笔者想感谢西峡罗优良的鱼类养殖和技术支持。这项工作是由美国国立卫生赠款R21EY019401（RT）和P30EY04068（RT），和启动资金RT，包括无限制的授予从韦恩州立大学，眼科防盲研究。 JT是由托马斯·C.隆隆韦恩州立大学研究生院提供的奖学金支持。

在韦恩州立大学医学院动物使用委员会批准，在本协议中所述的所有程序。 1。暗适应<ol><li>转让〜10成年白化或色素的鱼，从正常的住房制度进入一个黑暗的外壳。如果可用，建成斑马鱼的正常水流通过水箱外壳模块，它允许使用一个黑暗的外壳。 （如果这样的系统不可用，放置鱼缸在一个完全黑暗的外壳，确保通气鱼与氧气）。 </li><li>保持10天的鱼在黑暗中。当喂养动物或加入新鱼暗箱，确保尽可能快地移动，以避免将鱼暴露在相当长的一段光。 </li></ol> 2。紫外线照射<ol><li>确保紫外光源电源关闭。卸下UV光灯丝从荧光体视显微镜。 <ol><li>使用倒置直径15厘米的玻璃培养皿（或一些类似的2厘米的高度）作为一个独立的UV长丝。用胶带将培养皿中的实验室工作台上。 </li><li>带UV光长丝顶部倒培养皿。这样排列〜2毫米的灯丝的端部悬伸的陪替氏培养皿。 </li></ol></li><li>获得的250毫升的玻璃烧杯中。量½底部，侧面，和背面的烧杯中，用铝箔，确保"闪亮"的箔面一侧的内部烧杯。如果毕业烧杯，烧杯带箔覆盖措辞一半，留下清晰的烧杯中露出的一半。 </li><li>填充在250毫升烧杯中，用100毫升的水从鱼的设施系统。 </li><li>将250毫升烧杯中4升烧杯中。填充的4升烧杯中的水，直到水位甚至与100毫升水的250毫升烧杯中的线。 </li><li>添加最多10暗处理动物250毫升烧杯中。量的250毫升烧杯中，用一小片铝陪衬。 </li><li>将整个烧杯装置，紧邻的UV灯丝。 ，灯丝应接触的外侧的4升烧杯中，并面临着的250毫升烧杯中的暴露部分。确保集中在底部的4升烧杯中的250毫升烧杯中。 </li><li>后面的4L烧杯中的一个大的，不透明的屏幕的位置，允许暴露的动物，但是，为了防止任何从看到紫外线灯丝的前端的实验室工作人员。警告:确保这个障碍在接通电源前紫外光源。 </li><li>打开UV电源。将定时器设置为30分钟。放置以确保任何必要的警示标签，不知情的实验室的工作人员不小心把自己暴露于紫外线辐射。 </li><li> 30分钟后，切断紫外线的动力源。取出250毫升烧杯中的鱼。注:如果有多个回合的紫外线的照射是必需的，让电源降温（约20分钟），然后重复曝光的协议。确保替代T他每次曝光系统用新鲜水100毫升水。在4升烧杯中的水并不需要更换。 </li></ol> 3。卤素灯光曝光<ol><li>从250毫升烧杯中的鱼转移到一个1.8升的透明亚克力鱼缸。油箱加满到溢出标记。 </li><li>卤素灯的光处理区域。获得四个250瓦的卤素灯实用灯，从当地的五金商店。面对两个灯在同一个方向，在中央分开安排29厘米。以类似的方式排列的两个灯。 <ol><li>将第二组灯，使它们面对所述第一组灯，留下约73厘米之间的两组灯。这将创建一个29×73厘米的矩形光治疗区域。 </li></ol></li><li>获得一个小风扇，从当地的五金商店。将风扇两组灯之间的距离相等，外面的光的治疗区域。 </li><li>将两个装满水的1.8升罐的中心光治疗区域，两组灯之间的距离相等。灯和水箱的外壁上之间的距离应为〜29厘米。注意:即使只处理10鱼在一个1.8升的油箱，使用这样的安排。需要这两个罐，每个罐的水的温度保持在适当的范围之内。 </li><li>每罐放置增氧机增氧。 </li><li>每个水箱盖透明压克力盖，留1〜2厘米的缝隙年底最接近风扇。安排，使风扇将空气吹入这个差距。一个温度计放置在罐中的一者或两者。 </li><li>打开电源灯，风扇和通风装置。保持光治疗4天。鱼不应该被送入光治疗期间，因为这会影响水质和造成更多的压力的动物。 </li><li>每天监控温度和水位。的温度保持在30-33℃。如果有必要，调整风扇转速和/或坦克和李之间的距离亮灯。 <ol><li>每罐顶过水系统，通常需要每天。 </li><li>始终使用健康的成年斑马鱼（一般为6-9个月的年龄）保持近100％的成活率。然而，如果鱼被发现死在坦克，取出立即更换整个水箱中的水。 </li></ol></li></ol> 4。组织收集<ol><li>的卤光治疗在发病后48小时去除鱼从治疗区域。 <ol><li>准备新鲜乙醇甲醛固定液（1份37％的甲醛，9份100％的乙醇）。 </li><li>安乐死斑马鱼与麻醉剂过量的2 - 苯氧基乙醇（0.4毫克/升）。 </li><li>将安乐死斑马鱼的纸巾。用弯钳Enucleate眼睛。 </li><li>将去核的眼睛在固定液和存储O / N在4°C </li></ol></li><li> Cryoprotect眼睛。 <ol><li>在5％蔗糖的1倍PBS洗眼睛，在室温下30分钟，然后更换新鲜的5％蔗糖的1倍PBS 2小时。接下来，洗眼睛，30％的蔗糖1X PBSØ/ N在4°C。在4℃下以1:1的（相等的部分）的组织的冷冻保存液和30％蔗糖的1倍PBSö/ N洗眼睛</li></ol></li><li>嵌入眼睛在100％组织冷冻介质和存储于-80°C东方的眼睛，使的组织cryosectioning的背/腹轴。 </li><li>组织冰冻切片，在载玻片上。 2小时，在55℃的暖幻灯片商店幻灯片在-80°C或立即执行标准免疫组化。 </li><li>执行标准切片组织的免疫组化和荧光显微镜图像40,51。 </li></ol>

