Os autores gostariam de agradecer a Xixia Luo excelente para criação de peixes e suporte técnico. Este trabalho foi financiado pelo National Institutes of Health bolsas R21EY019401 (RT) e P30EY04068 (RT) e start-up fundos para RT, incluindo uma subvenção irrestrita de Pesquisa de Prevenção à Cegueira Wayne State University, Departamento de Oftalmologia. JT foi apoiado por um Thomas C. Rumble Fellowship fornecido pela Escola de Pós-Graduação Wayne State University.

Todos os procedimentos descritos neste protocolo foi aprovado pelo comitê de uso de animais na Wayne State University School of Medicine. 1. Adaptação ao escuro <ol><li> Transferência de ~ 10 adulto albino ou pigmentada peixes do sistema de habitação normal em um recinto escuro. Se possível, use uma caixa escura que está embutido no módulo habitacional peixe-zebra, que permite o fluxo normal da água através do tanque. (Se esse sistema não está disponível, coloque o tanque de peixes em um compartimento completamente escuro, certificando-se de arejar o peixe com oxigênio). </li><li> Manter o peixe no escuro durante 10 dias. Quando a alimentação dos animais, ou adicionando novos peixes para a caixa escura, certifique-se para mover tão rapidamente quanto possível para evitar expor o peixe a um longo período de luz. </li></ol> 2. Exposição à luz UV <ol><li> Certifique-se que a alimentação da fonte UV é OFF. Remover o filamento de luz UV a partir de estereomicroscópio fluorescente. <ol><li> Use uma invertida15 centímetros de diâmetro de vidro placa de Petri (ou algo semelhante a dois centímetros de altura) como um suporte para o filamento UV. Tape o prato de Petri para a bancada do laboratório. </li><li> Tape o filamento de luz UV para o topo da placa de Petri invertida. Dispor de modo que ~ 2 mm na extremidade das saliências de incandescência a placa de Petri. </li></ol></li><li> Obter uma proveta de vidro de 250 ml. Cubra ½ do fundo, nas laterais e parte de trás do copo com uma folha de alumínio, tornando-se o lado &quot;brilhante&quot; da folha voltada para o interior do copo. Se o recipiente é formado, cobrir a metade teor da proveta com a folha, deixando a metade transparente do copo exposta. </li><li> Encher o copo de 250 ml com 100 ml de água a partir do sistema de instalação de peixe. </li><li> Colocar o copo de 250 ml numa proveta de 4 litros. Encher a 4 L copo com água até o nível de água é, mesmo com a linha de 100 ml de água no copo de 250 ml. </li><li> Adicionar um máximo de 10 animais tratados com escuro à proveta de 250 ml. Cobrir a proveta de 250 ml com um pedaço pequeno de alumíniofrustrar. </li><li> Colocar todo o aparelho proveta imediatamente adjacente ao filamento de UV. O filamento deve estar a tocar o exterior do copo de 4 L e de frente para a parte exposta do copo de 250 ml. Certifique-se de que o copo de 250 ml é centrada no fundo da proveta de 4 litros. </li><li> Posicionar um grande tela opaca por trás da taça 4L, permitindo que os animais a serem expostas, mas evitando qualquer pessoal do laboratório de ver a ponta do filamento de UV. ATENÇÃO: certifique-se esta barreira está no lugar antes de ligar o aparelho à fonte de UV. </li><li> Ligue a fonte de energia UV. Definir temporizador para 30 min. Coloque os rótulos de advertência necessárias para garantir que o pessoal de laboratório desavisados ​​não possa se expor à radiação UV. </li><li> Após 30 min, desligue a fonte de energia UV. Remover o copo de 250 ml com o peixe. NOTA: se várias rodadas de exposição UV são necessárias, deixe a fonte de alimentação esfriar (~ 20 min) antes de repetir o protocolo de exposição. Certifique-se de substituir tele 100 ml de água com sistema de água fresca para cada exposição. A água no copo de 4 L não necessita de ser substituído. </li></ol> 3. Lâmpada de halogéneo Luz exposição <ol><li> Transfira o peixe do copo de 