Los autores desean agradecer a Xixia Luo excelente para la cría de peces y soporte técnico. Este trabajo fue financiado por los Institutos Nacionales de Salud subvenciones R21EY019401 (RT) y P30EY04068 (RT), y los fondos de puesta en marcha a la temperatura ambiente, incluida una subvención sin restricciones de Investigación para Prevención de la Ceguera a la Universidad Estatal de Wayne, Departamento de Oftalmología. JT fue apoyado por una beca Thomas C. Rumble proporcionado por la Escuela de Graduados de la Universidad Estatal Wayne.

Todos los procedimientos descritos en este protocolo fue aprobado por el comité de la utilización de animales en la Facultad de Medicina de Wayne State University. 1. Adaptación a la oscuridad <ol><li> Transferencia ~ 10 peces adultos albino o pigmentado del sistema de vivienda normal en un recinto oscuro. Si está disponible, utilizar un recinto oscuro que está integrado en el módulo de vivienda pez cebra, que permite el flujo normal de agua a través del tanque. (Si tal sistema no está disponible, colocar el tanque de peces en un recinto completamente oscuro, asegurándose de airear el pescado con el oxígeno). </li><li> Mantener a los peces en la oscuridad durante 10 días. Cuando la alimentación de los animales o la adición de nuevos peces a la caja oscura, asegúrese de avanzar lo más rápidamente posible para evitar la exposición de los peces a un largo período de luz. </li></ol> 2. Exposición a la luz UV <ol><li> Asegúrese de que la alimentación de la fuente de radiación UV es OFF. Retire el filamento de la luz UV fluorescentes estereoscópica. <ol><li> Use un invertida15 cm de diámetro de cristal placa de Petri (o algo parecido a 2 cm de altura) como soporte para el filamento UV. Tape la placa de Petri para la mesa del laboratorio. </li><li> Tape el filamento de luz UV al principio del plato invertido Petri. Organizar de manera que ~ 2 mm del extremo de los salientes de filamentos de la placa de Petri. </li></ol></li><li> Obtener un vaso de 250 ml de vidrio. Cubra la mitad de la parte inferior, lados, y la parte posterior del vaso de precipitados con papel de aluminio, asegurándose de que el lado &quot;brillante&quot; de la lámina se enfrenta al interior del vaso de precipitados. Si se graduó el vaso, cubrir la mitad redactado del vaso con papel de aluminio, dejando el claro de la mitad de la copa expuesta. </li><li> Llenar el vaso de precipitados de 250 ml con 100 ml de agua del sistema de instalación de peces. </li><li> Coloque el vaso de 250 ml en un vaso de precipitados de 4 l. Llenar el vaso con 4 L de agua hasta que el nivel del agua está aún con la línea de agua de 100 ml en el vaso de 250 ml. </li><li> Añadir un máximo de 10 animales tratados oscuro al vaso de 250 ml. Cubrir el vaso de 250 ml con una pequeña pieza de aluminiopapel de aluminio. </li><li> Colocar todo el aparato vaso de precipitados inmediatamente adyacente al filamento UV. El filamento debe estar en contacto con el exterior del vaso de precipitados de 4 L y frente a la parte expuesta del vaso de precipitados de 250 ml. Asegúrese de que el vaso de precipitados de 250 ml se centra en la parte inferior del vaso de precipitados de 4 l. </li><li> Coloque una grande, detrás de la pantalla opaca vaso de precipitados de 4 l, lo que permite a los animales a ser expuestos, pero la prevención de cualquier personal de laboratorio de ver la punta del filamento de UV. ADVERTENCIA: asegúrese de que esta barrera es en su lugar antes de conectar la alimentación a la fuente de UV. </li><li> Encienda la fuente de alimentación de UV. Ajuste del temporizador durante 30 min. Coloque lo que las etiquetas de advertencia necesarias para asegurar que el personal de laboratorio desprevenidos no querer exponerse a la radiación UV. </li><li> Después de 30 minutos, apague la fuente de energía UV. Retire el vaso de 250 ml con el pescado. NOTA: si se requieren múltiples rondas de exposición UV, vamos a la fuente de alimentación se enfríe (~ 20 min) antes de repetir el protocolo de exposición. Asegúrese de sustituir tque 100 ml de agua con sistema de agua dulce para cada