在这里，我们提供持续的强光曝晒普遍的感光损失和强大的再生反应的结果，结合短的紫外线照射。这个相结合的方法相比与个人LIRD方法，也是最有效的协议，损害双方的两部分的视网膜杆和视锥。重要的是，这种治疗是有效的颜料的动物，以及患白化病的动物。 虽然我们提供的证据表明与个别LIRD方法相比更为广泛和一致的损伤合并协议结果，科学的考虑，应该讨论...

光诱导的视网膜变性（LIRD）是常用的啮齿动物损伤杆和视锥细胞和斑马鱼。在成年斑马鱼，光感受器变性触发穆勒胶质细胞重新进入细胞周期和产生瞬态放大祖​​。这些祖细胞继续增殖，因为他们迁移到受损区域，在那里他们最终产生新的光感受器。目前，有两个广泛使用LIRD的范例，其中每个感光损失和相应的差异在再生反应在不同程度的结果。随着遗传学和药理学工具可用于测试单个基因在再生过程中的利益的作用，有必要制定一个强大的LIRD范式。这里我们描述一个LIRD协议，导致在杆和锥感光体，其中结合使用的两个以前建立LIRD技术的广泛和一致的损失。此外这个协议可以延长使用色素的动物，从而消除了需要保持白化背景LIRD研究的转基因株系的利息。