250 ml em uma L claro aquário 1,8 acrílico. Encha o tanque até a marca de transbordo. </li><li> Configure área de tratamento de luz a lâmpada halógena. Obter quatro de 250 W lâmpadas halógenas utilidade de uma loja de hardware local. Enfrentando duas lâmpadas na mesma direção, arranjar 29 centímetros de distância do centro. Dispor as outras duas lâmpadas de uma maneira semelhante. <ol><li> Coloque o segundo conjunto de lâmpadas, de modo que eles estão de frente para o primeiro conjunto de lâmpadas, deixando ~ 73 centímetros entre os dois conjuntos de lâmpadas. Isso cria uma área de tratamento de luz em forma de retângulo de 29 x 73 cm. </li></ol></li><li> Obter um pequeno ventilador de uma loja de hardware local. Coloque o ventilador equidistante entre os dois conjuntos de lâmpadas, apenas fora da área de tratamento de luz. </li><li> Colocar dois tanques cheios de 1,8 L de água no centro doárea de tratamento de luz, equidistante entre os dois conjuntos de lâmpadas. A distância entre as lâmpadas e a parede externa do tanque deve ser ~ 29 cm. Nota: mesmo que apenas o tratamento de 10 peixes em um tanque de 1,8 L, use este arranjo. Ambos os tanques são necessários para manter a temperatura da água de cada tanque no intervalo apropriado. </li><li> Coloque um aerador de oxigênio em cada tanque. </li><li> Cobrir cada tanque com tampas de acrílico transparente, deixando um intervalo de ~ 2 cm na região da extremidade mais próxima do ventilador. Organizar o ventilador de modo que ele vai soprar ar para esta lacuna. Coloque um termómetro em um ou ambos os tanques. </li><li> Ligue a alimentação para as luzes, ventilador, e aeradores. Manter o tratamento luz por até 4 dias. O peixe não deve ser alimentado durante o tratamento com luz, como isso vai afetar a qualidade da água e resultam em mais estresse para os animais. </li><li> Monitorar o nível de temperatura e água em uma base diária. Manter a temperatura a 30-33 ° C. Se necessário, ajustar a velocidade do ventilador e / ou a distância entre os tanques e o lights. <ol><li> Top fora de cada tanque com água do sistema, conforme necessário, geralmente diária. </li><li> Sempre use zebrafish adulto saudável (geralmente 6-9 meses de idade) para manter uma taxa de sobrevivência de quase 100%. No entanto, se um peixe é encontrado morto no tanque, removê-lo imediatamente e substituir a água por todo o tanque. </li></ol></li></ol> 4. Coleção de tecidos <ol><li> 48 horas após o início do tratamento com luz de halogéneo remover o peixe a partir da área de tratamento. <ol><li> Preparar fresco fixador formaldeído etanólico (1 parte de 37% de formaldeído, 9 partes de etanol a 100%). </li><li> Eutanásia peixe-zebra com uma sobredose de anestésico de 2-fenoxietanol (0,4 mg / L). </li><li> Transfira o peixe-zebra sacrificados a uma toalha de papel. Enuclear o olho usando uma pinça curva. </li><li> Coloque olhos enucleados em fixador e loja O / N a 4 ° C. </li></ol></li><li> Cryoprotect os olhos. <ol><li> Lavar os olhos em 5% de sacarose 1x PBS durante 30 min à temperatura ambiente, e depois substituir comfresco 5% de sacarose 1x PBS durante 2 h. Em seguida, lavar os olhos em sacarose a 30% PBS 1x O / N a 4 ° C. Lavar os olhos em uma mistura 1:1 (partes iguais) de meio de congelamento de tecidos e 30% de sacarose PBS 1x O / N a 4 ° C. </li></ol></li><li> Incorporar os olhos no meio de congelamento de 100% do tecido e armazenar a -80 ° C. Oriente os olhos, de modo que cryosectioning do tecido é realizada sobre o eixo dorsal / ventral. </li><li> Criocorte o tecido e coloque em lâminas de vidro. Lâminas quentes para 2 horas a 55 ° C. Loja lâminas a -80 ° C ou imediatamente executar imunohistoquímica padrão. </li><li> Realizar imunohistoquímica padrão em tecido seccionado e imagem com microscopia fluorescente 40,51. </li></ol>

Os autores não têm nada a revelar.

