We gratefully acknowledge support by the Harold and Pauline Price Chair in Ophthalmology and the Jules Stein Eye Institute to MBG, the NEI Core grant (EY00331-43) to SN. Research was supported in part by a generous gift from the Sakaria family to SN and MGB, and from an unrestricted grant from the Research to Prevent Blindness to the Department of Ophthalmology. We thank Charlotte Yiyi Wang at Berkeley School of Optometry for obtaining initial OCT images of subretinal injections.

动物:野生型C57BL / 6J小鼠在加州大学洛杉矶分校（UCLA）育成。所有动物为11之间 - 周17岁，并包括雄性和雌性小鼠。所有小鼠群养，保持与食品和水随意 12:12光照/黑暗周期。所有实验均按照加州大学洛杉矶分校和视觉的研究协会和眼科声明在眼科和视觉研究使用动物的机构准则进行的。 注:所有的药物和注射剂是美国药典（USP）级。 1.手术准备<ol><li>麻醉小鼠以100毫克/千克氯胺酮和8毫克/公斤赛拉嗪的盐水混合物腹膜内注射。辖麻醉的深度，使得鼠标有没有脚趾捏或角膜反射触摸。 </li><li>维持体温在37.0℃用循环水垫。 </li><li>扩张2.5％福林眼ð学生ROPS和修剪胡须，以方便可视化。晶须鼠标提供显著感觉输入，因此，晶须修整应该只删除部阻止明确前往眼睛，而不是与晶须的基极。根据我们的经验，小鼠显示该过程后正常恢复。应用甲基纤维素滴眼液，以防止干燥和减少麻醉药引起的瞬态白内障13。 </li><li>消毒手术前（ 即，聚维酮碘和乙醇或烫珠）仪器。 </li><li>制备稀释的荧光素（使用0.9％盐水0.01％）在无菌环境中（ 即 ，生物安全柜）如果可视化将被执行（见下文第3节）。 </li></ol> 2.注射整地<ol><li>准备的注射器（ 例如 ，5微升注射器）与适当的喷射量（ 例如 ，0.3〜1.0微升）。 </li><li>所以眼睛朝上，并在二清晰可见鼠标位置ssecting显微镜。 </li><li>轻轻一捏用细尖镊子时间结膜。使用弯曲Vannas剪刀大约90度的周向切口。 </li><li>重复步骤2.3与底层的眼球筋膜囊。 </li><li>而鼻旋转地球切除用细尖镊子周围的结缔组织。向注射部位大约0.5mm颞视神经工作。使用非常谨慎，以避免破坏眶窦。 </li></ol> 3.巩膜与视网膜下注射注:建议在0.9％盐水0.01％荧光素的注射可以用来协助可视化，同时学习此过程。荧光素的地形分布眼底成像（见下文第4节）进行有效记录。 <ol><li>通过用22.5度的眼用刀片轻轻搔抓眼罩使在注射部位的小巩膜切口。这个切口寿LD仅足够大以允许针的尖端穿过巩膜。 </li><li>插入斜面33克针（倾斜5 - 10°与该斜面面对并成角度平行于视网膜的巩膜注入所需量（ 例如，0.3〜1.0微升0.01％荧光素的用于学习的目的）。 注:通过每次注射前适当的溶剂和去离子水连续清洗彻底清洗维护注射器的无菌。 </li><li>压低柱塞缓慢（超过3〜秒）无需移动针，并用均匀的压力。 注意:当针在视网膜下腔，轻微的阻力将在按下柱塞可以感觉到。将没有对最小的阻力，如果针头穿刺视网膜，和高电阻，如果针不会刺入巩膜或视网膜色素上皮。 </li><li>抽出针头，以尽量减少回流前等待几秒钟。 </li><li>冲洗眼睛用无菌缓冲盐水和保证眼睛ħ作为旋转回其正常位置。 </li></ol> 4. OCT和眼底成像评估视网膜脱离<ol><li>注射来评价喷射的质量后立即和在适当的时间执行OCT成像点注射后根据需要来评估视网膜结构。 