1为蓝本。在这个研究中，我们证明了OKR可以用来计算两个单眼和在斑马鱼中双眼视力。

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			<td height="64" style="height:64px;width:209px;">Super Low Light Black White Video Security Camera</td>
			<td style="width:143px;">Super Circuits</td>
			<td style="width:119px;">PC164CEX-2</td>
			<td style="width:124px;"> Any low light camera may be used</td>
		</tr>
		<tr height="106">
			<td height="106" style="height:106px;width:209px;">Arduino Duemilanove microcontroller</td>
			<td style="width:143px;">Adafruit Industries</td>
			<td style="width:119px;"> </td>
			<td style="width:124px;"> The Arduino Uno is also compatible with the speed control software.</td>
		</tr>
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			<td height="20" style="height:20px;">Tricaine Methanesulfonate</td>
			<td>VWR</td>
			<td>101107-950</td>
			<td></td>
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the optokinetic response as a quantitative measure of visual acuity in zebrafish

斑马鱼是一种经过验证的模型视觉研究，但许多早期的方法，一般集中在仔鱼或者演示了一个简单的反应。最近在成年斑马鱼的视觉行为已成为一个有意义的，但方法来衡量具体的回应是新来的。为了解决这个差距，我们着手开发一种方法来反复，准确地利用视动反应（OKR）来衡量成年斑马鱼的视力。在这里，我们表明，成年斑马鱼的视力可以被测量，包括双眼和单眼acuities。因为鱼没有在手术过程中受损，视力可以测量和比较多的时间短或长周期。这里所描述的视力测量，也可进行快速，可用于高吞吐量和额外的视觉过程，如果需要的话。这种类型的分析，有利于药物干预研究或疾病进展的调查。

OKR设备功能 如上面所述和如图1所示的功能以最小的维护OKR设备。正如在步骤2.3，固定的尾巴是保持在录制过程中抑制了鱼的关键。充分地固定，鱼可以保持在OKR装置长时间存放。因此，应注意在最初的固定化步骤被采取，使得更多的数据可以被收集为每个单独的鱼。 视力测量 双目OKR很容易引起在正常成鱼。按照以上程序可以发现视力为许多不同年龄的斑马鱼（ 图2）。我们记录了最好的双眼视力空间为0.74 CPD。正如图2所指出的，双眼视力一般随着年龄的增大（N = 46，P = 0.002方差分析采用SPSS 14节），但各级关经过约12个月的年龄。当我们比较所测视力5个月大的野生型鱼年龄相匹配的lrp2/bugeye鱼，我们发现lrp2/bugeye鱼有显著视力降低（P &lt;0.001;野生型:0.49±0.05 CPD，N = 8，lrp2/bugeye:0.35±0.06，N = 10;学生T检验使用Excel）。 通过阻断右眼视力为左眼可以判定，反之亦然。时相比，双眼视力，双眼总和也同样观察到人类视觉17（ 图3）。双眼视力普遍比任何权利（OD）或向左独立测量（OS）acuities更好的5-10％。对于一些鱼个体差异有25％的改善。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50832/50832fig1.jpg" width="500px" /> 图1。 RECO OKR录制A）的OKR装置由14.5厘米直径的旋转鼓轮和显微镜可调节光强度设置（300-8,000勒克斯）的。一个摄像头提供了一个相邻的监视器上实时饲料。每度（CPD）的旋转鼓在顺时针和逆时针的方向和可互换的空间频率光栅不同的速度设置被插入和除去如需要。 乙 ）鱼被固定并放置在旋转滚筒的中心。℃）循环计算使用CPD = <img alt="" src="img2.png" /> <a href="https://www.jove.com/files/ftp_upload/50832/50832fig1highres.jpg" target="_blank">点击此处查看大图</a> 。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/50832/50832fig2.jpg" width="500px" /> 图2。随着年龄增长视力改变 。图表显示测量的5个独立的视力 -<html>年龄15个月。视力通常会增加整个发展的第一年，然后尾巴了一个位在15个月的年龄（P = 0.002方差分析采用SPSS 14节）。每个点可能不是代表1或几个鱼。视力是衡量周期/度（CPD）。 （N = 46总; 8 5个月; 4在6个月; 7为8个 ​​月; 6日9个月; 11日10个月; 2为11个月; 2在12个月和6个在15个月）。 <a href="https://www.jove.com/files/ftp_upload/50832/50832fig2highres.jpg" target="_blank">点击这里查看大图</a> 。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/50832/50832fig3.jpg" width="500px" /> 图3。双眼视力求和效果 。双眼（OU）视力普遍较高是比较合适的（OD）或左（OS）的眼睛acuities。在这个特定基团的OS acuities是显著更低。 （* p值= 0.006，N = 8）jove.com/files/ftp_upload/50832/50832fig3highres.jpg"目标="_blank"&gt;点击这里查看大图。

