This work was supported by grants to D.K. from the Medical Research Foundation of Oregon and from NIH/NINDS (NS047663). E.S. was supported by a training grant from the NIH (T32AG023477).

1. Fixação da cabeça em coleiras e inclusão em parafina Nota: Todos os passos no processo de fixação deve ser feito numa hotte. Metilbenzoato, embora não representam um risco para a saúde, tem um odor altamente distinta, que pode ser avassaladora se não for tratado em um exaustor. <ol><li> Antes de anestesiar as moscas, fazem-se 50 ml de solução de Carnoy por adição de 15 ml de clorofórmio e 5 ml de ácido acético glacial e 30 mL de etanol a 99% (não se misturam o clorofórmio e ácido acético). Derramá-lo num recipiente de vidro com um fundo plano, tal como um prato cristalizadas, para garantir que os colares podem estabelecer plana e são completamente cobertas com a solução. </li><li> Anestesiar as moscas com CO 2 ou éter. </li><li> moscas de rosca (até 20 com a maioria dos colares) por seus pescoços para os colares usando uma pinça. Recorde para alinhar todos os cabeças com a mesma orientação, como pode ser visto na Figura 1A, e ser suave para assegurar que não se verifiquem danos na cabeça ou olhos. </li><li> Incluir moscas seno Oculis (setas, Figura 1A) em posições aleatórias para que a ordem das moscas podem ser facilmente identificados nas secções. Além disso, se as moscas experimentais têm uma cor branca ou clara do olho, passe algumas moscas de olhos vermelhos, tais como o tipo selvagem, entre elas para assegurar que o pigmento está presente suficiente para corar a corrediça. Grave a ordem das moscas sobre a folha de protocolo em conjunto com o número colar se utilizar mais do que um anel. </li><li> Uma vez que um colar foi terminado, colocá-lo em solução de Carnoy preparado para 3,5-4 h. </li><li> Despejar a solução Carnoy dentro do recipiente eliminação adequada e começar as lavagens de etanol. Certifique-se lentamente para derramar, de modo a não perturbar a colocação dos colares no recipiente. </li><li> Lavar os colares durante 30 min em 99% de etanol duas vezes. </li><li> Lavar os colares em 100% de etanol durante 1 h. Certifique-se de alterar as lavagens a tempo para evitar overdehydration. </li><li> Coloque os colares em metilbenzoatoO / N à temperatura ambiente. Fecha-se o recipiente com parafilme para evitar a evaporação do metilbenzoato. </li><li> Verter a methylbenozate para dentro do recipiente descartável adequado na hotte. Adicionar uma mistura previamente preparada de 1: 1 de baixo ponto de fusão (56-57 ° C), cera de parafina e metilbenzoato de metilo. A partir deste ponto, os colares devem ser mantidas numa incubadora a 65 ° C para se certificar de que a parafina não endurece. </li><li> Deite a mistura de parafina e methylbenozate para dentro do recipiente descartável adequado, e derramar a cera de parafina puro fundido, mantido a 65 ° C, para os colares. </li><li> Alterar a parafina depois de 30 min e repita este pelo menos 5 vezes. Pelo menos 6 - 8 lavagens devem ser executadas. </li><li> Uma vez que as lavagens são completos, coloque os colares em uma bandeja de cubos de gelo de borracha com ranhuras de aproximadamente o tamanho dos colares. Despeje parafina derretida sobre eles até que esteja completamente coberto e deixe-a endurecer O / N (tentar evitar bolhas de ar). </li><li> Remova os blocos de parafina containing os colares da bandeja de cubos de gelo. Separa-se o bloco de parafina a partir da gola utilizando uma lâmina de barbear, suavemente romper o colar. As cabeças estará no bloco de parafina ao passo que os corpos vão ficar na gola. Os blocos podem ser mantidos à temperatura ambiente. </li><li> Para limpar os colares, mergulhe-os em um agente desparafinização a 65 ° C para remover a parafina, limpo esfregando-se ligeiramente, e lave em etanol antes de reutilizar. </li></ol> 2. corte e montagem <ol><li> Aquecer uma placa de aquecimento a 50 ° C. Coloque os titulares