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			<td height="21" style="height:21px;width:216px;">UV light source</td>
			<td style="width:215px;">Leica</td>
			<td style="width:155px;">EL600</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glass Petri dish (150 x 20 mm)</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS3160152BO</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 ml glass beaker</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS1000250</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4 L glass beaker</td>
			<td>Sigma-Aldrich/Pyrex</td>
			<td>CLS10004L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminum foil</td>
			<td>Fisher</td>
			<td>01-213-105</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">250 W halogen lamps</td>
			<td>Workforce</td>
			<td>265-669</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.8 L clear acrylic tanks</td>
			<td>Aquaneering</td>
			<td>ZT180T</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">1.8 L clear acrylic tank lids</td>
			<td>Aquaneering</td>
			<td>ZT180LCL</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Fan</td>
			<td>Honeywell</td>
			<td>HT-900</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Aerator</td>
			<td>Tetra</td>
			<td>77853-900</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Thermometer</td>
			<td>Cole-Parmer</td>
			<td>YO-08008-58</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bent forceps (5/45)</td>
			<td>World Precision Instruments</td>
			<td>504155</td>
		</tr>
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a novel light damage paradigm for use in retinal regeneration studies in adult zebrafish

光诱导的视网膜变性（LIRD）是常用的啮齿动物损伤杆和视锥细胞和斑马鱼。在成年斑马鱼，光感受器变性触发穆勒胶质细胞重新进入细胞周期和产生瞬态放大祖​​。这些前体细胞继续增殖，他们迁移到受损区域，他们最终产生新感光。目前，有两个广泛使用LIRD的范例，其中每个感光损失和相应的差异在再生反应在不同程度的结果。随着遗传学和药理学工具可用于测试单个基因在再生过程中的利益的作用，有必要制定一个强大的LIRD范式。这里我们描述一个LIRD协议，导致在杆和锥感光体，其中结合使用的两个以前建立LIRD技术的广泛和一致的损失。此外这个协议可以延长使用色素的动物，从而消除了需要保持白化背景LIRD研究的转基因株系的利息。

此前所述的光治疗方案相比，每一个人的LIRD方法。在暗处理成年的白化动物（ 图3-5），个人光疗法造成重大损失棒（ 图3）和锥感光器（ 图4）。然而，无论是单独的治疗方法主要损坏感光体在背侧视网膜一半，留下相对保护的光的治疗方法（ 图3和4）的腹侧视网膜。此外，相比与卤素灯处理，UV光处理损坏的视锥细胞在背视?...

Watch this Scientific Journal Video about A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish at JoVE.com

A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

多个光损伤协议已损坏感光细胞，从而诱发成年斑马鱼视网膜的再生反应。此协议描述了一种改进的方法，可以用在着色的动物，并在整个视网膜杆和视锥细胞，损害绝大多数。

成年斑马鱼视网膜再生研究中使用的一种新型的光损伤范式

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, ...

a-novel-light-damage-paradigm-for-use-retinal-regeneration-studies

Research

JoVE Journal

Neuroscience

10.1K 次观看.  Wayne State University School of Medicine. 多个光损伤协议已损坏感光细胞，从而诱发成年斑马鱼视网膜的再生反应。此协议描述了一种改进的方法，可以用在着色的动物，并在整个视网膜杆和视锥细胞，损害绝大多数。 

视频: A Novel Light Damage Paradigm for Use in Retinal Regeneration Studies in Adult Zebrafish

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

小鼠视网膜神经节细胞的转染在体内电穿孔

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural ...

科研

神经科学

Design and Assembly of an Ultra-light Motorized Microdrive for Chronic Neural Recordings in Small Animals

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of neural circuits underlying a variety of natural behaviors, including navigation1 , decision making 2,3, and the generation of complex motor sequences4,5,6. Advances in precision machining has allowed for the fabrication of light-weight devices suitable for chronic recordings in small animals, such as mice and songbirds. The ability to adjust the electrode position with small remotely controlled motors has further increased the recording yield in various behavioral contexts by reducing animal handling.6,7
 Here we describe a protocol to build an ultra-light motorized microdrive for long-term chronic recordings in small animals. Our design evolved from an earlier published version7, and has been adapted for ease-of use and cost-effectiveness to be more practical and accessible to a wide array of researchers. This proven design 8,9,10,11 allows for fine, remote positioning of electrodes over a range of ~ 5 mm and weighs less than 750 mg when fully assembled. We present the complete protocol for how to build and assemble these drives, including 3D CAD drawings for all custom microdrive components.