Watch this Scientific Journal Video about The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish at JoVE.com

The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

斑马鱼已经有很多研究领域的重要工具。在这里，我们展示了一个方法来引出一个视觉反应，并计算在成年斑马鱼功能性视力。

在视动反应在斑马鱼视力的定量测量

Zebrafish are a proven model for vision research, however many of the  earlier methods generally focused on larval fish or demonstrated a simple  ...

the-optokinetic-response-as-quantitative-measure-visual-acuity

Research

JoVE Journal

Neuroscience

20.4K 次观看.  Western University of Health Sciences. 斑马鱼已经有很多研究领域的重要工具。在这里，我们展示了一个方法来引出一个视觉反应，并计算在成年斑马鱼功能性视力。 

视频: The Optokinetic Response as a Quantitative Measure of Visual Acuity in Zebrafish

Double Fluorescence in situ Hybridization in Fresh Brain Sections

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh frozen brain sections. Our group has successfully used this approach to study gene co-regulation. More specifically, we have used this dFISH method to explore the anatomical organization, neurochemical properties, and the impact of sensory experience in central sensory circuits, at single cell resolution. This protocol has been validated in brain tissue from mice, rats and songbirds but is expected to be easily adaptable to other vertebrate species, as well as to an array of non-neural tissues. In this film we provide a detailed demonstration of the main steps of this procedure.

Double Fluorescence in situ Hybridization in Fresh Brain Sections

This protocol involves a non-radioactive in-situ hybridization procedure that enables the simultaneous identification of two transcript species, at a single cell resolution, in thin sections of the vertebrate brain.

双荧光原位杂交食脑切片

Here we describe a modified version of a double fluorescence in situ hybridization (dFISH) method optimized for detecting two mRNAs of interest in fresh ...

科研

神经科学

Hyponeophagia: A Measure of Anxiety in the Mouse

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often hampered by a phenomenon known as "bait shyness". Mice and rats cannot vomit, due to the tightness of the cardiac sphincter of the stomach, so to overcome the problem of potential food toxicity they have evolved a strategy of first ingesting only very small amounts of novel substances. The amounts ingested then gradually increase until the animal has determined whether the substance is safe and nutritious. So the old rat-catchers would first put a palatable substance such as oatmeal, which was to be the vehicle for the toxin, in the infested area. Only when large amounts were being readily consumed would they then add the poison, in amounts calculated not to affect the taste of the vehicle. The poisoned bait, which the animals were now readily eating in large amounts, would then swiftly perform its function.
Bait shyness is now used in the behavioural laboratory as a way of measuring anxiety. A highly palatable but novel substance, such as sweet corn, nuts or sweetened condensed milk, is offered to the mice (or rats) in a novel situation, such as a new cage. The latency to consume a defined amount of the new food is then measured.
Robert M.J. Deacon can be reach at <a href="mailto:robert.deacon@psy.ox.ac.uk">robert.deacon@psy.ox.ac.uk</a>

Mice and rats, due to their innate cautiousness, are initially slow in consuming a novel food, particularly in a novel place. This hyponeophagia can readily be measured in the laboratory, even though laboratory animals are much less anxious than their wild counterparts

Hyponeophagia:在小鼠的焦虑测量

Before the present day, when fast-acting and potent rodenticides such as alpha-chloralose were not yet in use, the work of pest controllers was often ...

