Os autores também desejam expressar graças a Kris Burner, Stephen McKinney, Laura Tobin, Jenny Gilbreath, Ashley Olley, Megan Hoffer, e Megan Hyde por seu trabalho em sintonia fina neste ensaio.

1. Preparação de Individual Moscas para Locomotion Assay <ol><li> Transferência voa batendo suavemente em frascos individuais vazias e tampe os frascos com um tampão de algodão. Permitir moscas para se sentar sem perturbações 20-30 min antes da observação. É importante não anestesiar as moscas dentro das horas antes da observação comportamental em estudos anteriores descobriram que a exposição a anestesia podem alterar o comportamento em relação a moscas não anestesiados 14. </li><li> Bata levemente a mosca do frasco e coloque moscas individuais sob a 5 cm de diâmetro tampa placa de Petri (5 mm de altura), com pequenas fendas para permitir a passagem de ar. Iluminar abaixo e observar as moscas usando uma webcam montada acima da placa de Petri. </li></ol> 2. Preparação de Individual Moscas para Apreensão Assay <ol><li> Alimente de dois dias de idade apreensão moscas sensíveis tanto media levedura / fubá / ágar padrão ou mídia padrão misturado diretamente com drogas. Para os dados aqui apresentados, as moscas foram alimentados com um movimentof mídia padrão ou 1 g de mídia padrão misturado com 25 mg de metformina. </li><li> Após as moscas têm alimentado por dois dias na mídia ou mídia além de drogas, transferi-los agitando ligeiramente em frascos individuais vazias e tampe os frascos com um tampão de algodão. Permitir moscas para se sentar sem ser perturbado por 20 min antes da observação. É importante não anestesiar as moscas dentro das horas antes da observação comportamental em estudos anteriores descobriram que a exposição a anestesia podem alterar o comportamento em relação a moscas não anestesiados 14. </li><li> Vortex frascos individuais contendo uma única mosca em uma vortexer laboratório usando a configuração mais alta por 10 segundos. Coloque a mosca imobilizada em uma folha branca de papel em branco logo abaixo de uma webcam montada acima do papel. </li></ol> 3. Gravação de Vídeo <ol><li> Registre o movimento ou SLA usando o programa de software HandyAvi <a href="http://www.azcendant.com/download.htm">http://www.azcendant.com/download.htm</a> . Este programa utiliza a webcam paracapturar imagens com base nas configurações do usuário. </li><li> Selecione a opção Time-Lapse imagens na aba captura, quando a janela abrir, selecione a webcam como o dispositivo de captura. Para a maioria dos experimentos o tamanho do quadro de vídeo de 640 x 480 é adequada. </li><li> Selecione Intel IYUV codec para a compressão e escolher 0,1 seg / frame para o ensaio apreensão ou escolher 0,1-0,5 seg / frame para o ensaio de movimento. Na aba Avançado, selecione criar um arquivo de imagem. Bmp para cada captura e selecione uma pasta para o programa para armazenar as imagens que leva (a pilha de imagem). </li><li> Coloque uma mosca morta na folha de papel. Clique na caixa de configurações de vídeo e ajuste as configurações na guia Configurações do dispositivo, para que haja um fundo branco brilhante e um claro contraste com a mosca mais escura. </li><li> Clique no botão Iniciar para começar a gravação. Uma vez que o período de gravação tiver terminado, clique no botão stop. A pilha de imagens deve estar agora na pasta selecionada. Nota: Antes de gravar, verifique se a folder selecionado para a pilha de imagens está vazio. </li></ol> 4. Análise de Dados Utilizando ImageJ <ol><li> Imagem livre ImageJ software de processamento aberto do NIH, que pode ser baixado em <a href="http://rsbweb.nih.gov/ij/download.html">http://rsbweb.nih.gov/ij/download.html</a> . Nota: Além de baixar o programa, o Multitracker plug-in deve ser baixado e instalado, conforme indicado no <a href="http://rsbweb.nih.gov/ij/plugins/index.html">http://rsbweb.nih.gov/ij/plugins/index.html</a> . </li><li> Importe a pilha de imagens em ImageJ clicando na guia Arquivo e, em seguida, selecionando Import → seqüência de imagens. <ol><li> Selecione a pasta que contém a pilha de imagens, realce uma das imagens e selecione Abrir. Uma caixa de diálogo Opções de seqüência será aberta. Clique nos nomes Ordenar numericamente e em seguida, selecione OK ea pilha de imagem será carregada no ImageJ. </li></ol></li><li> Limite de todas as imagens, de modo que cada um constituído por um fundo branco com a mosca serconvertida para um ponto negro. Para iniciar este processo, converter a imagem para um formato de 8 bits, selecionando Tipo → 8 bit sob a guia Imagem. <ol><li> Clique na aba Imagem novamente e selecione Ajustar → Threshold. Isto irá abrir a caixa de diálogo Limiar em que o limiar ponto de corte pode ser ajustado. </li><li> Digitalizar através de imagens para ver se há pontos pretos extras ou se uma fatia carece de um ponto preto. Se este for o caso, ajustar o limiar para aliviar o problema. Normalmente, o limite não precisa ser ajustado como o programa padrão para um valor adequado. Nota: todos os slides na pilha deve ter apenas um ponto preto que representa a posição da mosca. </li><li> Selecione aplicar na caixa de diálogo Limiar e um convertido ao Máscara caixa de diálogo será aberta. Não clique em qualquer uma das opções, mas basta clicar em OK para limiar da pilha de imagem. </li></ol></li><li> Após limiarizar a pilha de imagens, selecione o Multitracker plug-in sob a aba Plug-ins, a fim demedir o movimento da mosca em pixels. A caixa de diálogo Rastreador de objeto aparecerá. <ol><li> Clique em todos os quatro opções dadas na caixa de diálogo e, em seguida, selecione OK. O plug-in Multitracker então anexar coordenadas para o ponto preto em cada fatia e listá-los na janela de resultados, bem como o comprimento total do caminho, em pixels. A janela Paths dá uma visualização gráfica do caminho a mosca pegou. </li><li> Copie a lista x-e coordenada y da janela Resultados e colar os dados em uma planilha do Excel. Se qualquer slide na pilha não tem um ponto preto na mesma, o programa Multitracker parará em que slide. Se isso ocorrer, é necessário voltar ao passo 4.2 e re-limite a pilha para garantir que os registos voar como um ponto preto no slide em questão. </li></ol></li><li> Para converter o caminho comprimentos de pixels para cm, dê uma imagem separada de dois pontos que estão espaçados um centímetro de distância em uma parte branca de papel em branco. <ol><li> Siga o acimapassos (3.1-4.4.2), para determinar as coordenadas x, y do pixel para os dois pontos e usar esta informação para determinar o número de pixels correspondentes a um centímetro. Este fator de conversão é usado na etapa cinco para gerar dados de movimento em cm. Nota: Uma vez feito isso, é importante para manter a câmera fixa para todas as gravações futuras. Cada vez que a câmera é movida ou modificada, é preciso repetir este passo para converter os pixels para cm. </li><li> O comprimento do percurso que é gerado pelo Multitracker plug-in sobrestima movimento porque pequenas flutuações na posição do corpo e intensidade de luz / reflexão vai afectar o tamanho e no centro do ponto negro que é gerado quando limiarizar as imagens. Este ruído deve ser removido a fim de obter uma estimativa precisa do movimento mosca. </li></ol></li></ol> 5. Análise de Dados Usando o Excel <ol><li> Remover o ruído dos dados gravados, importando a série de coordenadas x, y gerado pelo pr MultitrackerOgram em Análise Fly, um programa do Visual Basic Excel criado pelos autores. (O programa está disponível em contato com os autores.) </li><li> Clique no Click Me botão e selecione um valor de limite de corte. Selecione um valor limite de 0,5 cm / s para análise SLA ou selecione um valor entre 0,1-0,3 cm / s para análise do movimento. Clique em OK eo programa irá calcular a velocidade, tempo e distância do movimento. Nota: O programa usa uma análise de janela deslizante para avaliar os dados seqüenciais 0,5-1,0 caixas seg e calcula a velocidade durante essa janela. Qualquer coisa acima do limite definido pelo usuário é tratado como movimento mosca. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Ao examinar locomotora ou padrões de movimento em Drosophila, que é útil para ser capaz de extrair a informação sobre a distância percorrida, a velocidade do movimento e o padrão de movimento. Para extrair esta informação, equipamentos caros tem sido tradicionalmente empregada que é muitas vezes proibitivo para laboratórios menores ou laboratórios que desejam utilizar tais ensaios com moderação 4,8,9. O ensaio aqui descrito pode ser utilizado para determinar...