注:在类似的研究中使用OCT的实例先前已描述7,14。 <ol><li>调整并对准OCT图像定位到注射部位。注射部位应是中线为0.5mm颞视神经头。或根据需要，如果脱离超出框架不能很好地集中重复。 </li></ol></li><li>可视化视网膜脱离和染料有连接面眼底成像7,14注射区域。 注意:如果一个OCT成像系统不可用时，为实践的载体少量荧光素的注射将允许与执行荧光素血管任何眼底照相机的可视化造影使用相同的激发波长和阻塞过滤器。超荧光的局部区域将会出现在脉管系统下方，如果视网膜下空间被正确地定位在脉管将具有尖锐和明显边界。从注射水泡的边缘将由hyper-到低渗荧光过渡划定。几台仪器提供此功能的鼠标;这里使用的仪器是其他地方14条所描述。 </li></ol> 5.术后护理<ol><li>应用三联抗生素眼药霜厚厚的棉衣，以注入眼睛的角膜表面。 </li><li>将小鼠恢复干净孤笼。请勿将已经接受了手术，直到他们完全康复小鼠。 </li><li>监控麻醉恢复过程中呼吸和温度。监测动物，直到他们能保持胸骨斜卧。 </li><li>执行其他适当的术后监测和治疗，包括卡布洛芬为手术后疼痛管理一个皮下注射（5毫克/千克）。 </li></ol> 6.评估视网膜功能的电描记所（ERG） <ol><li>进行ERG分析预喷射和在适当的时间后喷射作为需要评估视网膜的功能。如果注射制成视网膜下腔，视网膜脱离应72小时内解决。 <ol><li>使用标准的ERG技术如前所述14,15前和注射后评估视网膜的功能。 </li></ol></li></ol> 7.三维重建和水泡量化卷注:OCT具有高对比度包围的视图帧内的整个脱离扫描是最佳的使用。 ImageJ的/斐济17,18和了Imaris被使用，但也可以使用其它的软件。 <ol><li>导出感兴趣的B超扫描，进口的ImageJ /斐济和作物（图像&gt;裁剪）华侨城SC的部分一个使用矩形选择工具来建模。 <ol><li>调整对比度（图像&gt;调整&gt;亮度/对比度），并用一条线连接两个部分划定任何丢失的边界。 </li><li>绘制与线工具（保持移）跨越RPE到感光层的直线。测量（分析&gt;测量）的行的长度，以获得最大分离尺寸为步骤7.8。 </li></ol></li><li>进口裁剪帧的三维重建软件（见材料表）使用"RGB到灰色"插件和MATLAB编译器运行时。 </li><li>设置使用校准参数从OCT的扫描（X，Y，Z）的体素尺寸（下图片属性）。 </li><li>执行"RGB格雷"插件（在图像属性），具有相同的权重给每个通道，创建第四个通道。删除原始红 - 绿 - 蓝通道。 </li><li>使用反转对比变化的灰色通道。店铺形象。 </li><li>点击"添加EW面"中的3D视图选项卡按钮，开始创建面的引导下4个步骤。 <ol><li>设置表面水平细节（步骤4 1）。 注意:根据我们的经验8.0至12.0是最有效的范围。 </li><li>设置最大球体大小（背景下的选择）比7.1.2测得的最大尺寸支队略少。创建面和撤消灰色通道反转（步骤4 2）。 </li><li>阈值设定为最大值，使视网膜外的负空间和脱离的表面不接触（4步骤3）来。 </li><li>设置过滤器类型的体素的数目和按大小分离在分离部位的负空间。完成表面（步骤4 4）。 注:该支队面的体积位于下的统计标签的体积。 </li></ol></li></ol>

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None of the authors have any commercial disclosures.

视网膜下注射是选择的方法为病毒载体的递送和干细胞衍生的疗法用于操纵光感受器和在基础研究和临床治疗视网膜色素上皮。在患者中，视网膜下注射是通常用前巩膜通过用直接可视化的针进行在玻璃体，视网膜的后部芯玻璃体切割术及渗透。如同大多数玻璃体切割程序，是很常见的，除非眼睛已经晶状体过早发生白内障形成。在小鼠中，视网膜下注射传统上与巩膜前到视网膜，经常与透镜，?...

