Agradecemos Christian Landry para o calor de design da sonda, Daniel Babcock para o desenvolvimento do ensaio sonda larval calor, Sean Sweeney para sugerindo o ensaio de calor de placas, o Bloomington Drosophila Stock Center para as populações da mosca, e membros Galko laboratório para a leitura crítica do manuscrito. Este trabalho foi financiado pelo NIH R01 NS069828 para MJG e um NIH MARC U-STAR Training Grant (T34GM079088 para a Universidade de Houston Downtown-Scholars Academy) para o acesso das minorias às carreiras de investigação (AVG).

<ol>
	<li>Babcock, D. T., Landry, C., Galko, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytokine+signaling+mediates+UV-induced+nociceptive+sensitization+in+Drosophila+larvae.">Cytokine signaling mediates UV-induced nociceptive sensitization in Drosophila larvae.</a> Curr. Biol. 19, 799-806 (2009).</li><li>Tracey, W. D., Wilson, R. I., Laurent, G., Benzer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=painless,+a+Drosophila+gene+essential+for+nociception.">painless, a Drosophila gene essential for nociception.</a> Cell. 113, 261-273 (2003).</li><li>Oswald, M., Rymarczyk, B., Chatters, A., Sweeney, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+thermosensitive+escape+behavior+in+Drosophila+larvae.">A novel thermosensitive escape behavior in Drosophila larvae.</a> Fly (Austin). 5, 17810 (2011).</li><li>Grueber, W. B., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tiling+of+the+Drosophila+epidermis+by+multidendritic+sensory+neurons.">Tiling of the Drosophila epidermis by multidendritic sensory neurons.</a> Development. 129, 2867-2878 (2002).</li><li>Moore, A. W., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=hamlet,+a+binary+genetic+switch+between+single-+and+multiple-+dendrite+neuron+morphology.">hamlet, a binary genetic switch between single- and multiple- dendrite neuron morphology.</a> Science. 297, 1355-1358 (2002).</li><li>Gao, F. B., Brenman, J. E., Jan, L. Y., Jan, Y. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genes+regulating+dendritic+outgrowth,+branching,+and+routing+in+Drosophila.">Genes regulating dendritic outgrowth, branching, and routing in Drosophila.</a> Genes Dev. 13, 2549-2561 (1999).</li><li>Walters, E. T., Illich, P. A., Weeks, J. C., Lewin, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defensive+responses+of+larval+Manduca+sexta+and+their+sensitization+by+noxious+stimuli+in+the+laboratory+and+field.">Defensive responses of larval Manduca sexta and their sensitization by noxious stimuli in the laboratory and field.</a> J. Exp. Biol. 204, 457-469 (2001).</li><li>Woolf, C. J., Ma, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptors--noxious+stimulus+detectors.">Nociceptors--noxious stimulus detectors.</a> Neuron. 55, 353-364 (2007).</li><li>Hwang, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nociceptive+neurons+protect+Drosophila+larvae+from+parasitoid+wasps.">Nociceptive neurons protect Drosophila larvae from parasitoid wasps.</a> Curr. Biol. 17, 2105-2116 (2007).</li><li>Xiang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-avoidance-mediating+photoreceptors+tile+the+Drosophila+larval+body+wall.">Light-avoidance-mediating photoreceptors tile the Drosophila larval body wall.</a> Nature. 468, 921-926 (2010).</li><li>Grueber, W. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Projections+of+Drosophila+multidendritic+neurons+in+the+central+nervous+system:+links+with+peripheral+dendrite+morphology.">Projections of Drosophila multidendritic neurons in the central nervous system: links with peripheral dendrite morphology.</a> Development. 134, 55-64 (2007).</li><li>Sweeney, N. T., Li, W., Gao, F. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+manipulation+of+single+neurons+in+vivo+reveals+specific+roles+of+flamingo+in+neuronal+morphogenesis.">Genetic manipulation of single neurons in vivo reveals specific roles of flamingo in neuronal morphogenesis.</a> Dev. Biol. 247, 76-88 (2002).</li><li>Kernan, M., Cowan, D., Zuker, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+dissection+of+mechanosensory+transduction:+mechanoreception-defective+mutations+of+Drosophila.">Genetic dissection of mechanosensory transduction: mechanoreception-defective mutations of Drosophila.</a> Neuron. 12, 1195-1206 (1994).</li><li>Nitabach, M. N., Blau, J., Holmes, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrical+silencing+of+Drosophila+pacemaker+neurons+stops+the+free-running+circadian+clock.">Electrical silencing of Drosophila pacemaker neurons stops the free-running circadian clock.</a> Cell. 109, 485-495 (2002).</li></ol>

Não temos nada a divulgar.