Dada a sua curta vida útil e as ferramentas genéticas robustos disponíveis, Drosophila melanogaster é um sistema excelente modelo para investigar a etiologia de várias doenças ea fisiologia subjacente de vários processos biológicos. Em muitos casos, é vantajoso para medir os efeitos que as manipulações comportamentais, genéticos ou farmacológicos têm em locomoção nestes organismos modelo 1,2. Há uma variedade de métodos que são geralmente utilizados para medir o movimento da mosca em duas dimensões 3-7. Estes sistemas podem apoiar o acompanhamento de várias moscas ao mesmo tempo e pode medir velocidade, comprimento do passo de registro e registrar a porcentagem de tempo que uma mosca passou em movimento. Eles têm sido utilizados para estudar o movimento numa variedade de contextos, incluindo os efeitos de drogas sobre a locomoção e a natureza sexual dimórfico de movimento mosca 6-9. A principal desvantagem desses sistemas é o custo dos sistemas de rastreamentotemperatura ou o software correspondente e câmera. Em alguns casos, isso pode ser executado em milhares de dólares. O custo é uma preocupação particular para um laboratório que só precisa de um uso limitado de tal sistema, por exemplo, quantificar os padrões de locomotivas de um mutante recém isolado. Um método mais simples e menos robustos é a utilização de sistemas que o movimento ficha base em quantas vezes uma mosca atravessa um percurso de feixe de luz infravermelha, que está colocado no meio de um tubo fechado 10,11. Embora tais sistemas podem dar informações valiosas sobre ciclos de movimento e de sono-vigília, eles podem sobre ou sob estimativa movimento, porque eles não conseguem captar o caminho real da mosca. Por exemplo, moscas que apresentam movimento considerável nas extremidades do tubo vai registrar um preço tão baixo movimento voa embora os métodos de alta resolução adicionais foram usados ​​para tentar contornar essas limitações 12. Mais simples e mais barato ainda estão subindo dispositivos que medem geotaxis, o movimento ascendente das moscas, através de um único tubo ou uma série de tubos de 2,13. Embora esses sistemas são baratos e podem facilmente identificar defeitos geotaxis, eles não conseguem captar muitos outros aspectos de movimento que seria de interesse para os investigadores. Para muitos laboratórios, um sistema de análise robusta de baixo custo que é simples de configurar e operar seria uma ferramenta vantajosa para a caracterização de diferenças comportamentais em D. melanogaster cepas. Aqui nós descrevemos um ensaio que pode ser set-up para menos de duzentos dólares e é capaz de dar informações sobre o caminho, velocidade e duração do movimento da mosca. Para demonstrar a eficácia do ensaio, foram apresentados dados mostrando que pode ser utilizado para identificar, 1) um defeito locomotor em um mutante Bang-sensível (BS), que é susceptível a ataques e 2) a capacidade da metformina, a qual é comumente usado para tratar a diabetes do tipo II, para reduzir a intensidade da atividade de apreensão semelhante (SLA) in dois mutantes BS.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="68">
			<td height="68" style="height:68px;width:251px;">Name of the Reagent/Equipment</td>
			<td style="width:152px;">Company</td>
			<td style="width:169px;">Catalog Number/Model</td>
			<td style="width:545px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">WebCam</td>
			<td>Logitech</td>
			<td>Pro900</td>
			<td>Any quality webcam will suffice.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HandiAVI time-lapse software</td>
			<td>Azcendant software</td>
			<td></td>
			<td>Latest version can be found at http://www.azcendant.com/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ImageJ</td>
			<td>NIH</td>
			<td></td>
			<td>Latest version can be found at http://rsbweb.nih.gov/ij/download.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multiracker Plug-In</td>
			<td>NIH</td>
			<td></td>
			<td>Latest version can be found at http://rsbweb.nih.gov/ij/plugins/index.html</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortexer</td>
			<td>VWR</td>
			<td>Vortex Genie 2</td>
			<td>Most standard size vortexers such as the Vortex Genie 2 will suffice.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5cm petri dish cover</td>
			<td>LabM Limited</td>
			<td>D011</td>
			<td>Smaller or larger petri dish covers can be used for an arena in movement assay.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Light box</td>
			<td colspan="2">Built from scrap material.</td>
			<td>Illumination is used for the locomotion assay. Depending on the room lighting, it is possible to perform the assay without the light box.&#160;</td>
		</tr>
	</tbody>
</table>