视网膜下注射是递送细胞和病毒剂的小鼠的视网膜研究其对感光体和下面的视网膜色素上皮1,2-效果的主要手段。在小鼠中大多数视网膜下注射协议使用transcorneal或经巩膜注射部位前到赤道（ 图1）。这种方法可能会导致在包括切口和透镜的所得混浊时，神经视网膜和虹膜，视网膜出血，大量视网膜脱离和持久的视网膜下水肿3-9的玻璃体，渗透的完整性的破坏固有的附带损害。实验操作必须以评价治疗性干预3,7,10,11的作用克服这些影响。本研究提供了一种避免这些并发症的后经巩膜注射方法的详细描述和验证，减少创伤和具有定位子的高成功率视网膜空间。 注射小鼠靶向视网膜下空间往往非常难以执行并且多数研究者遇到失败的尝试，其中该载体被递送到不正确的位置或有显著视网膜损伤，例如，在一个完整的视网膜脱离6的高频。眼睛从分析中排除，因为注射的并发症的数量一般不报道在小鼠研究中，但在我们自己的经验，并与其他研究者的讨论中，失败的注射次数可以是高达50％，并且变化依赖于经验和谁正在执行注射研究者的能力。注射的成功通常通过直接眼底成像和/或光学相干断层扫描（OCT）7,9评估。在小鼠高成功率视网膜下注射容易掌握的方法可以加速实验，减少特雷的临床前研究的成本atments视网膜疾病是在美国失明的主要原因。 后部，这里描述经巩膜视网膜下注射技术是从临床和临床前协议9,12的适应。在注射的小鼠进行的非侵入性诊断评估论证温和，高度本地化的破坏和缺乏额外的抵押镜头，视网膜和视网膜色素上皮的损伤。此外，以相对较少的实践中，实验者可以实现这些结果以高成功率（80 - 90％或更好），从而降低了与这类研究相关的成本。此过程可用于递送细胞，病毒，或药理学治疗干预到光感受器和/或视网膜色素上皮中的临床前研究，并轻松地评估实验介入。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Hamilton Model 62 RN SYR</td>
			<td>Hamilton</td>
			<td>87942</td>
			<td>Syringe x 1</td>
		</tr>
		<tr>
			<td>Hamilton Needle 33G, 1.0&#34;, 20 DEG, point 3 (304 stainless steel)</td>
			<td>Hamilton</td>
			<td>7803-05</td>
			<td>Needles x 6</td>
		</tr>
		<tr>
			<td>Vannas Curved Scissors</td>
			<td>Ted Pella, INC.</td>
			<td>1347</td>
			<td>5mm Blade</td>
		</tr>
		<tr>
			<td>22.5 Degree Microsurgery Knife</td>
			<td>Wilson Ophthalmic Corp.</td>
			<td>91204</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketaject&#160;</td>
			<td>Phoenix</td>
			<td>NDC 57319-609-02</td>
			<td>Ketamine</td>
		</tr>
		<tr>
			<td>Anased</td>
			<td>Lloyd Laboratories</td>
			<td>NDC 61311-482-10</td>
			<td>Xylazine</td>
		</tr>
		<tr>
			<td>Fluorescein 10% AK-Fluor</td>
			<td>Akorn</td>
			<td>NDC 17478-253-10</td>
			<td>100mg/ml</td>
		</tr>
		<tr>
			<td>0.9% Saline USP</td>
			<td>Hospira</td>
			<td>NDC 0409-4888-50</td>
			<td>0.9% NaCl</td>
		</tr>
		<tr>
			<td>Antibiotic Ointment</td>
			<td>Akorn</td>
			<td>NDC 17478-235-35</td>
			<td>Ophthalmic</td>
		</tr>
		<tr>
			<td>Water Circulating Pump</td>
			<td>Gaymar</td>
			<td>TP-500 T/Pump&#160;</td>
			<td>P/N 07999-000</td>
		</tr>
		<tr>
			<td>sd-OCT</td>
			<td>Bioptigen</td>
			<td>R-Series</td>
			<td>Commercial</td>
		</tr>
		<tr>
			<td>Fundus Camera</td>
			<td>Phoenix Research Laboratories</td>
			<td>MICRON III</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers Type 3</td>
			<td>Ted Pella, INC.</td>
			<td>5385-3SU</td>
			<td></td>
		</tr>
		<tr>
			<td>2.5% Phenylephrine</td>
			<td>Paragon BioTeck</td>
			<td>NDC 42702-102-15</td>
			<td>Ophthalmic</td>
		</tr>
		<tr>
			<td>IMARIS8</td>
			<td>Bitplane</td>
			<td></td>
			<td>Version 8.1.2</td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>V1.8.0_77</td>
		</tr>
		<tr>
			<td>Hypromellose&#160;2.5%</td>
			<td>Goniovisc</td>
			<td>AX0401</td>
			<td>Methylcellulose</td>
		</tr>
		<tr>
			<td>Eye Drops (Rinse)</td>
			<td>Bausch &#38; Lomb</td>
			<td></td>
			<td>Saline Solution</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Stemi 2000</td>
			<td>Microscope</td>
		</tr>
		<tr>
			<td>Light source</td>
			<td>Fostec</td>
			<td>P/N 20520</td>
			<td>Light source</td>
		</tr>
	</tbody>
</table>