Os ensaios aqui descritos podem ser usados ​​para avaliar qualitativamente e quantitativamente as larvas de genótipos diferentes para a capacidade de resposta a estímulos térmicos nocivos. Uma característica principal do ensaio sonda de calor é que o estímulo é dada apenas com um único locus. Este presumivelmente leva à demissão de apenas um pequeno subconjunto de classe IV de neurônios as do segmento contactados pela sonda e, talvez, aqueles que imediatamente segmentos adjacentes 11. Devido à estimulação local, o ensaio de sonda de calor imita a experiência comum sensorial de detecção de um estímulo nocivo que está localizada a uma região do corpo em particular, como uma mão contactando um fogão quente. Uma desvantagem do ensaio sonda de calor é que tem alguma variabilidade utilizador para utilizador que pode provavelmente ser atribuída a três factores: i) a pressão com a qual o utilizador aplica a sonda para a larva, ii) a localização precisa da sonda sobre a larva em relação aos neurónios subjacentes nociceptiva, e iii) o ang precisole em que a sonda contacta com a superfície da larva. Nós relatado anteriormente uma estratégia quantitativa de larvas de categorização em não-respondedores, respondedores e respondedores lentos, rápidos baseados em sua latência de retirada a uma dada temperatura 1. Aqui nós relatamos em resposta larval a temperaturas ainda mais elevadas. Curiosamente, descobrimos que existe um limite máximo para larvais térmicas respostas nociceptivas e que este limite máximo situa-se entre 52 e 54 ° C. Isso pode indicar que as larvas não possuem um potencial receptor transiente (TRP) canais capaz de modular a temperaturas superiores a 52 ° C. Alternativamente, poderia sugerir que os neurônios ou músculos usados ​​para iniciar ou executar a resposta do motor danificado antes mesmo de funcionar na retirada aversivo. Relatamos também uma análise diferente da amplitude da retirada-resposta utilizando-se o número de rolos como um indicador da &quot;robustez&quot; da resposta. Ingenuamente, umaSeria de esperar que estes parâmetros aumentaria com o aumento da temperatura ou do tempo de estimulação. Surpreendentemente, descobrimos que este não é o caso. As larvas estimuladas durante um tempo mais longo a uma temperatura na extremidade baixa da gama nociva (42 ° C) mostram rolos mais e mais tempo gasto de rolamento do que as larvas sondado a temperaturas mais elevadas (48-52 ° C). Isto sugere que dentro da janela de temperatura nocivo é sobretudo a duração da exposição que determina a amplitude da resposta. Uma vez que as larvas expostas a temperaturas altamente nocivo (48-52 ° C) respondem, em média, muito rapidamente, que não apresentem como rolos muitos como larvas exposto a uma temperatura inferior a nociva durante um longo período de tempo. A análise da amplitude da resposta aqui relatados acrescenta uma outra dimensão quantitativa ao longo do qual os diferentes genótipos ou manipulações ambientais podem ser comparados. Uma característica principal do ensaio em placa de calor é que envolve uma exposição global para o nocivocalor. Como tal, é mais parecido com o animal sentado em um caldeirão de aquecimento do que tocar um fogão quente. Embora não seja claro quando uma larva pode experimentar um estímulo nocivo globalmente na natureza, no laboratório as respostas comportamentais a esta exposição global são mais complexos do que aqueles observados com a estimulação local. Uma força de ensaio em placa de calor, também observado por outros 3, é que tem variabilidade utilizador para utilizador pouco desde tocar a larva não é um componente do protocolo. A variância apenas substancial parece ser na definição quando cada comportamento começa e isto pode ser minimizado com visualização repetido / familiaridade. Uma diferença interessante entre os ensaios é as temperaturas às quais os comportamentos aversivos começar. Estes são muito mais baixa no ensaio em placa de calor do que com a sonda de calor. O comportamento preliminar exibido por larvas em contacto com a sonda de calor (cabeça e aumento da cauda) pode estar correlacionado com o thrash cabeça observada a ~ 27 ° C, na plataforma de calorensaio e. É possível que esta resposta reflete &quot;desconforto&quot; mais de &quot;dor&quot;. Nós não observaram uma correlação de chicotear, convulsões, paralisia e mesmo em concentrações elevadas (até 48 ° C), as temperaturas no ensaio de sonda de calor e pode ser que uma massa crítica de neurónio sensorial de queima a partir de mais do que uma região do corpo é necessário para trazer a esses comportamentos. Curiosamente, os comportamentos convulsivos e paralisia são observadas a temperaturas (~ 34 - 37 ° C) abaixo da extremidade inferior do limiar nociceptivo observado com a sonda de calor indicando que a estimulação global pode envolver soma das respostas neuronais que são insuficientes para provocar o comportamento com locais aplicação da sonda de calor. Que estas temperaturas são realmente percebida como nocivo para as larvas é suportada pela observação de que as larvas que começam o comportamento paralisia e são subsequentemente deixados a recuperar em alimentos mosca não na maioria dos casos sobreviver (Figura 5C). Favor da tese de que o pl calorensaio comeu é a leitura de respostas nociceptivas é o facto de que o bloqueio da transmissão sináptica na conhecidos nociceptivos neurónios sensoriais aumenta a latência da maioria dos comportamentos observados (Figura 6). A observação de que não há um aumento na latência para o comportamento temperatura mais elevada-chicotear sugere que outros neurónios sensoriais que não expressam md-Gal4 pode ser necessário para este comportamento. Em suma, ambos os ensaios envolvem expondo uma larva indivíduo a um estímulo nocivo térmica da temperatura definida - a ponta quente de uma sonda de metal pequeno no ensaio de local e de imersão em uma gota de água aquecendo rapidamente no ensaio global. Respostas comportamentais de Drosophila larvas de diferentes origens genéticas e / ou expostos a diferentes condições ambientais (por exemplo mais ou menos danos no tecido), pode ser estudada e quantificada utilizando estes ensaios. Em última análise, os resultados destes ensaios nos ajudará a entender melhor as redes genéticas que controlam nociception em Drosophila e outras espécies relacionadas.