a low-cost method for analyzing seizure-like activity and movement in drosophila

Sistemas de monitoramento de vídeo têm sido amplamente utilizados para analisar Drosophila melanogaster movimento e detectar várias anormalidades no comportamento locomotiva. Embora esses sistemas podem fornecer uma riqueza de informações comportamentais, o custo ea complexidade desses sistemas pode ser proibitivo para muitos laboratórios. Nós desenvolvemos um teste de baixo custo para medir o comportamento locomotiva e movimento apreensão em D. melanogaster. O sistema usa uma web-cam para capturar imagens que podem ser processados ​​usando uma combinação de software de baixo custo e livre para seguir a distância percorrida, a velocidade média de circulação ea duração do movimento durante um período de tempo especificado. Para demonstrar a utilidade deste sistema, que analisou um grupo de D. melanogaster mutantes, os paralíticos (BS) Bang-sensíveis, que são 3-10 vezes mais suscetíveis a atividade de apreensão semelhante (SLA) de moscas de tipo selvagem. Usando este novo sistema, pudemos detectar que o BS mutanat sem sentido estrondo (BSS) apresenta níveis mais baixos de locomoção exploratória em um ambiente de romance que tipo selvagem moscas. Além disso, o sistema foi utilizado para identificar que a metformina, que é vulgarmente utilizada para tratar a diabetes do tipo II, reduz a intensidade do SLA nos mutantes BS.