an alternative and validated injection method for accessing the subretinal space via a transcleral posterior approach

视网膜下注射已在人类和啮齿类动物被成功地用于递送蛋白质的治疗性干预，抗病毒剂，以及细胞到具有直接暴露于光感受器和视网膜色素上皮细胞（RPE）的interphotoreceptor /视网膜下舱。纤维蛋白溶酶原的视网膜下注射，以及最近的临床前和临床试验已经证明递送病毒载体和干细胞的个体具有先进视网膜疾病的安全性和/或功效。视网膜疾病，特别是遗传性视网膜营养不良的小鼠模型，用于测试这些治疗是必不可少的。在啮齿类动物中最常见的注射程序是用小transcorneal或经巩膜切口用前路到视网膜。通过这种方法，注射针头穿透视网膜神经上皮层破坏底层RPE和插入可以轻松尼克镜头，引起晶状体混浊及无创意马减值准备NG。通过经巩膜访问视网膜下的空间，后路避免了这些问题:针穿过巩膜从视神经约为0.5毫米，没有视网膜渗透并避免破坏玻璃体。附带损害仅限于与焦点巩膜和一过性，浆液性视网膜脱离的影响有关。该方法的简单性最大限度地减少眼外伤，确保快速视网膜复位和恢复，并具有低故障率。视网膜和RPE的损害最小允许的疗效明确评估和治疗剂本身的直接影响。该原稿描述了一种新颖的视网膜下注射技术，可用于靶向病毒载体，药剂，干细胞或诱导的多能干细胞（iPS细胞），以在具有高效力，最小的损伤，快恢复小鼠的视网膜下间隙。

后路经巩膜视网膜下注射了31健康的眼睛用0.3微升（N = 18），0.5微升（N = 8）和0.01％荧光素的1.0微升（N = 5）注射执行16个野生型小鼠。一只眼睛由于该防止结构和功能分析一个预先存在的角膜混浊排除注射。每次注射的眼睛是包括本报告。没有意外的视网膜脱离，检测也没有透镜切口，炎症反应，葡萄膜炎，或外科手术后的感染中观察到的任何眼睛的任何证据的神经视网?...

Watch this Scientific Journal Video about An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach at JoVE.com

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

用于访问视网膜下腔的另类和验证注射法<i&gt;通过</i&gt;一经巩膜后路

Subretinal injections have been successfully used in both humans and rodents to deliver therapeutic interventions of proteins, viral agents, and cells to ...

an-alternative-validated-injection-method-for-accessing-subretinal

Universidad de Cartagena UDEC

Research

JoVE Journal

Neuroscience

An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

13.1K 次观看.  University of California, Los Angeles. Subretinal injections are the most common technique for delivering large therapeutic agents such as proteins and viral vectors to photoreceptors and the retinal pigment epithelium. An alternative method in mice that successfully targets the subretinal space with minimal collateral damage and fast recovery times is described here.