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Sonda térmica </td><td> Pro-Dev Engenharia </td><td> Custom-construído sob demanda </td><td> As informações de contato podem ser fornecidas a pedido </td></tr><tr><td> Banho seco Incubadora </td><td> Fisher Scientific </td><td> 11-718 </td><td> 1 bloco de aquecimento sólida e 1 bloco de aquecimento com poços de 16mm </td></tr><tr><td> Leica DFC290 12v/400mA Câmera colorida </td><td> Leica Microsystems </td><td> 12730080 </td><td> Qualquer equivalente câmara irá fazer. </td></tr><tr><td> Leica MZ6 microscópio </td><td> Leica Microsystems </td><td> Número da peça de corpo MZ6 zoom (portadora óptica) é 10445614 </td><td></td></tr><tr><td> Schott Ace Unidade Modulamp </td><td> Schott North America, Inc. </td><td> A20500 </td><td></td></tr><tr><td> Schott dupla Gooseneck de 23 polegadas de Fibra Óptica Guia de Luz </td><td> Schott North America, Inc. </td><td> Schott A08575 </td><td></td></tr><tr><td> Unidade de Controle Térmico </td><td> TSCI corp. </td><td> Custom Built </td><td> Detalhes podem ser fornecidas a pedido </td></tr><tr><td> Zeiss Stemi 2000 microscópio </td><td> Zeiss </td><td> NT55-605 </td><td> Qualquer equivalente microscópiovai fazer. </td></tr><tr><td> Fórceps </td><td> FST </td><td> FS-1670 </td><td></td></tr><tr><td> Um milímetro de malha </td><td> Genesee Científico </td><td> 57-101 </td><td></td></tr><tr><td> Paintbrush </td><td> Dick Blick Materiais de arte </td><td> 06762-1002 </td><td></td></tr><tr><td> Reticulador UV </td><td> Fisher Scientific </td><td> 1199289 </td><td></td></tr><tr><td> Coplin Jars </td><td> Fisher Scientific </td><td> 08-816 </td><td></td></tr><tr><td> Copo 10ml </td><td> Fisher Scientific </td><td> 02-540C </td><td></td></tr><tr><td> Éter dietílico </td><td> Fisher Scientific </td><td> E138-500 </td><td></td></tr><tr><td> 35 X 10 mm de poliestireno prato de Petri </td><td> Falcão </td><td> 351008 </td><td> Nós não testamos pratos alternativos. </td></tr><tr><td> Lâmina de vidro </td><td> Corning </td><td> 26003 </td><td></td></tr><tr><td> Termopar </td><td> Omega Engineering, Inc. </td><td> HH802U </td><td></td></tr><tr><td> Pedaço de vinil </td><td> Office Depot </td><td> 480009 </td><td></td></tr><tr><td> Tubo de microcentrífuga </td><td> Denville CientíficoInc. </td><td> C-2170 </td><td></td></tr></tbody></table>