objeto (ou blocos de montagem de metal) e lâminas de barbear sobre a placa e deixá-los aquecer. </li><li> Dependendo da orientação desejada para a secção transversal, e anexar o bloco de parafina, quer com as cabeças para o lado (para secções horizontais) ou virada para cima (para secções frontais) a um bloco de montagem aquecida (derretendo brevemente o bloco no lado do contacto). Remover o bloco da placa de aquecimento e deixe-o esfriar por pelo menos 10 min para assegurar que a parafina é endurecido o suficiente para uma vedação adequada para o bloco de montagem. Manter a fila de cabeças alinhadas em paralelo com a superfície do bloco, tanto quanto possível para evitar desnivelamentos. </li><li> Pegue uma lâmina de barbear e aparar o excesso de parafina longe das cabeças de mosca de modo que apenas uma pequena fila com os chefes incorporados permanece (a lâmina de barbear pode ser aquecido para corte mais fácil). Certifique-se de não cortar muito para que a parafina não quebra durante o corte (mais corte pode ser feito durante o corte). </li><li> Colocar o bloco de montagem no suporte do objecto do micrótomo e assegurar que o alinhamento da fila de cabeças é tão paralela quanto possível da aresta da lâmina. </li><li> Preparar lâminas de microscópio, cobrindo-os com uma camada fina de-L-lisina de poli (PLL) solução e deixar secar durante 5 min. Cubra-os com água imediatamente antes da utilização. </li><li> Cortar 7 uM secções e transferir a fita de secções para a corrediça que flutua na água. &lt;br /&gt; NOTA: Para obter o cérebro inteiro para seções horizontais, recolhemos a fita de quando iniciar o corte no olho até que a cabeça foi completamente cortado (corte a partir a tromba para dentro do cérebro). Mais de um slide pode ser necessário para toda a cabeça. </li><li> Colocar a lâmina numa placa de calor a 37 ° C e permitir que a fita para expandir durante cerca de 1 min. </li><li> Retire o excesso de água (por vazamento-lo ou usando um tecido) e secar a diapositivos O / N. </li><li> Remover a cera de parafina a partir do slide, colocando os diapositivos numa altura, vertical frasco deslizante de coloração com um agente desparafinização (cobrindo completamente as secções). Realizar 3 lavagens de 30 min - 60 min cada. </li><li> Remover o slide da lavagem final. Coloque 2 gotas de meios de incorporação para o slide e cobri-lo com uma lamela grande. </li></ol> 3. fotografando e analisando as Secções <ol><li> Permitir que as lâminas preparadas para secar por 1-2 d. Em seguida, examiná-los em um microsc fluorescênciaope sob a luz azul. </li><li> Utilize uma ampliação menor para determinar a orientação das moscas e para encontrar a região de interesse se concentrando-se em uma região específica. NOTA: Para as moscas SWS (ver Figura 2), encontramos a seção que contém a grande comissura e tomar uma imagem (geralmente em 40X). Ao analisar todo o cérebro (como na Figura 3), que percorrer todas as secções de uma cabeça e quer fotografar a secção com o fenótipo mais grave ou todas as secções que mostram vacúolos. </li><li> Para uma análise duplo-cego, tomar e imagens numéricas sem saber o genótipo e anote o número colarinho e posição da cabeça na linha de identificá-los mais tarde. </li><li> Depois que as imagens foram tomadas, analisá-los usando um software de imagem. </li><li> Contar o número de vacúolos por secção ou por cabeça. Para medir o tamanho vacúolo, abrir as imagens em um programa de software e selecionar os vacúolos com uma seleção muitoeu. Determinar a quantidade de pixels nos vacúolos seleccionados. </li><li> Para a conversão para mm 2, tirar uma foto de um slide de calibração estágio micrômetro na ampliação usada para a aquisição de fotos. Determinar a quantidade de pixels em 100 uM 2 para calcular um factor de conversão. </li><li> Converter o número total de pixels em 2 uM dividindo o número de pixels de acordo com o factor de conversão calculado no passo acima. </li></ol>