A Dual Tracer PET-MRI Protocol for the Quantitative Measure of Regional Brain Energy Substrates Uptake in the Rat

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the rat. The developed method is a small-animal positron emission tomography (PET) protocol, in which&#160;11C-AcAc and 18F-fluorodeoxyglucose (18F-FDG) are injected sequentially in each animal. This dual tracer PET acquisition is possible because of the short half-life of 11C (20.4 min). The rats also undergo a magnetic resonance imaging (MRI) acquisition seven days before the PET protocol. Prior to image analysis, PET and MRI images are coregistered to allow the measurement of regional cerebral uptake (cortex, hippocampus, striatum, and cerebellum). A quantitative measure of 11C-AcAc and 18F-FDG brain uptake (cerebral metabolic rate; &#956;mol/100 g/min) is determined by kinetic modeling using the image-derived input function (IDIF) method. Our new dual tracer PET protocol is robust and flexible; the two tracers used can be replaced by different radiotracers to evaluate other processes in the brain. Moreover, our protocol is applicable to the study of brain fuel supply in multiple conditions such as normal aging and neurodegenerative pathologies such as Alzheimer&#39;s and Parkinson&#39;s diseases.

Small-animal positron emission tomography enables the assessment of the brain&#39;s two main energy substrates:&#160;glucose and ketones. In the present method, 11C-acetoacetate and 18F-fluorodeoxyglucose are injected sequentially in each animal, and their uptake is measured quantitatively in specific brain regions determined from the magnetic resonance images.

We present a method for comparing the uptake of the brain&#39;s two key energy substrates: glucose and ketones (acetoacetate [AcAc] in this case) in the ...

Electroretinogram Analysis of the Visual Response in Zebrafish Larvae

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the surface of the cornea, electrical activity generated in response to light can be measured and used to assess the activity of retinal cells in vivo. This manuscript describes the use of the ERG to measure visual function in zebrafish. Zebrafish have long been utilized as a model for vertebrate development due to the ease of gene suppression by morpholino oligonucleotides and pharmacological manipulation. At 5-10 dpf, only cones are functional in the larval retina. Therefore, the zebrafish, unlike other animals, is a powerful model system for the study of cone visual function in vivo. This protocol uses standard anesthesia, micromanipulation and stereomicroscopy protocols that are common in laboratories that perform zebrafish research. The outlined methods make use of standard electrophysiology equipment and a low light camera to guide the placement of the recording microelectrode onto the larval cornea. Finally, we demonstrate how a commercially available ERG stimulator/recorder originally designed for use with mice can easily be adapted for use with zebrafish. ERG of larval zebrafish provides an excellent method of assaying cone visual function in animals that have been modified by morpholino oligonucleotide injection as well as newer genome engineering techniques such as Zinc Finger Nucleases (ZFNs), Transcription Activator-Like Effector Nucleases (TALENs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9, all of which have greatly increased the efficiency and efficacy of gene targeting in zebrafish. In addition, we take advantage of the ability of pharmacological agents to penetrate zebrafish larvae to evaluate the molecular components that contribute to the photoresponse. This protocol outlines a setup that can be modified and used by researchers with various experimental goals.

We present a method for the electroretinographic (ERG) analysis of zebrafish larvae utilizing micromanipulation and electroretinography techniques. This is a simple and straightforward method for assaying visual function of zebrafish larvae in vivo.

在斑马鱼幼虫的视觉反应的视网膜电图分析

The electroretinogram (ERG) is a noninvasive electrophysiological method for determining retinal function. Through the placement of an electrode on the ...