exposición. El agua en el vaso de precipitados de 4 L no necesita ser reemplazado. </li></ol> 3. Halógena luz de la lámpara de exposición <ol><li> Transferir el pescado de 250 ml vaso en un claro L pecera 1.8 acrílico. Llenar el depósito hasta la marca de desbordamiento. </li><li> Configure la lámpara de luz halógena área de tratamiento. Obtener cuatro 250 W Lámparas halógenas de servicios públicos en una ferretería local. Frente a dos lámparas en la misma dirección, organizar 29 cm de distancia en el centro. Colocar los otros dos lámparas en una manera similar. <ol><li> Coloque el segundo conjunto de lámparas de modo que se enfrentan a la primera serie de lámparas, dejando ~ 73 cm entre los dos conjuntos de lámparas. Esto crea un área de tratamiento de la luz en forma de rectángulo de 29 x 73 cm. </li></ol></li><li> Obtener un pequeño ventilador en una ferretería local. Coloque el ventilador equidistante entre los dos conjuntos de lámparas, a las afueras de la zona de tratamiento de la luz. </li><li> Coloque dos 1.8 L tanques llenos de agua en el centro de laárea de tratamiento de la luz, a medio camino entre los dos conjuntos de lámparas. La distancia entre las lámparas y la pared exterior del tanque debe ser ~ 29 cm. Nota: aunque sólo el tratamiento de 10 peces en un tanque de 1.8 L, utilice este arreglo. Se necesitan tanto los tanques para mantener la temperatura del agua de cada tanque dentro del rango apropiado. </li><li> Coloque un aireador de oxígeno en cada tanque. </li><li> Cubra cada tanque con tapas de acrílico transparente, dejando una brecha de ~ 2 cm en el extremo más cercano al ventilador. Organizar el ventilador para que sople aire dentro de este vacío. Coloque un termómetro en uno o ambos de los tanques. </li><li> Encienda la energía a las luces, ventiladores, y aireadores. Mantener el tratamiento de la luz para un máximo de 4 días. El pescado no debe ser alimentado durante el tratamiento de la luz, ya que esto afectará la calidad del agua y dar lugar a más estrés a los animales. </li><li> Controle la temperatura y del nivel del agua sobre una base diaria. Mantener la temperatura a 30-33 ° C. Si es necesario, ajustar la velocidad del ventilador y / o la distancia entre los tanques y la lilos dere. <ol><li> Remate de cada tanque de agua de la instalación, según sea necesario, por lo general todos los días. </li><li> Siempre use pez cebra adulto sano (típicamente 6-9 meses de edad) para mantener una tasa de supervivencia de cerca de 100%. Sin embargo, si un pez es encontrado muerto en el tanque, retire inmediatamente y reemplazar el agua en todo el tanque. </li></ol></li></ol> 4. Colección de tejidos <ol><li> 48 horas después de la aparición del tratamiento con luz halógeno eliminar el pescado de la zona de tratamiento. <ol><li> Preparar fijador formaldehído etanólico fresca (1 parte de 37% de formaldehído, 9 partes de etanol 100%). </li><li> La eutanasia de pez cebra con una sobredosis anestésica de 2-fenoxietanol (0,4 mg / L). </li><li> Transferir el pez cebra eutanasia a una toalla de papel. Enucleación del ojo utilizando pinzas curvas. </li><li> Coloque ojos enucleados en fijador y almacenar O / N a 4 ° C. </li></ol></li><li> Cryoprotect los ojos. <ol><li> Lavar los ojos en 5% de sacarosa 1x PBS durante 30 min a temperatura ambiente, a continuación, reemplazar confresca 5% de sacarosa 1x PBS durante 2 hr. A continuación, lavar los ojos en el 30% de sacarosa 1x PBS O / N a 4 ° C. Lavar los ojos en una mezcla 1:1 (partes iguales) de medio de congelación tisular y 30% de sacarosa 1x PBS O / N a 4 ° C. </li></ol></li><li> Insertar los ojos en el 100% de medio de congelación de tejido y se almacenan a -80 ° C. Oriente los ojos de manera que cryosectioning del tejido se realiza en el eje dorsal / ventral. </li><li> Cryosection el tejido y el lugar en portaobjetos de vidrio. Diapositivas caliente durante 2 horas a 55 ° C. Diapositivas Almacenar a -80 ° C o realizar inmediatamente inmunohistoquímica estándar. </li><li> Realizar inmunohistoquímica estándar sobre el tejido seccionado y la imagen con microscopía de fluorescencia 40,51. </li></ol>