local and global methods of assessing thermal nociception in drosophila larvae

Neste artigo, demonstramos ensaios para estudar a nocicepção térmica em larvas de Drosophila. Um ensaio envolve a estimulação espacialmente restrita (local) de 1,2 nociceptores térmica, enquanto o segundo envolve uma ativação (global) no atacado da maioria ou todos os neurônios tais 3. Juntas, essas técnicas permitem a visualização e quantificação das funções comportamentais de Drosophila nociceptivas dos neurônios sensitivos. A larva Drosophila é um sistema modelo estabelecido para estudar a nocicepção térmica, uma resposta sensorial para temperaturas potencialmente prejudiciais que é evolutivamente conservada entre espécies 1,2. As vantagens de Drosophila para tais estudos são relativa simplicidade do seu sistema nervoso ea sofisticação das técnicas genéticas que podem ser usados ​​para dissecar a base molecular da biologia subjacente 4-6 Em Drosophila, como em todos os metazoários, os response para estímulos térmicos nocivos geralmente envolve um &quot;nocifensive&quot; retirada aversivo ao estímulo apresentado 7. Tais estímulos são detectados através de terminações nervosas livres ou nociceptores e da amplitude da resposta organismal depende do número de nociceptores que recebem o estímulo nocivo 8. Em Drosophila, são os da classe IV, os neurónios sensoriais dendríticas arborização que detectam nocivas estímulos térmicos e mecânicos 9 em adição ao seu papel recentemente descoberto como fotorreceptores 10. Esses neurônios, que foram muito bem estudadas ao nível de desenvolvimento, arborize sobre a folha de barreira epidérmica e fazer contatos com quase todas as células epidérmicas 11,12. O axônio único de cada classe IV projectos de neurónios na medula ventral do nervo do sistema nervoso central 11, onde eles podem ligar a segunda ordem neurónios que projecto para o cérebro. Em condições basais, neuro sensorial nociceptivans não dispara até um limiar relativamente elevado for atingido. Os ensaios aqui descritos permitem que o investigador para quantificar as respostas comportamentais da linha de base ou, presumivelmente, a sensibilização que se segue danos nos tecidos a seguir. Cada ensaio provoca distintas, porém relacionadas locomotores respostas comportamentais a estímulos térmicos nocivos e permite ao pesquisador visualizar e quantificar diversos aspectos da nocicepção térmica em larvas de Drosophila. Os ensaios podem ser aplicados a larvas de genótipos desejados ou para larvas criadas em diferentes condições ambientais que podem afectar a nocicepção. Uma vez que a nocicepção térmica é conservada entre espécies, os resultados recolhidos a partir de dissecção genética em Drosophila provavelmente informar a nossa compreensão de nocicepção térmica em outras espécies, incluindo vertebrados.

