This is supported by 973 MOST grant (Grant No. 2011CB504402, 2012CB947602), National Natural Science Foundation of China (Grant No. 91132724, U1332136) and the 'Hundred Talents Project' of Chinese Academy of Science. The protocol was approved by the Committee on the Ethics of Animal Experiments of the USTC (Permit Number: USTCACUC1103013).

1. Construir o Aparelho Cirurgia NOTA: Para assegurar o peixe permanece vivo durante e após a operação, a anestesia de gotejamento uma solução de acetato de metanossulfonato de 3-aminobenzoato de metilo (MS-222, ou tricaina) a uma metade da concentração (0,015%), com uma velocidade de 1 gota / seg através da boca do peixe utilizando um sistema de gotejamento homebuilt mostrado na Figura 1. <ol><li> Fazer uma caixa, como mostrado na Figura 1A (comprimento é de 28 cm, a largura é de 10 cm, a altura é de 5 cm), colocar uma esponja (comprimento é de 6 cm, a largura é de 9 cm, altura de 4 cm) no meio e abrir um lacuna nele. </li><li> Fazer uma superfície lisa globular na extremidade da agulha, com ligação de cura de luz (Figura 1B), o que irá então ser inserido na boca de peixe. Certifique-se de que a superfície da agulha é suave para evitar que a boca do peixe seja danificada. </li><li> Adicionar a metade da concentração de MS-222 (câmara esquerda, o comprimento é de 6 cm, largura é de 10 cm, a altura é de 8 cm) e solução salina (câmara direita, o comprimento de 4 cm, a largura é de 10 cm, a altura é de 8 cm) para os reservatórios como mostrado na Figura 1C. Nota: fresco de MS-222 é recomendado. </li><li> Anestesiar peixes em 0,03% MS-222 por 2 min. </li><li> Coloque o peixe na abertura da esponja e corrigir o peixe com dois pares de pinos, inserida atrás da barbatana peitoral e ao lado do poro cloacal, respectivamente 11. </li><li> Ligue boca do peixe, o MS-222, e água, com a criação de um tubo de tipo T, como mostrado na Figura 1C. </li><li> Ligar o tubo ligado a MS-222. </li></ol> 2. CGR rótulo retrógrados do Tectum Direito NOTA: Antes de começar, desinfetar todos os aparelhos com etanol 70-75%, garantindo que nenhum etanol permanece. <ol><li> Rossing: ligeiramente remover a pele ao longo de todo o crânio com sclerectome (vista dorsal do crânio é mostrado nas Figuras 2A e 2B </strong&gt;). </li><li> Compensação: lavar o crânio com soro fisiológico e, em seguida, seque-o com a orelha lâmpada de lavar. </li><li> Corrosão inicial: corroer o crânio com o etchant por 10 seg. </li><li> Compensação: limpar completamente afastado o etchant com um cotonete e, em seguida, lavar a área com soro fisiológico e seque-o. </li><li> Proteção: a fim de proteger o crânio em outras áreas, exceto o tectum direito de excesso de corrosão, cobrir essas áreas com ligação de luz de cura e depois curá-lo pela exposição à luz azul com iluminante portátil. </li><li> Corrosão completa: colocar o etchant na área central do tectum direita novamente para gravação completa para 2 min. Nota: Como crânios de peixes mais velhos são calcificada, corrosão vai demorar mais tempo. </li><li> Compensação: limpar completamente afastado o etchant com um cotonete e, em seguida, lavar a área com soro fisiológico e seque-o