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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glucocerebrosidase+Deficiency+in+Drosophila+Results+in+alpha-Synuclein-Independent+Protein+Aggregation+and+Neurodegeneration.">Glucocerebrosidase Deficiency in Drosophila Results in alpha-Synuclein-Independent Protein Aggregation and Neurodegeneration.</a> PLoS Genet. 12, e1005944 (2016).</li><li>Dias-Santagata, D., Fulga, T. A., Duttaroy, A., Feany, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oxidative+stress+mediates+tau-induced+neurodegeneration+in+Drosophila.">Oxidative stress mediates tau-induced neurodegeneration in Drosophila.</a> J Clin Invest. 117, 236-245 (2007).</li><li>Kretzschmar, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glial+and+neuronal+expression+of+polyglutamine+proteins+induce+behavioral+changes+and+aggregate+formation+in+Drosophila.">Glial and neuronal expression of polyglutamine proteins induce behavioral changes and aggregate formation in Drosophila.</a> Glia. 49, 59-72 (2005).</li><li>Li, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Drosophila+model+for+TDP-43+proteinopathy.">A Drosophila model for TDP-43 proteinopathy.</a> Proc Natl Acad Sci U S A. 107, 3169-3174 (2010).</li></ol>

Os autores não têm nada para revelar.

O método descrito fornece um meio para quantificar a neurodegeneração no cérebro de Drosophila. Enquanto que outros métodos, tais como contagem de um tipo celular específico, pode ser usado para identificar a neurodegeneração, a vantagem deste método é que ele pode ser aplicada de modo mais geral. Contar as células requer que estas células podem ser identificadas com fiabilidade utilizando quer um anticorpo específico ou a expressão de um marcador específico de células, a qual não está sempre ...