Watch this Scientific Journal Video about Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae at JoVE.com

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae | Neuroscience | Video

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Neste artigo, demonstramos ensaios para estudar a nocicepção térmica em<em&gt; Drosophila</em&gt; Larvas. Um ensaio envolve a estimulação espacialmente restrita (local) de nociceptores térmicos<sup&gt; 1,2</sup&gt; Enquanto o segundo envolve uma ativação (global) no atacado da maioria ou todos os neurônios tais<sup&gt; 3</sup&gt;. Juntas, essas técnicas permitem a visualização e quantificação das funções comportamentais de<em&gt; Drosophila</em&gt; Nociceptivas dos neurônios sensitivos.

Métodos locais e globais de Avaliação nocicepção térmica em<em&gt; Drosophila</em&gt; Larvas

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of ...

local-global-methods-assessing-thermal-nociception-drosophila

Research

Education

JoVE Journal

JoVE Core

Biology

Neuroscience

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Human Biology

Sensory Systems

SCIENTISTS IN ACTION

15.7K visualizações.  The University of Texas MD Anderson Cancer Center. Neste artigo, demonstramos ensaios para estudar a nocicepção térmica em Drosophila Larvas. Um ensaio envolve a estimulação espacialmente restrita (local) de nociceptores térmicos 1,2 Enquanto o segundo envolve uma ativação (global) no atacado da maioria ou todos os neurônios tais 3. Juntas, essas técnicas permitem a visualização e quantificação das funções comportamentais de Drosophila Nocicep...

Vídeo: Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

Electrophysiological Measurements and Analysis of Nociception in Human Infants

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are based on behavioural and physiological body reactions, such as crying, body movements or changes in facial expression. While these measures demonstrate that infants mount a response following noxious stimulation, they are limited: they are based on activation of subcortical somatic and autonomic motor pathways that may not be reliably linked to central sensory processing in the brain. Knowledge of how the central nervous system responds to noxious events could provide an insight to how nociceptive information and pain is processed in newborns.
The heel lancing procedure used to extract blood from hospitalised infants offers a unique opportunity to study pain in infancy. In this video we describe how electroencephalography (EEG) and electromyography (EMG) time-locked to this procedure can be used to investigate nociceptive activity in the brain and spinal cord.
This integrative approach to the measurement of infant pain has the potential to pave the way for an effective and sensitive clinical measurement tool.

The assessment and treatment of pain in infants is difficult because infants cannot verbally report their experience. In this video we describe quantitative electrophysiological methods and analysis techniques that can be used to measure the response to noxious events from the infant nervous system.

Medidas eletrofisiológicas e Análise de nocicepção em bebês humanos

Pain is an unpleasant sensory and emotional experience. Since infants cannot verbally report their experiences, current methods of pain assessment are ...

Neurociência

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 ...

Appetitive Associative Olfactory Learning in Drosophila Larvae

In the following we describe the methodological details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with genetic interference, provides a handle to analyze the neuronal and molecular fundamentals of specifically associative learning in a simple larval brain.
Organisms can use past experience to adjust present behavior. Such acquisition of behavioral potential can be defined as learning, and the physical bases of these potentials as memory traces1-4. Neuroscientists try to understand how these processes are organized in terms of molecular and neuronal changes in the brain by using a variety of methods in model organisms ranging from insects to vertebrates5,6. For such endeavors it is helpful to use model systems that are simple and experimentally accessible. The Drosophila larva has turned out to satisfy these demands based on the availability of robust behavioral assays, the existence of a variety of transgenic techniques and the elementary organization of the nervous system comprising only about 10,000 neurons (albeit with some concessions: cognitive limitations, few behavioral options, and richness of experience questionable)7-10.
Drosophila larvae can form associations between odors and appetitive gustatory reinforcement like sugar11-14. In a standard assay, established in the lab of B. Gerber, animals receive a two-odor reciprocal training: A first group of larvae is exposed to an odor A together with a gustatory reinforcer (sugar reward) and is subsequently exposed to an odor B without reinforcement 9. Meanwhile a second group of larvae receives reciprocal training while experiencing odor A without reinforcement and subsequently being exposed to odor B with reinforcement (sugar reward). In the following both groups are tested for their preference between the two odors. Relatively higher preferences for the rewarded odor reflect associative learning - presented as a performance index (PI). The conclusion regarding the associative nature of the performance index is compelling, because apart from the contingency between odors and tastants, other parameters, such as odor and reward exposure, passage of time and handling do not differ between the two groups9.