视频: An Alternative and Validated Injection Method for Accessing the Subretinal Space via a Transcleral Posterior Approach

Novel Apparatus and Method for Drug Reinforcement

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction. Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in animal research to model various components of drug reinforcement, consumption, and addiction in humans. For this study, we used a novel approach to studying drug reinforcement in rats by combining traditional CPP and self-administration methodologies. We assembled an apparatus using two Med Associate operant chambers, sensory stimuli, and a Plexiglas-constructed neutral zone. These modifications allowed our experiments to encompass motivational aspects of drug intake through self-administration and drug-free assessment of drug/cue conditioning strength with the CPP test. In our experiments, rats self-administered cocaine (0.75 mg/kg/inj, i.v.) during either four (e.g., the "short-term") or eight (e.g., the "long-term") alternating-day sessions in an operant environment containing distinctive sensory cues (e.g., olfactory and visual). On the alternate days, in the other (differently-cued) operant environment, saline was available for self-infusion (0.1 ml, i.v.). Twenty-four hours after the last self-administration/cue-pairing session, a CPP test was conducted. Consistent with typical CPP findings, there was a significant preference for the chamber associated with cocaine self-administration. In addition, in animals undergoing the long-term experiment, a significant positive correlation between CPP magnitude and the number of cocaine-reinforced lever responses. In conclusion, this apparatus and approach is time and cost effective, can be used to examine a wide array of topics pertaining to drug abuse, and provides more flexibility in experimental design than CPP or self-administration methods alone.

Operant drug self-administration and conditioned place preference (CPP) procedures are expansively used in research to model various components of drug reinforcement, consumption, and addiction in humans. In this report, we combined traditional CPP and self-administration methods as a novel approach to studying drug reinforcement and addiction in rats.

新型仪器和药物加固方法

Animal models of reinforcement have proven to be useful for understanding the neurobiological mechanisms underlying drug addiction.  Operant drug ...

科研

神经科学

A Method of Nodose Ganglia Injection in Sprague-Dawley Rat

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located in the organs of the abdomen and thorax. The vagus nerve communicates information from stimuli such as heart rate, blood pressure, bronchopulmonary irritation, and gastrointestinal distension to the nucleus of solitary tract of the medulla. The cell bodies of the vagus nerve are located in the nodose and petrosal ganglia, of which the majority are located in the former. The nodose ganglia contain a wealth of receptors for amino acids, monoamines, neuropeptides, and other neurochemicals that can modify afferent vagus nerve activity. Modifying vagal afferents through systemic peripheral drug treatments targeted at the receptors on nodose ganglia has the potential of treating diseases such as sleep apnea, gastroesophageal reflux disease, or chronic cough. The protocol here describes a method of injection neurochemicals directly into the nodose ganglion. Injecting neurochemicals directly into the nodose ganglia allows study of effects solely on cell bodies that modulate afferent nerve activity, and prevents the complication of involving the central nervous system as seen in systemic neurochemical treatment. Using readily available and inexpensive equipment, intranodose ganglia injections are easily done in anesthetized Sprague-Dawley rats.

Afferent vagal signaling transmits important information to central nervous system from receptors located in organs of the abdomen and thorax. The nodose ganglia of vagus nerves contain many types of receptors that modulate vagal activity. This protocol describes a method of local injections of neurochemicals into the nodose ganglia.

结状神经节注射液只SD大鼠的方法

Afferent signaling via the vagus nerve transmits important general visceral information to the central nervous system from many diverse receptors located ...