Com o aumento da expectativa de vida, doenças neurodegenerativas, como a doença de Alzheimer ou de Parkinson tornaram-se uma ameaça crescente de saúde para a população em geral. De acordo com os Institutos Nacionais de Saúde, 115 milhões de pessoas em todo o mundo estão previstos para ser afetada pela demência em 2050. Embora um progresso significativo tem sido feito na identificação de genes e fatores de risco envolvidos em pelo menos algumas dessas doenças, para muitos deles, o subjacente mecanismos moleculares ainda são desconhecidos ou não bem compreendida. Simples organismos modelo de invertebrados como Caenorhabditis elegans e Drosophila melanogaster oferecem uma variedade de vantagens experimentais para estudar os mecanismos de doenças neurodegenerativas, incluindo um ciclo de vida curta, grande número de descendentes, e da disponibilidade de métodos genéticos e moleculares bem estabelecidas e, por vezes únicas 1 -12. Além disso, estes organismos são passíveis de imparcialtelas de interação que podem identificar fatores que contribuem para estas doenças por seus efeitos agravantes ou melhorar em fenótipos neurodegenerativas. Analisar tais interações genéticas e avaliar os efeitos do envelhecimento requer protocolos quantitativos para detectar neurodegeneração e medir a sua gravidade. Esta avaliação pode ser feita de forma relativamente fácil quando se mede aspectos comportamentais em Drosophila, como a aprendizagem olfactiva, geotaxis negativos, ou fototaxia rápido, que fornecem um valor de desempenho numérico 13-21. É também possível determinar os efeitos sobre a sobrevivência neuronal contando os neurónios. No entanto, isso só é possível quando o foco em uma população específica que é claramente identificável, como os neurónios dopaminérgicos que são afectados na doença de Parkinson, e mesmo assim, os resultados têm sido controversos 22-24. O protocolo aqui descrito utiliza o método de colar para realizar cortes seriados de parafina, um métodoque foi originalmente desenvolvido por Heisenberg e Böhl, que o usou para isolar mutantes cerebrais anatômicas em Drosophila 25. A utilização do método de colar foi subsequentemente adaptado, incluindo em criocortes, vibratome secções, e secções de plástico 26-28. Aqui, este método é empregue para obter secções em série de toda a cabeça da mosca, que pode então ser utilizado para medir os vacúolos que se desenvolvem em moscas com fenótipos 16,21,29-32 neurodegenerativas. Estas medições podem ser feitas em regiões específicas do cérebro, ou pode cobrir todo o cérebro; esta última abordagem permite um para identificar os fenótipos fracos mesmo degenerativas, como observado durante o envelhecimento. Finalmente, quando se utiliza os colares, até 20 moscas pode ser processada como uma preparação, que não só é menos demorado, mas também permite a análise de controlo e as moscas experimentais na mesma preparação, minimizando artefactos devido a ligeiras alterações na a preparação.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Name of the Reagent/Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td></td>
		</tr>
		<tr>
			<td>Collar</td>
			<td>Genesee Scientific</td>
			<td>TS 48-100</td>
			<td>We are using custom made collars that are made from one piece of metal instead of layers as the ones by Genesee. A discription to make collars can be found at http://flybrain.neurobio.arizona.edu/Flybrain/html/atlas/fluorescent/index.html&#160;</td>
		</tr>
		<tr>
			<td>Rubber ice cube tray for embedding</td>
			<td>Household store</td>
			<td></td>
			<td>The size can be made to fit by glueing in additional walls&#160;</td>
		</tr>
		<tr>
			<td>Crystallizing dish</td>
			<td>Fisher Scientific company</td>
			<td>08-762-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Ether</td>
			<td>Fisher Scientific Company</td>
			<td>E138-1</td>
			<td></td>
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			<td>Ethanol</td>
			<td>Decon Laboratories Inc.</td>
			<td>2701</td>
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			<td>Choloroform</td>
			<td>Fisher Scientific Company</td>
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			<td>Fisher Scientific Company</td>
			<td>A38-212</td>
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			<td>Methylbenzoate</td>
			<td>Fisher Scientific Company</td>
			<td>M205-500</td>
			<td>Distinct Odor</td>
		</tr>
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			<td>Use in fume hood</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
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			<td>Low Melting Point Paraffin Wax</td>
			<td>Fisher Scientific Company</td>
			<td>T565</td>
			<td>Make sure to keep extra melted in a 65&#176;C waterbath</td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica Biosystems</td>
			<td>Reichert Jung 2040 Autocut</td>
			<td></td>
		</tr>
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			<td>Microscope Slide</td>
			<td>Fisher Scientific Company</td>
			<td>12-550D</td>
			<td></td>
		</tr>
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			<td>Microscope Cover Glass</td>
			<td>Fisher Scientific Company</td>
			<td>12-545-M</td>
			<td></td>
		</tr>
		<tr>
			<td>SafeClear</td>
			<td>Fisher Scientific Company</td>
			<td>314-629</td>
			<td>Three different containers for washes</td>
		</tr>
		<tr>
			<td>Vertical Staining Jar with Cover</td>
			<td>Ted Pella Inc.&#160;</td>
			<td>432-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Permount</td>
			<td>Fisher Scientific Company</td>
			<td>SP15-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-lysine Solution</td>
			<td>Sigma Life Science</td>
			<td>P8290-500</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass histology to quantify neurodegeneration in drosophila