Appetitive Associative Olfactory Learning in Drosophila Larvae

Drosophila larvae are able to associate odor stimuli with gustatory reward. Here we describe a simple behavioral paradigm that allows the analysis of appetitive associative olfactory learning.

Aprendizagem associativa apetitiva olfativa em<em&gt; Drosophila</em&gt; As larvas

In the following we describe the methodological  details of appetitive associative olfactory learning in Drosophila larvae. The setup, in combination with ...

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer basic questions on how light information is processed and shared between rapid and circadian behaviors. Drosophila larvae display a stereotypical avoidance behavior when exposed to light. To investigate light dependent behaviors comparably simple light-dark preference tests can be applied. In vertebrates and arthropods the neural pathways involved in sensing and processing visual inputs partially overlap with those processing photic circadian information. The fascinating question of how the light sensing system and the circadian system interact to keep behavioral outputs coordinated remains largely unexplored. Drosophila is an impacting biological model to approach these questions, due to a small number of neurons in the brain and the availability of genetic tools for neuronal manipulation. The presented light-dark preference assay allows the investigation of a range of visual behaviors including circadian control of phototaxis.

Light Preference Assay to Study Innate and Circadian Regulated Photobehavior in Drosophila Larvae

Here we describe a light-dark preference test for Drosophila larva. This assay provides information about innate and circadian regulation of light sensing and processing photobehavior.

Luz Ensaio Preferências para estudar inata e circadiano Photobehavior Regulamentado<em&gt; Drosophila</em&gt; As larvas

Light acts as environmental signal to control animal behavior at various levels. The Drosophila larval nervous system is used as a unique model to answer ...

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the underlying neuronal components in the circuits1-6. Application of optogenetics7,8 in the larval neural circuit allows us to manipulate neuronal activity in spatially and temporally patterned ways9-13. Typically, specimens are broadly illuminated with a mercury lamp or LED, so specificity of the target neurons is controlled by binary gene expression systems such as the Gal4-UAS system14,15. In this work, to improve the spatial resolution to "sub-genetic resolution", we locally illuminated a subset of neurons in the ventral nerve cord using lasers implemented in a conventional confocal microscope. While monitoring the motion of the body wall of the semi-intact larvae, we interactively activated or inhibited neural activity with channelrhodopsin16,17 or halorhodopsin18-20, respectively. By spatially and temporally restricted illumination of the neural tissue, we can manipulate the activity of specific neurons in the circuit at a specific phase of behavior. This method is useful for studying the relationship between the activities of a local neural assembly in the ventral nerve cord and the spatiotemporal pattern of motor output.

Optogenetic Perturbation of Neural Activity with Laser Illumination in Semi-intact Drosophila Larvae in Motion

Here we describe a protocol for optogenetic manipulation of motoneuronal activity while monitoring changes in motor output (muscle contraction) in semi-intact Drosophila larvae using lasers within a conventional confocal microscope. This technique enables researchers to achieve local perturbation of neural activity in a few neuromeres to elucidate the dynamics of motor circuits.

Optogenetic Perturbação da atividade neural com Iluminação Laser no Semi-intacta<em&gt; Drosophila</em&gt; As larvas in Motion

Drosophila larval locomotion is a splendid model system in developmental and physiological neuroscience, by virtue of the genetic accessibility of the ...

Foraging Path-length Protocol for Drosophila melanogaster Larvae

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral variation. The larval path-length assay was developed to measure individual differences in foraging behavior that were later linked to the foraging gene. Larval path-length is an easily scored trait that facilitates the collection of large sample sizes, at minimal cost, for genetic screens. Here we provide a detailed description of the current protocol for the larval path-length assay first used by Sokolowski. The protocol details how to reproducibly handle test animals, perform the behavioral assay and analyze the data. An example of how the assay can be used to measure behavioral plasticity in response to environmental change, by manipulating feeding environment prior to performing the assay, is also provided. Finally, appropriate test design as well as environmental factors that can modify larval path-length such as food quality, developmental age and day effects are discussed.