novamente. </li><li> Abertura Crânio: remova cuidadosamente o crânio de malacia (Figura 2C) com uma pinça para expor o tectum direita (Figura 2D </strong&gt;). </li><li> Compensação: limpar o muco e sangue effusing do buraco com pedaços de Gelfoam e lavá-lo com soro fisiológico até que o sangramento parou. </li><li> Colocação Dye: coloque o corante (a rotulagem tecido colar) em todo o tectum direita e cobri-lo com gelfoam. </li><li> Segregando: cobrir o buraco com uma escala estéril do mesmo animal. </li><li> Vedação: vínculo lugar fotopolimerizador para a escala e depois curá-lo com luz azul novamente por 40 seg. </li><li> Revival: lavar a superfície do vínculo e reviver o animal com solução salina. </li><li> Enfermagem e reprodução: Mantenha o peixe em 28,5 ° C médio embrião (ZFIN) durante 5 dias e alimentar 2 vezes / dia. Não incluem peixe cujo corante não ficar no tectum por 5 dias no experimento. Nota: A solução de médio Embrião (EM) é vital para a sobrevivência dos peixes e da temperatura é o fator chave para a rotulagem DII. </li></ol> 3. Wholemounting Retina e Imagem <ol><li> Coloque o peixe em um campo escuro por 2 horas antes de retina quelemount. </li><li> Anestesiar os peixes com água gelada. </li><li> Perfurar um buraco na margem da córnea e faça um copo olho, removendo a córnea com uma tesoura Vênus. </li><li> Retire a lente e, em seguida, lave o centro da retina com gelado 1x PBS. </li><li> Lave a lacuna entre retina ea esclera com gelado 1x PBS até que nenhum pigmento é esquerda. </li><li> Corte o nervo óptico no disco. </li><li> Mantenha a camada RGC para cima e mantenha a retina com uma colher de vidro self-made (Figuras 3A e 3B). Um conta-gotas também pode ser usado para transferir a retina. </li><li> Coloque a retina em uma queda de gelo de 30% de glicerina em um slide com a camada RGC para cima (Figura 3C). </li><li> Retire a glicerina e depois achatar a retina na lâmina antes de cortar retina em quatro quadrantes (Figura 3D). Mantendo a retina se desenrolou com camada RGC para cima será útil para achatamento da retina. </li><li> Gotejamento gelada 50% de glicerina em tele retina para lavar os fragmentos de pigmento. </li><li> Retirar a glicerina e em seguida gota 20 mL de gelo de 75% de glicerina na retina. </li><li> Cobrir a amostra com 1,8 centímetros x 1,8 centímetros lamela quadrado. </li><li> Selar a lamela com unha polonês. Recomenda imagem imediata da retina. </li><li> Imagem não-entrelaçada toda a retina sob uma lente de objeto 20X (NA = 0,70) com DP72 CCD (cada imagem é 1360 x 1024 pixels). Cada campo tem três focos diferentes. </li><li> Estender a profundidade de cada campo com a ferramenta de &quot;profundidade de campo estendida&quot; no software. </li><li> Montar uma retina virtual com a função de photomerge. </li><li> Escolha três campos de 680 x 512 pixels (área efectiva é de 350 mM em 264 mM, 0,092 mm 2) em cada uma orientação da retina para analisar a densidade média das CGR (Figura 4A). </li><li> Obter a área com a função de medidas - criar um polígono na imagemsoftware. </li><li> Calcule o número total de CGR pela equação &quot;número de CGR = destino x área&quot; 9. </li></ol>