doenças neurodegenerativas progressivas como a doença de Alzheimer (AD) ou a doença de Parkinson (DP) é uma ameaça crescente para a saúde humana em todo o mundo. Embora os modelos de mamíferos forneceram importantes insights sobre os mecanismos subjacentes da patogenicidade, a complexidade dos sistemas de mamíferos, juntamente com seus altos custos estão limitando a sua utilização. Portanto, o simples, mas bem estabelecida modelo de sistema de Drosophila fornece uma alternativa para investigar as vias moleculares que são afetados nessas doenças. Além défices comportamentais, doenças neurodegenerativas são caracterizadas por fenótipos histológicos, tais como a morte neuronal e axonopatia. Para quantificar a degeneração neuronal e determinar como ela é afetada por fatores genéticos e ambientais, usamos uma abordagem histológica que é baseado na medição dos vacúolos no cérebro mosca adulta. Para minimizar os efeitos do erro sistemático e comparar diretamente as seções de controle e expmoscas erimental em uma preparação, usamos o método &quot;colar&quot; para cortes de parafina. A neurodegeneração é então avaliada medindo o tamanho e / ou o número de vacúolos que se desenvolveram no cérebro da mosca. Isto pode ser feito concentrando-se em uma região específica de interesse ou analisando todo o cérebro através da obtenção de cortes seriados que se estendem da cabeça completa. Por conseguinte, este método permite um para medir não só a degeneração severa mas também fenótipos relativamente suaves que só são detectáveis ​​em algumas secções, como ocorre durante o envelhecimento normal.

Usando os resultados do método descrito em cortes seriados corados pelo pigmento do olho 33, que abrangem toda a cabeça da mosca. Uma parte desta é mostrado na Figura 1B, em que as secções de uma cabeça individual são mostrados de cima para baixo. As secções de diferentes moscas são vistos esquerda para a direita neste exemplo. Para facilitar a orientação e a identificação das moscas, de uma mosca sem olhos (oculis seno) é inserido como...

Watch this Scientific Journal Video about Mass Histology to Quantify Neurodegeneration in Drosophila at JoVE.com

Mass Histology to Quantify Neurodegeneration in Drosophila

Drosophila é amplamente usado como um sistema modelo para estudar a neurodegeneração. Este protocolo descreve um método pelo qual a degeneração, como determinado pela formação de vacúolo no cérebro, podem ser quantificados. Ela também minimiza os efeitos devido ao procedimento experimental por meio de processamento e controlo de moscas e de seccionamento experimentais como uma amostra.

Mass Histologia para quantificar neurodegeneração em<em&gt; Drosophila</em

Progressive neurodegenerative diseases like Alzheimer&#39;s disease (AD) or Parkinson&#39;s disease (PD) are an increasing threat to human health ...

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Neuroscience

Mass Histology to Quantify Neurodegeneration in Drosophila

10.1K visualizações.  Oregon Health & Sciences University.  Drosophila é amplamente usado como um sistema modelo para estudar a neurodegeneração. Este protocolo descreve um método pelo qual a degeneração, como determinado pela formação de vacúolo no cérebro, podem ser quantificados. Ela também minimiza os efeitos devido ao procedimento experimental por meio de processamento e controlo de moscas e de seccionamento experimentais como uma amostra. 

Vídeo: Mass Histology to Quantify Neurodegeneration in Drosophila

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Sinapses quantificar: um ensaio baseado em Imunocitoquímica para a quantificação do número Synapse

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Neurociência

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal. Such analysis of two or more genes needs to be complemented with in situ hybridization of other genes, or immunohistochemistry of their proteins, both in whole mounted developing organs or sections for detailed resolution of the cellular and tissue expression alterations. Combining multiple gene alterations requires the use of cre or flipase to conditionally delete genes and avoid embryonic lethality. Required breeding schemes dramatically enhance effort and cost proportional to the number of genes mutated, with an outcome of very few animals with the full repertoire of genetic modifications desired. Amortizing the vast amount of effort and time to obtain these few precious specimens that are carrying multiple mutations necessitates tissue optimization. Moreover, investigating a single animal with multiple techniques makes it easier to correlate gene deletion defects with expression profiles. We have developed a technique to obtain a more thorough analysis of a given animal; with the ability to analyze several different histologically recognizable structures as well as gene and protein expression all from the same specimen in both whole mounted organs and sections. Although mice have been utilized to demonstrate the effectiveness of this technique it can be applied to a wide array of animals. To do this we combine lipophilic dye tracing, whole mount in situ hybridization, immunohistochemistry, and histology to extract the maximal possible amount of data.