Foraging Path-length Protocol for Drosophila melanogaster Larvae

We provide a detailed protocol for a Drosophila melanogaster foraging path-length assay. We discuss the preparation and handling of test animals, how to perform the assay and analyze the data.

Forrageamento Protocolo Caminho de comprimento para<em&gt; Drosophila melanogaster</em&gt; Larvas

The Drosophila melanogaster larval path-length phenotype is an established measure used to study the genetic and environmental contributions to behavioral ...

Novel Assay for Cold Nociception in Drosophila Larvae

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory machinery, such as in patients experiencing peripheral neuropathy or injury-induced sensitization, are not well characterized. The genetically tractable Drosophila model has been used to study the cells and genes required for noxious heat detection, which has yielded multiple conserved genes of interest. Little is known however about the cells and receptors important for noxious cold sensing. Although, Drosophila does not survive prolonged exposure to cold temperatures (&#8804;10 &#186;C), and will avoid cool, preferring warmer temperatures in behavioral preference assays, how they sense and possibly avoid noxious cold stimuli has only recently been investigated.
Here we describe and characterize the first noxious cold (&#8804;10 &#186;C) behavioral assay in Drosophila. Using this tool and assay, we show an investigator how to qualitatively and quantitatively assess cold nociceptive behaviors. This can be done under normal/healthy culture conditions, or presumably in the context of disease, injury or sensitization. Further, this assay can be applied to larvae selected for desired genotypes, which might impact thermosensation, pain, or nociceptive sensitization. Given that pain is a highly conserved process, using this assay to further study&#160;thermal nociception will likely glean important understanding of pain processes in other species, including vertebrates.

Novel Assay for Cold Nociception in Drosophila Larvae

Here we demonstrate a novel assay to study cold nociception in Drosophila larvae. This assay utilizes a custom-built Peltier probe capable of applying a focal noxious cold stimulus and results in quantifiable cold-specific behaviors. This technique will allow further cellular and molecular dissection of cold nociception.

Romance Ensaio para Fria nocicepção em<em&gt; Drosophila</em&gt; Larvas

How organisms sense and respond to noxious temperatures is still poorly understood. Further, the mechanisms underlying sensitization of the sensory ...

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which enables them to seek out their preferred thermal landscape. To define the temperature preferences of larvae over a defined linear range, we developed an assay using a temperature gradient. To establish a single-directional gradient, two aluminum blocks are connected to independent water baths, each of which controls the temperature of individual blocks. The two blocks set the lower and upper limits of the gradient. The temperature gradient is established by placing an agarose-coated aluminum plate over the two water-controlled blocks so that the plate spans the distance between them. The ends of the aluminum plate that is set on the top of the water blocks defines the minimum and maximum temperatures, and the regions in-between the two blocks form a linear temperature gradient. The gradient assay can be applied to&#160;larvae of different ages and can be used to identify mutants that exhibit phenotypes, such as those with mutations affecting genes encoding TRP channels and opsins, which are required for temperature discrimination.

A Temperature Gradient Assay to Determine Thermal Preferences of Drosophila Larvae

Here, we present a protocol to determine the preferred environmental temperature of Drosophila larvae using a continuous thermal gradient.

Um gradiente de temperatura de ensaio para determinar preferências térmicas de larvas de Drosophila

Many animals, including the fruit fly, Drosophila melanogaster, are capable of discriminating minute differences in&#160;environmental temperature, which ...

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors

We present a protocol to electrophysiologically characterize bi-stable photopigments: (i) exploiting the charge displacements within the photopigment molecules following photon-absorption and their huge amount in the photoreceptors, and (ii) exploiting the absorption-spectra differences of rhodopsin and metarhodopsin photopigment states. These protocols are useful to screen for mutations affecting bi-stable photopigment systems.&#160;

Métodos Eletrofisiológicos para Medir Níveis de Fotopigment em Fotorreceptores de Drosophila

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is ...