The design, fabrication and assembly of an ultra-light motorized microdrive is described. The device provides a cost-effective and easy-to-use solution for chronic recordings of single units in small behaving animals.

设计和组装的超轻型电动微型硬盘（Microdrive）慢性神经录音的小动物

The ability to chronically record from populations of neurons in freely behaving animals has proven an invaluable tool for dissecting the function of ...

Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, including rats, hamsters, and mice, are active during the night and relatively inactive during the day. Many other behavioral and physiological measures also exhibit daily rhythms, but in rodents, running-wheel activity serves as a particularly reliable and convenient measure of the output of the master circadian clock, the suprachiasmatic nucleus (SCN) of the hypothalamus. In general, through a process called entrainment, the daily pattern of running-wheel activity will naturally align with the environmental light-dark cycle (LD cycle; e.g. 12 hr-light:12 hr-dark). However circadian rhythms are endogenously generated patterns in behavior that exhibit a ~24 hr period, and persist in constant darkness. Thus, in the absence of an LD cycle, the recording and analysis of running-wheel activity can be used to determine the subjective time-of-day. Because these rhythms are directed by the circadian clock the subjective time-of-day is referred to as the circadian time (CT). In contrast, when an LD cycle is present, the time-of-day that is determined by the environmental LD cycle is called the zeitgeber time (ZT).
Although circadian rhythms in running-wheel activity are typically linked to the SCN clock6-8, circadian oscillators in many other regions of the brain and body9-14 could also be involved in the regulation of daily activity rhythms. For instance, daily rhythms in food-anticipatory activity do not require the SCN15,16 and instead, are correlated with changes in the activity of extra-SCN oscillators17-20. Thus, running-wheel activity recordings can provide important behavioral information not only about the output of the master SCN clock, but also on the activity of extra-SCN oscillators. Below we describe the equipment and methods used to record, analyze and display circadian locomotor activity rhythms in laboratory rodents.

Circadian rhythms in voluntary wheel-running activity in mammals are tightly coupled to the molecular oscillations of a master clock in the brain. As such, these daily rhythms in behavior can be used to study the influence of genetic, pharmacological, and environmental factors on the functioning of this circadian clock.

运行轮活动在啮齿类动物的昼夜节律的记录和分析

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs survival and regeneration experiments. Many studies have been performed in mammalian animals to research RGCs survival after optic nerve injury. However, retrograde labeling of RGCs in adult zebrafish has not yet been reported, though some alternative methods can count cell numbers in retinal ganglion cell layers (RGCL). Considering the small size of the adult zebrafish skull and the high risk of death after drilling on the skull, we open the skull with the help of acid-etching and seal the hole with a light curing bond, which could significantly improve the survival rate. After absorbing the dyes for 5 days, almost all the RGCs are labeled. As this method does not need to transect the optic nerve, it is irreplaceable in the research of RGCs survival after optic nerve crush in adult zebrafish. Here, we introduce this method step by step and provide representative results.

We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

视网膜神经节细胞逆行标记的成年斑马鱼与荧光染料

As retrograde labeling retinal ganglion cells (RGCs) can isolate RGCs somata from dying sites, it has become the gold standard for counting RGCs in RGCs ...

The Use of Trace Eyeblink Classical Conditioning to Assess Hippocampal Dysfunction in a Rat Model of Fetal Alcohol Spectrum Disorders

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain undergoes rapid organizational change and is similar to accelerated brain changes that occur during the third trimester in humans. This model of fetal alcohol spectrum disorders (FASDs) produces severe brain damage, mimicking the amount and pattern of binge-drinking that occurs in some pregnant alcoholic mothers. We describe the use of trace eyeblink classical conditioning (ECC), a higher-order variant of associative learning, to assess long-term hippocampal dysfunction that is typically seen in alcohol-exposed adult offspring. At 90 days of age, rodents were surgically prepared with recording and stimulating electrodes, which measured electromyographic (EMG) blink activity from the left eyelid muscle and delivered mild shock posterior to the left eye, respectively. After a 5 day recovery period, they underwent 6 sessions of trace ECC to determine associative learning differences between alcohol-exposed and control rats. Trace ECC is one of many possible ECC procedures that can be easily modified using the same equipment and software, so that different neural systems can be assessed. ECC procedures in general, can be used as diagnostic tools for detecting neural pathology in different brain systems and different conditions that insult the brain.