Combining Lipophilic dye, in situ Hybridization, Immunohistochemistry, and Histology

A combination of different techniques to maximize data collection from mouse tissue is presented.

Combinando corante lipofílico, In situ Hibridação, imunohistoquímica e histologia

Going beyond single gene function to cut deeper into gene regulatory networks requires multiple mutations combined in a single animal.  Such analysis of ...

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of these diseases, including Alzheimer's, Parkinson's and Huntington's disease are characterized by age-dependent deterioration in learning and memory functions and movement coordination2. Here we use behavioral assays, including the negative geotaxis assay3 and the aversive phototaxic suppression assay (APS assay)4,5, to show that some of the behavior characteristics associated with human neurodegeneration can be recapitulated in flies. In the negative geotaxis assay, the natural tendency of flies to move against gravity when agitated is utilized to study genes or conditions that may hinder locomotor capacities. In the APS assay, the learning and memory functions are tested in positively-phototactic flies trained to associate light with aversive bitter taste and hence avoid this otherwise natural tendency to move toward light. Testing these trained flies 6 hours post-training is used to assess memory functions. Using these assays, the contribution of any genetic or environmental factors toward developing neurodegeneration can be easily studied in flies.

Assaying Locomotor, Learning, and Memory Deficits in Drosophila Models of Neurodegeneration

Behavior assays for measuring locomotor functions, learning, and memory abilities in Drosophila.

Locomotor ensaio, Aprendizagem e Memória Os déficits no Drosophila Modelos de neurodegeneração

Advances in genetic methods have enabled the study of genes involved in human neurodegenerative diseases using Drosophila as a model system1. Most of ...

In vivo Laser Axotomy in C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron is damaged by injury or disease, it may regenerate. Cell-intrinsic and extrinsic factors influence the ability of a neuron to regenerate and restore function. Recently, the nematode C. elegans has emerged as an excellent model organism to identify genes and signaling pathways that influence the regeneration of neurons1-6. The main way to initiate neuronal regeneration in C. elegans is laser-mediated cutting, or axotomy. During axotomy, a fluorescently-labeled neuronal process is severed using high-energy pulses. Initially, neuronal regeneration in C. elegans was examined using an amplified femtosecond laser5. However, subsequent regeneration studies have shown that a conventional pulsed laser can be used to accurately sever neurons in vivo and elicit a similar regenerative response1,3,7.
We present a protocol for performing in vivo laser axotomy in the worm using a MicroPoint pulsed laser, a turnkey system that is readily available and that has been widely used for targeted cell ablation. We describe aligning the laser, mounting the worms, cutting specific neurons, and assessing subsequent regeneration. The system provides the ability to cut large numbers of neurons in multiple worms during one experiment. Thus, laser axotomy as described herein is an efficient system for initiating and analyzing the process of regeneration.

In vivo Laser Axotomy in C. elegans

A protocol to cut neurons in C. elegans with a MicroPoint pulsed laser is presented. We describe setting up the system, immobilizing worms, and severing labeled neurons. Advantages include a relatively low-cost system and the ability to sever neuronal processes or ablate cells in vivo.

In vivo Laser axotomia em C. elegans

Neurons communicate with other cells via axons and dendrites, slender membrane extensions that contain pre- or post-synaptic specializations. If a neuron ...