Limbal Approach-Subretinal Injection of Viral Vectors for Gene Therapy in Mice Retinal Pigment Epithelium

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy in retinopathies using non-viral and viral gene delivery to the retina and retinal pigment epithelium (RPE). Subretinal injection is the best approach to deliver viral vectors directly to RPE cells. Before the clinical trial of a gene therapy, it is inevitable to validate the efficacy of the therapy in animal models of various retinopathies. Thus, subretinal injection in mice becomes a fundamental technique for an ocular gene therapy. In this protocol, we provide the easy and replicable technique for subretinal injection of viral vectors to experimental mice. This technique is modified from the intravitreal injection, which is widely used technique in ophthalmology clinics. The representative results of RPE/choroid/scleral complex flat-mount will help to understand the efficacy of this technique and adjust the volume and titer of viral vectors for the extent of gene transduction.

Subretinal injection is a surgical technique for effective gene delivery to retinal pigment epithelium in the mouse eye. Here we describe an easy and replicable method for subretinal injection of viral vectors to retinal pigment epithelium in experimental mice.

病毒载体的基因治疗的小鼠视网膜色素上皮缘方针，视网膜下注射

The eye is a small and enclosed organ which makes it an ideal target for gene therapy. Recently various strategies have been applied to gene therapy 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 ...

A Simple Alternative to Stereotactic Injection for Brain Specific Knockdown of miRNA

MicroRNAs (miRNAs) are key regulators of gene expression. In the brain, vital processes like neurodevelopment and neuronal functions depend on the correct expression of microRNAs. Perturbation of microRNAs in the brain can be used to model neurodegenerative diseases by modulating neuronal cell death. Currently, stereotactic injection is used to deliver miRNA knockdown agents to specific location in the brain. Here, we discuss strategies to design antagomirs against miRNA with locked nucleotide modifications (LNA). Subsequently describe a method for brain specific delivery of antagomirs, uniformly across different regions of the brain. This method is simple and widely applicable since it overcomes the surgery, associated injury and limitation of local delivery in stereotactic injections. We prepared a complex of neurotropic, cell-penetrating peptide Rabies Virus Glycoprotein (RVG) with antagomir against miRNA-29 and injected through tail vein, to specifically deliver in the brain. The antagomir design incorporated features that allow specific targeting of the miRNA and formation of non-covalent complexes with the peptide. The knock-down of the miRNA in neuronal cells, resulted in apoptotic cell death and associated behavioural defects. Thus, the method can be used for acute models of neuro-degeneration through the perturbation of miRNAs.

MicroRNAs play crucial roles in the brain and are potential targets for modeling neuro-degeneration. However, perturbing miRNA levels is challenging due to the short length of miRNA and inaccessibility of the brain tissue. This video presents a method for antagomir design and brain specific delivery using a neuropeptide in mice.

一个简单的替代，以立体定向注射miRNA的脑特异性敲除

MicroRNAs (miRNAs) are key regulators of gene expression.  In the brain, vital processes like neurodevelopment and neuronal functions depend on the ...

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This complex behavior is influenced by genetic, hormonal and environmental factors. As in many organisms, previous fighting experience influences the fighting strategy of flies and the outcome of later contests: losing a fight increases the probability of losing later contests, revealing &#34;loser&#34; effects that likely involve learning and memory 2-4. The learning and memory that accompanies expression of complex social behaviors like aggression, is sensitive to pre-test handling of animals 5,6. Many experimental procedures are used in different laboratories to study aggression 7-9, however, no routinely used protocol that excludes handling of flies is currently available. Here, we report a new behavioral apparatus that eliminates handling of flies, using instead their innate negative geotactic responses to move animals into or out of fighting chambers. In this protocol, small circular fight arenas containing a food cup are divided into two equal halves by a removable plastic slider prior to introduction of flies. Flies enter chambers from their home isolation vials via sliding chamber doors and geotaxis. Upon removal of plastic sliders, flies are free to interact. After specified time periods, flies are separated again by sliders for subsequent experimentation. All of this is done easily without handling of individual flies. This apparatus offers a novel approach to study aggression and the associated learning and memory, including the formation of &#34;loser&#34; effects in fly fights. In addition, this new general-purpose behavioral apparatus can be employed to study other social behaviors of flies and should, in general, be of interest for investigating experience-related changes in fundamental behavioral processes.