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We have nothing to disclose.

Marcação retrógrada das CGR é importante para a investigação CGRs sobrevivência em animais mamíferos, mas não tem sido usado em peixes-zebra. Os métodos alternativos, coloração HE 7 e coloração de anticorpos 8, não são padrões de ouro para a contagem número CGR e linhagens transgênicas com todos os CGRs marcadas ainda não foi construído 12, 13. Neste vídeo, apresentamos um método para retrógrado CGR rótulo na retina do peixe-zebra adulto. Apesar de cerca de 1% do...

Como adulto zebrafish tem uma forte capacidade de regenerar os axônios após a lesão do nervo óptico 1, um método apropriado para contar toda a CGR é essencial para avaliar CGR sobrevivência e regeneração 2. Baseado nos métodos de CGRs marcação retrógrada em mamíferos e peixes dourados 3-5, construímos o método para rotular CGRs do tectum em peixes-zebra adultos. Para o adulto zebrafish, duas preocupações técnicos críticos devem ser observados: o crânio de peixe-zebra adulto é muito pequena 6; eles vivem em um ambiente de água. Aqui, tratamos o crânio com etchant o que minimiza os riscos associados com a perfuração 5. Em seguida, selar o buraco com vínculo fotoativação que melhora a sobrevida dos animais após a cirurgia. Anteriormente, foram adotadas diversas outras técnicas para contar o número CGR de forma indireta. Coloração HE nas secções de retina rótulos de todos os tipos de células no RGCL 7. Marcação de anticorpos em toda a retina, tais como ilhotas-1, também podem marcar células amacrine 8. Embora rótulo retrógrada do tronco do nervo óptico pode rotular todos os CGR na retina, não pode ser adotado no modelo de esmagamento porque provoca lesão extra para o nervo óptico. Aproveitando-se da etiqueta retrógrada do tectum, temos pesquisado CGR sobrevivência e regeneração no esmagamento do nervo óptico. Os resultados mostram que quase todas as CGRs sobreviveram e mais de 90% das CGR regenerado para o tectum na primeira semana de o modelo de esmagamento do 9. Para rotular com sucesso todos os CGR, DII pasta foi escolhido após a comparação com vários outros corantes comerciais 10. Em primeiro lugar, é especialmente projetado para in vivo rotulagem tecido. Em segundo lugar, é um corante lipofílico que não se pode difundir na água. Além disso, essa fluorescência pode persistir por um longo período de tempo que o torna um excelente candidato para CGR investigação sobrevivência.

<table border="0" cellpadding="0" cellspacing="0" width="608">
	<tbody>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">MS222</td>
			<td style="width:152px;">Sigma Aldrich</td>
			<td style="width:152px;">E10521</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">DiI</td>
			<td style="width:152px;">Invitrogen</td>
			<td style="width:152px;">N22880</td>
			<td style="width:152px;">USA</td>
		</tr>
		<tr height="37">
			<td height="37" style="height:37px;width:152px;">lightcuring bond</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Durafill bond</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="55">
			<td height="55" style="height:55px;width:152px;">Gluma Etch</td>
			<td style="width:152px;">Heraeus Kulzer&#160;</td>
			<td style="width:152px;">Gluma Etch 35 Gel</td>
			<td style="width:152px;">Germany</td>
		</tr>
		<tr height="73">
			<td height="73" style="height:73px;width:152px;">Blue LED</td>
			<td style="width:152px;">Shenruo Medical Equipment Co.</td>
			<td style="width:152px;">Power Blue Light Curing Unit</td>
			<td style="width:152px;">China</td>
		</tr>
	</tbody>
</table>

retrograde labeling of retinal ganglion cells in adult zebrafish with fluorescent dyes

Rotulagem células ganglionares da retina como retrógrados (RGCs) pode isolar CGR somata de morrer locais, tornou-se o padrão-ouro para a contagem de CGR em CGR sobrevivência e experimentos de regeneração. Muitos estudos têm sido realizados em animais mamíferos para pesquisar CGR sobrevivência após a lesão do nervo óptico. No entanto, a marcação retrógrada de CGR em peixe-zebra adulto ainda não foi relatada, embora alguns métodos alternativos pode contar o número de células em camadas da retina células ganglionares (RGCL). Considerando o pequeno tamanho do crânio adulto zebrafish eo alto risco de morte após a perfuração no crânio, abrimos o crânio com a ajuda de condicionamento ácido e selar o buraco com um vínculo de cura luz, o que pode melhorar significativamente a taxa de sobrevivência. Depois de absorver os corantes, durante 5 dias, quase todos os CGR são rotulados. Como este método não precisa transecto o nervo óptico, é insubstituível na pesquisa de CGR sobrevivência após esmagamento do nervo óptico em peixe-zebra adulto. Aqui, apresentamoseste método passo a passo e fornecer resultados representativos.

Como Figuras 4B-D mostra, o número de células DII + é de dois terços de células DAPI + em RGCL. Na retina normal, uma imagem de montagem de uma retina inteira (Figura 4E) mostra que DII + CGRs são distribuídos ao longo de toda a retina, mas em uma retina regenerada (Figura 4F), como CGRs na área central não regenerado para o seu alvo em Na primeira semana, eles não poderiam ser rotulados. <p class="jove_content" fo:keep-tog...

Watch this Scientific Journal Video about Retrograde Labeling of Retinal Ganglion Cells in Adult Zebrafish with Fluorescent Dyes at JoVE.com

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

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 ...

retrograde-labeling-retinal-ganglion-cells-adult-zebrafish-with

Research

JoVE Journal

Neuroscience

9.4K visualizações.  University of Science and Technology of China. We introduce an efficient method to retrograde label retinal ganglion cells (RGCs) in adult zebrafish.