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise from extra-retinal sources. Ex vivo ERG provides an efficient method to dissect the function of retinal cells directly from an intact isolated retina of animals or donor eyes. In addition, ex vivo ERG can be used to test the efficacy and safety of potential therapeutic agents on retina tissue from animals or humans. We show here how commercially available in vivo ERG systems can be used to conduct ex vivo ERG recordings from isolated mouse retinas. We combine the light stimulation, electronic and heating units of a standard in vivo system with custom-designed specimen holder, gravity-controlled perfusion system and electromagnetic noise shielding to record low-noise ex vivo ERG signals simultaneously from two retinas with the acquisition software included in commercial in vivo systems. Further, we demonstrate how to use this method in combination with pharmacological treatments that remove specific ERG components in order to dissect the function of certain retinal cell types.

Simultaneous ex vivo Functional Testing of Two Retinas by in vivo Electroretinogram System

Ex vivo ERG can be used to record electrical activity of retinal cells directly from isolated intact retinas of animals or humans. We demonstrate here how common in vivo ERG systems can be adapted for ex vivo ERG recordings in order to dissect the electrical activity of retinal cells.

同时<em&gt;体外</em两个由视网膜&gt;功能测试<em&gt;在体内</em&gt;电图系统

An In vivo electroretinogram (ERG) signal is composed of several overlapping components originating from different retinal cell types, as well as noise ...

Visual Evoked Potential Recording in a Rat Model of Experimental Optic Nerve Demyelination

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to monitor the disease course in the follow-up period. Changes in the VEP parameters closely correlate with pathological damage in the optic nerve. This protocol provides a detailed description about the rodent model of optic nerve microinjection, in which a partial demyelination lesion is produced in the optic nerve. VEP recording techniques are also discussed. Using skull implanted electrodes, we are able to acquire reproducible intra-session and between-session VEP traces. VEPs can be recorded on individual animals over a period of time to assess the functional changes in the optic nerve longitudinally. The optic nerve demyelination model, in conjunction with the VEP recording protocol, provides a tool to investigate the disease processes associated with demyelination and remyelination, and can potentially be employed to evaluate the effects of new remyelinating drugs or neuroprotective therapies.

Focal demyelination is induced in the optic nerve using lysolecithin microinjection. Visual evoked potentials are recorded via skull electrodes implanted over the visual cortex to examine the signal conduction along the visual pathway in vivo. This protocol details the surgical procedures underlying electrode implantation and optic nerve microinjection.

在实验视神经脱髓鞘模型大鼠视觉诱发电位记录

The visual evoked potential (VEP) recording is widely used in clinical practice to assess the severity of optic neuritis in its acute phase, and to ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

体内基因转移到雪旺细胞在啮齿动物坐骨神经电穿孔

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Drosophila Courtship Conditioning As a Measure of Learning and Memory

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model organisms such as the fruit fly, Drosophila melanogaster. Drosophila is useful for understanding the basic neurobiology underlying cognitive deficits resulting from mutations in genes associated with human cognitive disorders, such as intellectual disability (ID) and autism. This work describes a methodology for testing learning and memory using a classic paradigm in Drosophila known as courtship conditioning. Male flies court females using a distinct pattern of easily recognizable behaviors. Premated females are not receptive to mating and will reject the male&#39;s copulation attempts. In response to this rejection, male flies reduce their courtship behavior. This learned reduction in courtship behavior is measured over time, serving as an indicator of learning and memory. The basic numerical output of this assay is the courtship index (CI), which is defined as the percentage of time that a male spends courting during a 10 min interval. The learning index (LI) is the relative reduction of CI in flies that have been exposed to a premated female compared to na&#239;ve flies with no previous social encounters. For the statistical comparison of LIs between genotypes, a randomization test with bootstrapping is used. To illustrate how the assay can be used to address the role of a gene relating to learning and memory, the pan-neuronal knockdown of Dihydroxyacetone phosphate acyltransferase (Dhap-at) was characterized here. The human ortholog of Dhap-at, glyceronephosphate O-acyltransferase (GNPT), is involved in rhizomelic chondrodysplasia punctata type 2, an autosomal-recessive syndrome characterized by severe ID. Using the courtship conditioning assay, it was determined that Dhap-at is required for long-term memory, but not for short-term memory. This result serves as a basis for further investigation of the underlying molecular mechanisms.