A New Approach that Eliminates Handling for Studying Aggression and the "Loser" Effect in Drosophila melanogaster

During fruit fly fights, the behavioral patterns observed, fight dynamics, and associated learning and memory are influenced by experimental conditions. The protocol presented here describes a novel procedure that entirely eliminates handling of flies during experiments. This improves fight dynamics and allows formation of strong &#34;loser&#34; effects.

一种新的方法，省去了处理的研究侵略和"失败者"的影响<em&gt;果蝇</em

Aggressive behavior in Drosophila melanogaster is composed of the sequential expression of stereotypical behavioral patterns (for analysis see 1). This ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

用优化的n-甲基 d-葡乙胺保护恢复法制备急性脑切片

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method described here enables the investigator to measure long-lasting (from minutes to hours) high-quality intracellular responses from single Drosophila R1-R6 photoreceptors and Large Monopolar Cells (LMCs) to light stimuli. Because the recording system has low noise, it can be used to study variability among individual cells in the fly eye, and how their outputs reflect the physical properties of the visual environment. We outline all key steps in performing this technique. The basic steps in constructing an appropriate electrophysiology set-up for recording, such as design and selection of the experimental equipment are described. We also explain how to prepare for recording by making appropriate (sharp) recording and (blunt) reference electrodes. Details are given on how to fix an intact fly in a bespoke fly-holder, prepare a small window in its eye and insert a recording electrode through this hole with minimal damage. We explain how to localize the center of a cell's receptive field, dark- or light-adapt the studied cell, and to record its voltage responses to dynamic light stimuli. Finally, we describe the criteria for stable normal recordings, show characteristic high-quality voltage responses of individual cells to different light stimuli, and briefly define how to quantify their signaling performance. Many aspects of the method are technically challenging and require practice and patience to master. But once learned and optimized for the investigator's experimental objectives, it grants outstanding in vivo neurophysiological data.

Electrophysiological Method for Recording Intracellular Voltage Responses of Drosophila Photoreceptors and Interneurons to Light Stimuli In Vivo

Sharp microelectrodes enable accurate electrophysiological characterization of photoreceptor and visual interneuron output in living Drosophila. Here we show how to use this method to record high-quality voltage responses of individual cells to controlled light stimulation. This method is ideal for studying neural information processing in insect compound eyes.

为记录细胞内电压响应电法<em&gt;果蝇</em&gt;光感受器和中间对光刺激<em&gt;在体内</em

Voltage responses of insect photoreceptors and visual interneurons can be accurately recorded with conventional sharp microelectrodes. The method ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

利用体外大鼠脑切片研究变性主要事件的一种替代方法

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Induction of Leptomeningeal Cells Modification Via Intracisternal Injection

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the underlying parenchyma. Previously published protocols recommend using straight needles that should be lowered to a maximum of 1-2 mm from the dural surface. The sudden drop in resistance once the dural membrane has been punctured makes it difficult to maintain the needle in a steady position. Our method, instead, employs a needle bent at the tip that can be stabilized against the occipital bone of the skull, thus preventing the syringe from penetrating into the tissue after perforation of the dural membrane. The procedure is straightforward, reproducible, and does not cause long-lasting discomfort in the operated animals. We describe the intracisternal injection strategy in the context of genetic fate mapping of vascular leptomeningeal cells. The same technique can, furthermore, be utilized to address a wide range of research questions, such as probing the role of leptomeninges in neurodevelopment and the spreading of bacterial meningitis, through genetic ablation of genes putatively implicated in these phenomena. Additionally, the procedure can be combined with an automatized infusion system for a constant delivery and used for tracking cerebrospinal fluid movement via injection of fluorescently labelled molecules.

We describe an intracisternal injection that employs a needle bent at the tip that can be stabilized to the skull, thus eliminating the risk of damage to the underlying parenchyma. The approach can be used for genetic fate mapping and manipulations of leptomeningeal cells and for tracking cerebrospinal fluid movement.

通过宫内注射诱导利普托门宁皮细胞修饰

The protocol outlined here describes how to safely and manually inject solutions through the cisterna magna while eliminating the risk of damage to the ...

用于访问视网膜下腔的另类和验证注射法

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