Aqui nós mostramos que a combinação de uma curta exposição UV com uma luz brilhante contínua exposição resulta em perda de fotorreceptores generalizada e uma resposta de regeneração robusta. Comparado com os métodos LIRD individuais, este método também combinado é o protocolo mais eficaz para danificar os dois cones e bastonetes em ambas as partes da retina. Importante, este tratamento é eficaz em animais pigmentadas, bem como animais albinos. Apesar de apresentar pro...

Degeneração da retina induzida pela luz (LIRD) é comumente usado em roedores e peixe-zebra para haste danos e cones fotorreceptores. No peixe-zebra adulto, degeneração de fotorreceptores provoca células gliais de Müller para re-entrar no ciclo celular e produzir progenitores transitória-amplificação. Estes progenitores continuam a proliferar como eles migram para a área danificada, onde, finalmente, dar origem a novos fotorreceptores. Actualmente, existem dois paradigmas LIRD amplamente utilizados, cada um dos quais resulta em graus variáveis ​​de perda de fotorreceptores e as diferenças correspondentes na resposta regenerativa. Como ferramentas mais genéticos e farmacológicos estão disponíveis para testar o papel dos genes individuais de interesse durante a regeneração, existe uma necessidade de desenvolver um paradigma LIRD robusto. Aqui, nós descrevemos um protocolo LIRD que resulta em perda generalizada e consistente de fotorreceptores cone e da haste na qual se combinaram o uso de duas técnicas LIRD previamente estabelecidos. Além disso, Este protocolo pode ser estendido para o uso em animais pigmentadas, o que elimina a necessidade de manter as linhagens transgênicas de interesse no fundo albino para estudos LIRD.

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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

Degeneração da retina induzida pela luz (LIRD) é comumente usado em roedores e peixe-zebra para haste danos e cones fotorreceptores. No peixe-zebra adulto, degeneração de fotorreceptores provoca células gliais de Müller para re-entrar no ciclo celular e produzir progenitores transitória-amplificação. Estes progenitores continuam a proliferar como eles migram para a área danificada, onde, finalmente, dar origem a novos fotorreceptores. Actualmente, existem dois paradigmas LIRD amplamente utilizados, cada um dos quais resulta em graus variáveis ​​de perda de fotorreceptores e as diferenças correspondentes na resposta regenerativa. Como ferramentas mais genéticos e farmacológicos estão disponíveis para testar o papel dos genes individuais de interesse durante a regeneração, existe uma necessidade de desenvolver um paradigma LIRD robusto. Aqui, nós descrevemos um protocolo LIRD que resulta em perda generalizada e consistente de fotorreceptores cone e da haste na qual se combinaram o uso de duas técnicas LIRD previamente estabelecidos. Além disso, Este protocolo pode ser estendido para o uso em animais pigmentadas, o que elimina a necessidade de manter as linhagens transgênicas de interesse no fundo albino para estudos LIRD.

O protocolo de tratamento de luz foi anteriormente descrito em relação a cada método individual de LIRD. Nos animais tratados com albinos adultos escuro (Figuras 3-5), os tratamentos de luz individuais resultou em perda significativa de haste (Figura 3) e do cone (Figura 4) fotorreceptores. No entanto, ambos os tratamentos individuais fotorreceptores danificados, principalmente na metade dorsal da retina, deixando a retina ventral relativamente protegida dos...

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

Vários protocolos de danos provocados pela luz têm sido descritas para fotorreceptores danos e, consequentemente, induzir uma resposta de regeneração da retina adulta em peixes-zebra. Este protocolo descreve um método melhorado, que pode ser utilizada em animais e pigmentados que danifica a grande maioria dos fotorreceptores cone e da haste ao longo de toda a retina.

A Light Novel danos Paradigma para uso em estudos de regeneração da retina em Zebrafish Adulto

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 visualizações.  Wayne State University School of Medicine.  Vários protocolos de danos provocados pela luz têm sido descritas para fotorreceptores danos e, consequentemente, induzir uma resposta de regeneração da retina adulta em peixes-zebra. Este protocolo descreve um método melhorado, que pode ser utilizada em animais e pigmentados que danifica a grande maioria dos fotorreceptores cone e da haste ao longo de toda a retina. 