Los autores no tienen nada que revelar.

Aquí nos muestran que la combinación de una exposición a UV corta con una luz brillante continua exposición resulta en la pérdida de fotorreceptores extendida y una respuesta robusta regeneración. En comparación con los métodos LIRD individuales, este método combinado es también el protocolo más eficaz para dañar los dos conos y bastones en ambas mitades de la retina. Es importante destacar que este tratamiento es eficaz en animales pigmentadas, así como animales albinos. <p class="jove_content...

La degeneración retiniana inducida por la luz (LIRD) se utiliza comúnmente en roedores y en pez cebra y daños a la varilla fotorreceptores de los conos. En el pez cebra adulto, degeneración de los fotorreceptores activa las células gliales de Müller para volver a entrar en el ciclo celular y la producción de progenitores transitoria-amplificación. Estos progenitores continúan proliferando a medida que migran a la zona dañada, donde se dan lugar en última instancia a nuevos fotorreceptores. Actualmente, hay dos paradigmas LIRD ampliamente utilizados, cada uno de los cuales da como resultado diversos grados de pérdida de fotorreceptores y las correspondientes diferencias en la respuesta de la regeneración. Como herramientas más genéticos y farmacológicos están disponibles para probar el papel de los genes individuales de interés durante la regeneración, hay una necesidad de desarrollar un paradigma LIRD robusta. Aquí se describe un protocolo LIRD que resulta en la pérdida generalizada y coherente de ambos fotorreceptores de los bastones y conos en el que hemos combinado el uso de dos técnicas LIRD previamente establecidos. Además, Este protocolo se puede extender para su uso en animales pigmentados, que elimina la necesidad de mantener las líneas transgénicas de interés en el fondo albino para estudios 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>
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		<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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			<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>
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		<tr height="21">
			<td height="21" style="height:21px;">Thermometer</td>
			<td>Cole-Parmer</td>
			<td>YO-08008-58</td>
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		<tr height="21">
			<td height="21" style="height:21px;">Bent forceps (5/45)</td>
			<td>World Precision Instruments</td>
			<td>504155</td>
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a novel light damage paradigm for use in retinal regeneration studies in adult zebrafish

La degeneración retiniana inducida por la luz (LIRD) se utiliza comúnmente en roedores y en pez cebra y daños a la varilla fotorreceptores de los conos. En el pez cebra adulto, degeneración de los fotorreceptores activa las células gliales de Müller para volver a entrar en el ciclo celular y la producción de progenitores transitoria-amplificación. Estos progenitores continúan proliferando a medida que migran a la zona dañada, donde se dan lugar en última instancia a nuevos fotorreceptores. Actualmente, hay dos paradigmas LIRD ampliamente utilizados, cada uno de los cuales da como resultado diversos grados de pérdida de fotorreceptores y las correspondientes diferencias en la respuesta de la regeneración. Como herramientas más genéticos y farmacológicos están disponibles para probar el papel de los genes individuales de interés durante la regeneración, hay una necesidad de desarrollar un paradigma LIRD robusta. Aquí se describe un protocolo LIRD que resulta en la pérdida generalizada y coherente de ambos fotorreceptores de los bastones y conos en el que hemos combinado el uso de dos técnicas LIRD previamente establecidos. Además, Este protocolo se puede extender para su uso en animales pigmentados, que elimina la necesidad de mantener las líneas transgénicas de interés en el fondo albino para estudios LIRD.

El protocolo de tratamiento de la luz hasta ahora descrito se comparó con cada método de LIRD. En los animales albinos adultos tratados con oscuro (Figuras 3-5), los tratamientos de luz individuales dieron como resultado una pérdida significativa de la varilla (figura 3) y el cono (Figura 4) fotorreceptores. Sin embargo, ambos tratamientos individuales dañados principalmente fotorreceptores en la mitad dorsal de la retina, dejando la retina ventral relativa...

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

Múltiples protocolos de daño de la luz se han descrito a daños fotorreceptores y, por consiguiente inducir una respuesta de la regeneración de la retina en adultos de pez cebra. Este protocolo describe un método mejorado que se puede utilizar en animales pigmentados y que daña la gran mayoría de la varilla y fotorreceptores de los conos a través de toda la retina.

Una novela ligera Paradigm daños para su uso en estudios de regeneración retiniana en adultos de pez cebra

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 vistas.  Wayne State University School of Medicine.  Múltiples protocolos de daño de la luz se han descrito a daños fotorreceptores y, por consiguiente inducir una respuesta de la regeneración de la retina en adultos de pez cebra. Este protocolo describe un método mejorado que se puede utilizar en animales pigmentados y que daña la gran mayoría de la varilla y fotorreceptores de los conos a través de toda la retina. 

Video: 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 y análisis de los ritmos circadianos en funcionamiento las ruedas Actividad en 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, ...

Investigación

Neurociencias

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.

Etiquetado retrógrada de células ganglionares retinianas en adultos de pez cebra con tintes 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.

Puñalada de Lesiones de la herida del pez cebra Telencéfalo adultos: un método para investigar Vertebrados Cerebro neurogénesis y Regeneración

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.

El uso de condicionamiento de clásico de Eyeblink rastro para evaluar disfunción hipocampal en un modelo de rata de trastornos del espectro alcohólico 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.

Toxicidad del amiloide β42 de modelado y la neurodegeneración en cerebro de pez cebra adulto

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.

Activación de la célula de Müller glía en un modelo de regeneración en el pez cebra y degeneración retiniana inducida por láser

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.

Preparación frescas rebanadas retinales de pez cebra adulto para Ex Vivo de experimentos

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.

Ablación de una población Neuronal utilizando un láser de dos fotones y su evaluación mediante grabación comportamiento y proyección de imagen de calcio en las larvas de pez cebra

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.

Un modelo de novela In Vitro de lesión cerebral traumática por explosión

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.

Un paradigma de objetivo emergente para evocar respuestas visuomotoras rápidas en los músculos humanos de las extremidades superiores

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, ...