Trace eyeblink classical conditioning (ECC) was used to assess hippocampal-dependent associative learning in adult rats that were administered a high concentration (11.9% v/v) of alcohol during early neonatal brain development. In general, ECC procedures are sound diagnostic tools for detecting brain dysfunction across many psychological and biomedical settings.

使用的跟踪经典条件反射，评估胎儿酒精谱系障碍大鼠模型海马功能障碍

Neonatal rats were administered a relatively high concentration of ethyl alcohol (11.9% v/v) during postnatal days 4-9, a time when the fetal brain ...

Modeling Amyloid-&#946;42 Toxicity and Neurodegeneration in Adult Zebrafish Brain

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to synaptic degeneration, inflammation, neuronal death, and learning deficits. Humans cannot regenerate lost neurons in the case of AD in part due to impaired proliferative capacity of the neural stem/progenitor cells (NSPCs) and reduced neurogenesis. Therefore, efficient regenerative therapies should also enhance the proliferation and neurogenic capacity of NSPCs. Zebrafish (Danio rerio) is a regenerative organism, and we can learn the basic molecular programs with which we could design therapeutic approaches to tackle AD. For this reason, the generation of an AD-like model in zebrafish was necessary. Using our methodology, we can introduce synthetic derivatives of A&#946;42 peptide with tissue penetrating capability into the adult zebrafish brain, and analyze the disease pathology and the regenerative response. The advantage over the existing methods or animal models is that zebrafish can teach us how a vertebrate brain can naturally regenerate, and thus help us to treat human neurodegenerative diseases better by targeting endogenous NSPCs. Therefore, the amyloid-toxicity model established in the adult zebrafish brain may open new avenues for research in the field of neuroscience and clinical medicine. Additionally, the simple execution of this method allows for cost-effective and efficient experimental assessment. This manuscript describes the synthesis and injection of A&#946;42 peptides into zebrafish brain.

This protocol describes the synthesis, characterization, and injection of monomeric amyloid-&#946;42 peptides for generating amyloid toxicity in adult zebrafish to establish an Alzheimer&#39;s disease model, followed by histological analyses and detection of aggregations.

成年斑马鱼脑 Amyloid-β42毒性和神经的模型研究

Alzheimer's disease (AD) is a debilitating neurodegenerative disease in which accumulation of toxic amyloid-&#946;42 (A&#946;42) peptides leads to ...

M&#252;ller Glia Cell Activation in a Laser-induced Retinal Degeneration and Regeneration Model in Zebrafish

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after severe damage. Investigating the regeneration pathways in zebrafish might bring new insights to develop innovative strategies for the treatment of retinal degenerative diseases in mammals. Herein, we focused on the induction of a focal lesion to the outer retina in adult zebrafish by means of a 532 nm diode laser. A localized injury allows investigating biological processes that take place during retinal degeneration and regeneration directly at the area of damage. Using non-invasive optical coherence tomography (OCT), we were able to define the location of the damaged area and monitor subsequent regeneration in vivo. Indeed, OCT imaging produces high-resolution, cross-sectional images of the zebrafish retina, providing information which was previously only available with histological analyses. In order to confirm the data from real-time OCT, histological sections were performed and regenerative response after the induction of the retinal injury was investigated by immunohistochemistry.

The zebrafish is a popular animal model to study mechanisms of retinal degeneration/regeneration in vertebrates. This protocol describes a method to induce localized injury disrupting the outer retina with minimal damage to the inner retina. Subsequently, we monitor in vivo the retinal morphology and the M&#252;ller glia response throughout retinal regeneration.

激光诱导的斑马鱼视网膜变性及再生模型中的 m ü ller 胶质细胞活化

A fascinating difference between teleost and mammals is the lifelong potential of the teleost retina for retinal neurogenesis and regeneration after ...