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

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.

Registro e análise dos ritmos circadianos em Running rodas Atividade em Roedores

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, ...

Neurociência

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.

Marcação retrógrada de células ganglionares da retina em Zebrafish Adulto com corantes fluorescentes

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 ...

Stab Wound Injury of the Zebrafish Adult Telencephalon: A Method to Investigate Vertebrate Brain Neurogenesis and Regeneration

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury.
In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

To shed light on the cellular and molecular mechanisms of zebrafish adult neurogenesis and regeneration, we developed a protocol for invasive surgery causing mechanical injuries in the zebrafish adult telencephalon and subsequent monitoring of changes in the stabbed hemisphere by immunohistochemistry or in situ hybridization.

Stab ferida ferimento do peixe-zebra adulto Telencéfalo: Um método para investigar Vertebrados Cérebro Neurogênese e Regeneração

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular ...

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.

O uso do condicionamento de rastreamento Eyeblink clássico para avaliar disfunção Hippocampal em um modelo do rato de transtornos do espectro do álcool Fetal

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.

Modeling Amyloid-&amp;#946;42 Toxicity and Neurodegeneration in Adult 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.

Toxicidade do Amyloid-β42 de modelagem e neurodegeneração no cérebro adulto de Zebrafish

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.

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

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.

Ativação de células da Müller Glia em um modelo de regeneração no Zebrafish e degeneração retiniana induzida por Laser

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.

Prepara frescas fatias da retina de adultos do Zebrafish para Ex Vivo experimentos de imagem

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.

Ablação de uma população Neuronal, usando um Laser de dois fotões e sua avaliação usando imagens de cálcio e gravação comportamental em larvas de Zebrafish

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.

A Novel In Vitro Model of Blast Traumatic Brain Injury

This paper describes a novel model of primary blast traumatic brain injury. A compressed-air driven shock tube is used to expose in vitro mouse hippocampal slice cultures to a single shock wave. This is a simple and rapid protocol generating a reproducible brain tissue injury with a high throughput.

Um modelo de romance In Vitro de explosão traumatismo crânio-encefálico

Traumatic brain injury is a leading cause of death and disability in military and civilian populations. Blast traumatic brain injury results from the ...

An Emerging Target Paradigm to Evoke Fast Visuomotor Responses on Human Upper Limb Muscles

To reach towards a seen object, visual information has to be transformed into motor commands. Visual information such as the object&#8217;s color, shape, and size are processed and integrated within numerous brain areas, then ultimately relayed to the motor periphery. In some instances, a reaction is needed as fast as possible. These fast visuomotor transformations, and their underlying neurological substrates, are poorly understood in humans as they have lacked a reliable biomarker. Stimulus-locked responses (SLRs) are short latency (&#60;100 ms) bursts of electromyographic (EMG) activity representing the first wave of muscle recruitment influenced by visual stimulus presentation. SLRs provide a quantifiable output of rapid visuomotor transformations, but SLRs have not been consistently observed in all subjects in past studies. Here we describe a new, behavioral paradigm featuring the sudden emergence of a moving target below an obstacle that consistently evokes robust SLRs. Human participants generated visually guided reaches toward or away from the emerging target using a robotic manipulandum while surface electrodes recorded EMG activity from the pectoralis major muscle. In comparison to previous studies that investigated SLRs using static stimuli, the SLRs evoked with this emerging target paradigm were larger, evolved earlier, and were present in all participants. Reach reaction times (RTs) were also expedited in the emerging target paradigm. This paradigm affords numerous opportunities for modification that could permit systematic study of the impact of various sensory, cognitive, and motor manipulations on fast visuomotor responses. Overall, our results demonstrate that an emerging target paradigm is capable of consistently and robustly evoking activity within a fast visuomotor system.

Presented here is a behavioral paradigm that elicits robust fast visuomotor responses on human upper limb muscles during visually guided reaches.

Um paradigma de alvo emergente para evocar respostas rápidas do visuomotor sobre músculos dos membros superiores humanos

To reach towards a seen object, visual information has to be transformed into motor commands. Visual information such as the object&#8217;s color, shape, ...