Drosophila Courtship Conditioning As a Measure of Learning and Memory

This protocol describes a Drosophila learning and memory assay called courtship conditioning. This classic assay is based on a reduction of male courtship behavior after sexual rejection by a non-receptive premated female. This natural form of behavioral plasticity can be used to test learning, short-term memory, and long-term memory.

果蝇调理作为学习和记忆的措施

Many insights into the molecular mechanisms underlying learning and memory have been elucidated through the use of simple behavioral assays in model ...

Using Optical Coherence Tomography and Optokinetic Response As Structural and Functional Visual System Readouts in Mice and Rats

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for the investigation of processes of neurodegeneration, neuroprotection and neuro-repair involving the visual system, as these often correlate well with retinal changes. As a functional readout, visually evoked compensatory eye and head movements are commonly used in experimental models involving the visual function. Combining both techniques allows a quantitative in vivo investigation of structure and function, which can be used to investigate the pathological conditions or to evaluate the potential of novel therapeutics. A great benefit of the presented techniques is the possibility to perform longitudinal analyses allowing the investigation of dynamic processes, reducing variability and cuts down the number of animals needed for the experiments. The protocol described aims to provide a manual for acquisition and analysis of high quality retinal scans of mice and rats using a low cost customized holder with an option to deliver inhalational anesthesia. Additionally, the proposed guide is intended as an instructional manual for researchers using optokinetic response (OKR) analysis in rodents, which can be adapted to their specific needs and interests.

A detailed protocol for the assessment of structural and visual readouts in rodents by optical coherence tomography and optokinetic response is presented. The results provide valuable insights for ophthalmologic as well as neurologic research.

光学相干层析成像和光运动响应在小鼠和大鼠中的结构和功能视觉系统读数

Optical coherence tomography (OCT) is a fast, non-invasive, interferometric technique allowing high-resolution retinal imaging. It is an ideal tool for ...

Double Direct Injection of Blood into the Cisterna Magna as a Model of Subarachnoid Hemorrhage

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% of the total stroke-related mortality with an important morbidity in terms of neurological outcome. A delayed cerebral vasospasm (CVS) may occur most often in association with a delayed cerebral ischemia. Different animal models of SAH are now being used including endovascular perforation and direct injection of blood into the cisterna magna or even the prechiasmatic cistern, each exhibiting distinct advantages and disadvantages. In this article, a standardized mouse model of SAH by double direct injection of determined volumes of autologous whole blood into the cisterna magna is presented. Briefly, mice were weighed and then anesthetized by isoflurane inhalation. Then, the animal was placed in a reclining position on a heated blanket maintaining a rectal temperature of 37 &#176;C and positioned in a stereotactic frame with a cervical bend of about 30&#176;. Once in place, the tip of an elongated glass micropipette filled with the homologous arterial blood taken from carotid artery of another mouse of the same age and gender (C57Bl/6J) was positioned at a right angle in contact with the atlanto-occipital membrane by means of a micromanipulator. Then 60 &#181;L of blood was injected in the cisterna magna followed by a 30&#176; downward tilt of the animal for 2 minutes. The second infusion of 30 &#181;L of blood into the cisterna magna was performed 24 h after the first one. The individual follow-up of each animal is carried out daily (careful evaluation of weight and well-being). This procedure allows a predictable and highly reproducible distribution of blood, likely accompanied by intracranial pressure elevation that can be mimicked by an equivalent injection of an artificial cerebral spinal fluid (CSF), and represents an acute to mild-model of SAH inducing low mortality.

We described in this protocol a standardized subarachnoid hemorrhage (SAH) mouse model by a double injection of autologous whole-blood into the cisterna magna. The high degree of standardization of the double-injection procedure represents a middle-to-acute model of SAH with relative safety regarding mortality.

双直接注射血液到西斯特纳麦格作为苏巴拉奇内德出血的模型

Among strokes, subarachnoid hemorrhage (SAH) consecutive to the rupture of a cerebral arterial aneurysm represents 5-9% but is responsible for about 30% ...