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

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Rotulagem e Células de imagem no rombencéfalo Zebrafish

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neurociência

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Uma preparação isolada da retina para gravar Resposta Luz geneticamente identificadas células ganglionares da retina

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

Optical Imaging of Neurons in the Crab Stomatogastric Ganglion with Voltage-sensitive Dyes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous activity of participating neurons leads to the emergence of the integral functionality of the network. Here we present the methodology of application of this technique to identified pattern generating neurons in the crab stomatogastric ganglion. We demonstrate the loading of these neurons with the fluorescent voltage-sensitive dye Di-8-ANEPPQ and we show how to image the activity of dye loaded neurons using the MiCAM02 high speed and high resolution CCD camera imaging system. We demonstrate the analysis of the recorded imaging data using the BVAna imaging software associated with the MiCAM02 imaging system. The simultaneous voltage-sensitive dye imaging of the detailed activity of multiple neurons in the crab stomatogastric ganglion applied together with traditional electrophysiology techniques (intracellular and extracellular recordings) opens radically new opportunities for the understanding of how central pattern generator neural networks work.

Here we present the methodology for fast and high resolution fluorescent voltage-sensitive dye imaging of detailed activity of neurons in the crab stomatogastric ganglion.

Imagem óptica de neurônios no gânglio de Caranguejo Stomatogastric com Tensão sensível Corantes

Voltage-sensitive dye imaging of neurons is a key methodology for the understanding of how neuronal networks are organised and how the simultaneous ...

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.

Transfecção de células de camundongo por Ganglionares da Retina In vivo Eletroporação

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

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Retrograde Loading of Nerves, Tracts, and Spinal Roots with Fluorescent Dyes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. Generally, the dyes are applied as solid crystals or by local pressure injection using glass pipettes. However, this can result in dilution of the dye and reduced labeling intensity, particularly when several hours are required for dye diffusion. Here we demonstrate a simple and low-cost technique for introducing fluorescent and ion-sensitive dyes into neurons using a polyethylene suction pipette filled with the dye solution. This method offers a reliable way for maintaining a high concentration of the dye in contact with axons throughout the loading procedure.

We describe a simple and low cost technique for introducing high concentration of fluorescent and calcium-sensitive dyes into neurons or any neuronal tract using a polyethylene suction pipette.

Carregando retrógrado de Nervos, folhetos, e raízes nervosas com corantes fluorescentes

Retrograde labeling of neurons is a standard anatomical method1,2 that has also been used to load calcium and voltage-sensitive dyes into neurons3-6. ...

Genetic Study of Axon Regeneration with Cultured Adult Dorsal Root Ganglion Neurons

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability to support axon growth and a hostile environment in the mature CNS1,2. In contrast, mature neurons in the peripheral nervous system (PNS) regenerate readily after injuries3. Adult dorsal root ganglion (DRG) neurons are well known to regenerate robustly after peripheral nerve injuries. Each DRG neuron grows one axon from the cell soma, which branches into two axonal branches: a peripheral branch innervating peripheral targets and a central branch extending into the spinal cord. Injury of the DRG peripheral axons results in substantial axon regeneration, whereas central axons in the spinal cord regenerate poorly after the injury. However, if the peripheral axonal injury occurs prior to the spinal cord injury (a process called the conditioning lesion), regeneration of central axons is greatly improved4. Moreover, the central axons of DRG neurons share the same hostile environment as descending corticospinal axons in the spinal cord. Together, it is hypothesized that the molecular mechanisms controlling axon regeneration of adult DRG neurons can be harnessed to enhance CNS axon regeneration. As a result, adult DRG neurons are now widely used as a model system to study regenerative axon growth5-7.
Here we describe a method of adult DRG neuron culture that can be used for genetic study of axon regeneration in vitro. In this model adult DRG neurons are genetically manipulated via electroporation-mediated gene transfection6,8. By transfecting neurons with DNA plasmid or si/shRNA, this approach enables both gain- and loss-of-function experiments to investigate the role of any gene-of-interest in axon growth from adult DRG neurons. When neurons are transfected with si/shRNA, the targeted endogenous protein is usually depleted after 3-4 days in culture, during which time robust axon growth has already occurred, making the loss-of-function studies less effective. To solve this problem, the method described here includes a re-suspension and re-plating step after transfection, which allows axons to re-grow from neurons in the absence of the targeted protein. Finally, we provide an example of using this in vitro model to study the role of an axon regeneration-associated gene, c-Jun, in mediating axon growth from adult DRG neurons9.