Methods to Assay Drosophila Behavior

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular disease and various neurological diseases1. We have optimized simple and robust behavioral assays for determining larval locomotion, adult climbing ability (RING assay), and courtship behaviors of Drosophila. These behavioral assays are widely applicable for studying the role of genetic and environmental factors on fly behavior. Larval crawling ability can be reliably used for determining early stage changes in the crawling abilities of Drosophila larvae and also for examining effect of drugs or human disease genes (in transgenic flies) on their locomotion. The larval crawling assay becomes more applicable if expression or abolition of a gene causes lethality in pupal or adult stages, as these flies do not survive to adulthood where they otherwise could be assessed. This basic assay can also be used in conjunction with bright light or stress to examine additional behavioral responses in Drosophila larvae. Courtship behavior has been widely used to investigate genetic basis of sexual behavior, and can also be used to examine activity and coordination, as well as learning and memory. Drosophila courtship behavior involves the exchange of various sensory stimuli including visual, auditory, and chemosensory signals between males and females that lead to a complex series of well characterized motor behaviors culminating in successful copulation. Traditional adult climbing assays (negative geotaxis) are tedious, labor intensive, and time consuming, with significant variation between different trials2-4. The rapid iterative negative geotaxis (RING) assay5 has many advantages over more widely employed protocols, providing a reproducible, sensitive, and high throughput approach to quantify adult locomotor and negative geotaxis behaviors. In the RING assay, several genotypes or drug treatments can be tested simultaneously using large number of animals, with the high-throughput approach making it more amenable for screening experiments.

Methods to Assay Drosophila Behavior

Drosophila melanogaster is a genetically and behaviorally tractable model system that has been used to understand the molecular and cellular basis of many important biological processes for over a century 1. Drosophila has been well exploited to gain insights into the genetic basis of fly behavior.

Métodos de Ensaio para Drosophila Comportamento

Drosophila melanogaster, the fruit fly, has been used to study molecular mechanisms of a wide range of human diseases such as cancer, cardiovascular ...

Visualization of Proprioceptors in Drosophila Larvae and Pupae

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other insects proprioception is provided by specialized sensory organs termed chordotonal organs (ChOs) 2. Like many other organs in Drosophila, ChOs develop twice during the life cycle of the fly. First, the larval ChOs develop during embryogenesis. Then, the adult ChOs start to develop in the larval imaginal discs and continue to differentiate during metamorphosis.
The development of larval ChOs during embryogenesis has been studied extensively 10,11,13,15,16. The centerpiece of each ChO is a sensory unit composed of a neuron and a scolopale cell. The sensory unit is stretched between two types of accessory cells that attach to the cuticle via specialized epidermal attachment cells 1,9,14. When a fly larva moves, the relative displacement of the epidermal attachment cells leads to stretching of the sensory unit and consequent opening of specific transient receptor potential vanilloid (TRPV) channels at the outer segment of the dendrite 8,12. The elicited signal is then transferred to the locomotor central pattern generator circuit in the central nervous system.
Multiple ChOs have been described in the adult fly 7. These are located near the joints of the adult fly appendages (legs, wings and halters) and in the thorax and abdomen. In addition, several hundreds of ChOs collectively form the Johnston's organ in the adult antenna that transduce acoustic to mechanical energy 3,5,17,4.
In contrast to the extensive knowledge about the development of ChOs in embryonic stages, very little is known about the morphology of these organs during larval stages. Moreover, with the exception of femoral ChOs 18 and Johnston's organ, our knowledge about the development and structure of ChOs in the adult fly is very fragmentary.
Here we describe a method for staining and visualizing ChOs in third instar larvae and pupae. This method can be applied together with genetic tools to better characterize the morphology and understand the development of the various ChOs in the fly.

Visualization of Proprioceptors in Drosophila Larvae and Pupae

A method to immunostain and visualize chordotonal organs in larvae and pupae of Drosophila melanogaster is described.

Visualização de Propriocepção em<em&gt; Drosophila</em&gt; As larvas e pupas

Proprioception is the ability to sense the motion, or position, of body parts by responding to stimuli arising within the body. In fruitflies and other ...