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding diseases, and future therapies hinge on fundamental understandings about how different retinal cells function normally. Gaining such information with biochemical methods has proven difficult because contributions of particular cell types are diminished in the retinal cell milieu. Live retinal imaging can provide a view of numerous biological processes on a subcellular level, thanks to a growing number of genetically encoded fluorescent biosensors. However, this technique has thus far been limited to tadpoles and zebrafish larvae, the outermost retinal layers of isolated retinas, or lower resolution imaging of retinas in live animals. Here we present a method for generating live ex vivo retinal slices from adult zebrafish for live imaging via confocal microscopy. This preparation yields transverse slices with all retinal layers and most cell types visible for performing confocal imaging experiments using perfusion. Transgenic zebrafish expressing fluorescent proteins or biosensors in specific retinal cell types or organelles are used to extract single-cell information from an intact retina. Additionally, retinal slices can be loaded with fluorescent indicator dyes, adding to the method&#39;s versatility. This protocol was developed for imaging Ca2+ within zebrafish cone photoreceptors, but with proper markers it could be adapted to measure Ca2+ or metabolites in M&#252;ller cells, bipolar and horizontal cells, microglia, amacrine cells, or retinal ganglion cells. The retinal pigment epithelium is removed from slices so this method is not suitable for studying that cell type. With practice, it is possible to generate serial slices from one animal for multiple experiments. This adaptable technique provides a powerful tool for answering many questions about retinal cell biology, Ca2+, and energy homeostasis.

Preparing Fresh Retinal Slices from Adult Zebrafish for Ex Vivo Imaging Experiments

Imaging retinal tissue can provide single-cell information that cannot be gathered from traditional biochemical methods. This protocol describes preparation of retinal slices from zebrafish for confocal imaging. Fluorescent genetically encoded sensors or indicator dyes allow visualization of numerous biological processes in distinct retinal cell types.

从成年斑马鱼中制备新鲜视网膜切片用于前体成像实验

The retina is a complex tissue that initiates and integrates the first steps of vision. Dysfunction of retinal cells is a hallmark of many blinding ...

Ablation of a Neuronal Population Using a Two-photon Laser and Its Assessment Using Calcium Imaging and Behavioral Recording in Zebrafish Larvae

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ablation of neurons is an effective method for this purpose when neurons are selectively labeled with fluorescent probes. In the present study, protocols for laser ablating a subpopulation of neurons using a two-photon microscope and testing of its functional and behavioral consequences are described. In this study, prey capture behavior in zebrafish larvae is used as a study model. The pretecto-hypothalamic circuit is known to underlie this visually-driven prey catching behavior. Zebrafish pretectum were laser-ablated, and neuronal activity in the inferior lobe of the hypothalamus (ILH; the target of the pretectal projection) was examined. Prey capture behavior after pretectal ablation was also tested.

Here, we present a protocol to ablate a genetically labeled subpopulation of neurons by a two-photon laser from Zebrafish larvae.

用双光子激光消融神经种群及其对斑马鱼幼虫钙成像和行为记录的评价

To identify the role of a subpopulation of neurons in behavior, it is essential to test the consequences of blocking its activity in living animals. Laser ...

A Novel In Vitro Model of Blast Traumatic Brain Injury

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the detonation of explosive devices, however, the mechanisms that underlie the brain damage resulting from blast overpressure exposure are not entirely understood and are believed to be unique to this type of brain injury. Preclinical models are crucial tools that contribute to better understand blast-induced brain injury. A novel in vitro blast TBI model was developed using an open-ended shock tube to simulate real-life open-field blast waves modelled by the Friedlander waveform. C57BL/6N mouse organotypic hippocampal slice cultures were exposed to single shock waves and the development of injury was characterized up to 72 h using propidium iodide, a well-established fluorescent marker of cell damage that only penetrates cells with compromised cellular membranes. Propidium iodide fluorescence was significantly higher in the slices exposed to a blast wave when compared with sham slices throughout the duration of the protocol. The brain tissue injury is very reproducible and proportional to the peak overpressure of the shock wave applied.