An in vitro model for genetic study of axon regeneration using cultured adult mouse dorsal root ganglion neurons is described. The method includes a re-suspension/re-plating step to allow axon re-growth from neurons undergoing genetic manipulation. This approach is especially useful for loss-of-function studies of axon regeneration using RNAi-based protein knockdown.

Estudo genético da regeneração do axônio com cultivadas Adulto neurônios do gânglio da raiz dorsal

It is well known that mature neurons in the central nervous system (CNS) cannot regenerate their axons after injuries due to diminished intrinsic ability ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Implementação da braçadeira dinâmico com Condutâncias Synaptic e Artificial em células ganglionares da retina do rato

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

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

Light-induced retinal degeneration (LIRD) is commonly used in both rodents and zebrafish to damage rod and cone photoreceptors. In adult zebrafish, photoreceptor degeneration triggers M&#252;ller glial cells to re-enter the cell cycle and produce transient-amplifying progenitors. These progenitors continue to proliferate as they migrate to the damaged area, where they ultimately give rise to new photoreceptors. Currently, there are two widely-used LIRD paradigms, each of which results in varying degrees of photoreceptor loss and corresponding differences in the regeneration response. As more genetic and pharmacological tools are available to test the role of individual genes of interest during regeneration, there is a need to develop a robust LIRD paradigm. Here we describe a LIRD protocol that results in widespread and consistent loss of both rod and cone photoreceptors in which we have combined the use of two previously established LIRD techniques. Furthermore, this protocol can be extended for use in pigmented animals, which eliminates the need to maintain transgenic lines of interest on the albino background for LIRD studies.

Multiple light damage protocols have been described to damage photoreceptors and consequently induce a retinal regeneration response in adult zebrafish. This protocol describes an improved method that can be used in pigmented animals and that damages the vast majority of rod and cone photoreceptors across the entire 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, ...

Single-cell RNA-Seq of Defined Subsets of Retinal Ganglion Cells

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for the improved understanding of neuronal diversity, it is important to identify genes whose expression defines various subpopulations of cells. The retina serves as an excellent model for the study of central nervous system diversity, as it is composed of multiple major cell types. The study of each major class of cells has yielded genetic markers that facilitate the identification of these populations. However, multiple subtypes of cells exist within each of these major retinal cell classes, and few of these subtypes have known genetic markers, although many have been characterized by morphology or function. A knowledge of genetic markers for individual retinal subtypes would allow for the study and mapping of brain targets related to specific visual functions and may also lend insight into the gene networks that maintain cellular diversity. Current avenues used to identify the genetic markers of subtypes possess drawbacks, such as the classification of cell types following sequencing. This presents a challenge for data analysis and requires rigorous validation methods to ensure that clusters contain cells of the same function. We propose a technique for identifying the morphology and functionality of a cell prior to isolation and sequencing, which will allow for the easier identification of subtype-specific markers. This technique may be extended to non-neuronal cell types, as well as to rare populations of cells with minor variations. This protocol yields excellent-quality data, as many of the libraries have provided read depths greater than 20 million reads for single cells. This methodology overcomes many of the hurdles presented by Single-cell RNA-Seq and may be suitable for researchers aiming to profile cell types in a straightforward and highly efficient manner.

Here, we present a combinatorial approach for classifying neuronal cell types prior to isolation and for the subsequent characterization of single-cell transcriptomes. This protocol optimizes the preparation of samples for successful RNA Sequencing (RNA-Seq) and describes a methodology designed specifically for the enhanced understanding of cellular diversity.

Single-cell RNA-Seq de subgrupos definidos de células de gânglio retiniano

The discovery of cell type-specific markers can provide insight into cellular function and the origins of cellular heterogeneity. With a recent push for ...