Utilizing 3D Printing Technology to Merge MRI with Histology: A Protocol for Brain Sectioning

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume three-dimensionally. While MRI is an extremely sensitive tool for detecting tissue abnormalities, association of signal changes with an underlying pathological process is usually not straightforward. In the central nervous system, for example, inflammation, demyelination, axonal damage, gliosis, and neuronal death may all induce similar findings on MRI. As such, interpretation of MRI scans depends on the context, and radiological-histopathological correlation is therefore of the utmost importance. Unfortunately, traditional pathological sectioning of brain tissue is often imprecise and inconsistent, thus complicating the comparison between histology sections and MRI. This article presents novel methodology for accurately sectioning primate brain tissues and thus allowing precise matching between histology and MRI. The detailed protocol described in this article will assist investigators in applying this method, which relies on the creation of 3D printed brain slicers. Slightly modified, it can be easily implemented for brains of other species, including humans.

The overall goal of this protocol is to accurately align magnetic resonance imaging (MRI) image volumes with histology sections via the creation of customized 3D-printed brain holders and slicer boxes.

Utilizando tecnologia de impressão 3D para mesclar MRI com Histologia: Um protocolo para o Cérebro Seccionamento

Magnetic resonance imaging (MRI) allows for the delineation between normal and abnormal tissue on a macroscopic scale, sampling an entire tissue volume ...

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic Brain Injury (mTBI). The mechanisms responsible for its complex pathological changes remain largely elusive, despite a recent consensus to define the neuropathological criteria. Here, we describe a novel method to develop a model of CTE in Drosophila melanogaster&#160;(Drosophila&#160;)&#160;in an attempt to identify the key genes and pathways that lead to the characteristic hyperphosphorylated tau accumulation and neuronal death in the brain. Adjustable-strength impacts to inflict mild closed injury are delivered directly to the fly head, subjecting the head to rapid acceleration and deceleration. Our method eliminates the potential problems inherent with other Drosophila mTBI models (e.g.,animal death might be induced by damage to other parts of the body or to internal organs). The less labor- and cost-intensive animal care, short life span, and extensive genetic tools make the fruit fly ideal to study CTE pathogenesis and make it possible to perform large-scale, genome-wide forward genetic and pharmacological screens. We anticipate that the ongoing characterization of the model will generate important mechanistic insights on disease prevention and therapeutic approaches.

A Novel Method to Model Chronic Traumatic Encephalopathy in Drosophila

Here, we describe a new approach to inflict closed-head traumatic brain injury in Drosophila melanogaster. Our method has the advantage of directly delivering repetitive impacts with adjustable strength to the head alone. Further exploration of the invertebrate system will help to illuminate the pathogenesis of chronic traumatic encephalopathy.

Um novo método para modelar a encefalopatia traumática crônica em<em&gt; Drosophila</em

Chronic Traumatic Encephalopathy (CTE) is an established neurodegenerative disease that is closely associated with exposure to repetitive mild Traumatic ...

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

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

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

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 ...

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. Drosophila larval neuronal dendritic arborization (da) is an ideal model for studying morphogenesis of neural dendrites and gene function in the development of nervous system. There are four classes of da neurons. Class IV is the most complex with a branching pattern that covers almost the entire area of the larval body wall. We have previously characterized the effect of silencing the Drosophila ortholog of SOX5 on class IV neuronal dendritic arborization complexity (NDAC) using four parameters: the length of dendrites, the surface area of dendrite coverage, the total number of branches, and the branching structure. This protocol presents the workflow of NDAC quantitative analysis, consisting of larval dissection, confocal microscopy, and image analysis procedures using ImageJ software. Further insight into da neuronal development and its underlying mechanisms will improve the understanding of neuronal function and provide clues about the fundamental causes of neurological and neurodevelopmental disorders.

Quantitative Analysis of Neuronal Dendritic Arborization Complexity in Drosophila

This protocol focuses on quantitative analysis of neuronal dendritic arborization complexity (NDAC) in Drosophila, which can be used for studies of dendritic morphogenesis.

Análise quantitativa da arborização dendrítica Neuronal complexidade em Drosophila

Dendrites are the branched projections of a neuron, and dendritic morphology reflects synaptic organization during the development of the nervous system. ...