A técnica aqui descrita foi utilizada anteriormente para analisar diferenças de SLA nas mutantes BS Drosophila 1. Os resultados aqui apresentados envolvem as cepas BS facilmente chocado (EAS), golpe sem sentido (BSS) e nocaute técnico (TKO). O locus eas codifica uma cinase de etanolamina envolvido na síntese de fosfatidil etanolamina 15 e o locus TKO codifica um riboprotein mitocondrial 16. A mutação BSS é um alelo do paral...

Watch this Scientific Journal Video about A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila at JoVE.com

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Usando uma webcam e uma combinação de programas de software livre e de baixo custo, os padrões de locomotivas em Drosophila melanogaster podem ser analisadas para detectar diferenças na velocidade, distância e tempo de várias atividades motoras.

Um método de baixo custo para análise de Apreensão-como atividade e movimento em<em&gt; Drosophila</em

Video tracking systems have been used widely to analyze Drosophila melanogaster movement and detect various abnormalities in locomotive behavior. While ...

a-low-cost-method-for-analyzing-seizure-like-activity-movement

Research

JoVE Journal

Neuroscience

A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

13.5K visualizações.  Franciscan University of Steubenville.  Usando uma webcam e uma combinação de programas de software livre e de baixo custo, os padrões de locomotivas em Drosophila melanogaster podem ser analisadas para detectar diferenças na velocidade, distância e tempo de várias atividades motoras. 

Vídeo: A Low-cost Method for Analyzing Seizure-like Activity and Movement in Drosophila

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) pacemakers or clocks. Malfunctions in the human circadian system are associated with numerous diseases or disorders. Much progress towards our understanding of the mechanisms underlying circadian rhythms has emerged from genetic screens whereby an easily measured behavioral rhythm is used as a read-out of clock function. Studies using Drosophila have made seminal contributions to our understanding of the cellular and biochemical bases underlying circadian rhythms. The standard circadian behavioral read-out measured in Drosophila is locomotor activity. In general, the monitoring system involves specially designed devices that can measure the locomotor movement of Drosophila. These devices are housed in environmentally controlled incubators located in a darkroom and are based on using the interruption of a beam of infrared light to record the locomotor activity of individual flies contained inside small tubes. When measured over many days, Drosophila exhibit daily cycles of activity and inactivity, a behavioral rhythm that is governed by the animal's endogenous circadian system. The overall procedure has been simplified with the advent of commercially available locomotor activity monitoring devices and the development of software programs for data analysis. We use the system from Trikinetics Inc., which is the procedure described here and is currently the most popular system used worldwide. More recently, the same monitoring devices have been used to study sleep behavior in Drosophila. Because the daily wake-sleep cycles of many flies can be measured simultaneously and only 1 to 2 weeks worth of continuous locomotor activity data is usually sufficient, this system is ideal for large-scale screens to identify Drosophila manifesting altered circadian or sleep properties.

Assaying Locomotor Activity to Study Circadian Rhythms and Sleep Parameters in Drosophila

We describe procedures for recording daily locomotor activity rhythms of Drosophila and subsequent data analysis. Locomotor activity rhythms are a reliable behavioral output of animal circadian clocks and are used as the standard readout of clock function when studying circadian mutants or examining how the environment regulates the circadian system.

Assaying atividade locomotora para estudar ritmos circadianos e sono Parâmetros em Drosophila</em

Most life forms exhibit daily rhythms in cellular, physiological and behavioral phenomena that are driven by endogenous circadian (&equiv;24 hr) ...

Neurociência

A Low Cost Setup for Behavioral Audiometry in Rodents

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the experiments. Such parameters may be physiological response characteristics of the auditory pathway, e.g. via brainstem audiometry (BERA). But these methods allow only indirect and uncertain extrapolations about the auditory percept that corresponds to these physiological parameters. To assess the perceptual level of hearing, behavioral methods have to be used. A potential problem with the use of behavioral methods for the description of perception in animal models is the fact that most of these methods involve some kind of learning paradigm before the subjects can be behaviorally tested, e.g. animals may have to learn to press a lever in response to a sound. As these learning paradigms change perception itself 1,2 they consequently will influence any result about perception obtained with these methods and therefore have to be interpreted with caution. Exceptions are paradigms that make use of reflex responses, because here no learning paradigms have to be carried out prior to perceptual testing. One such reflex response is the acoustic startle response (ASR) that can highly reproducibly be elicited with unexpected loud sounds in na&iuml;ve animals. This ASR in turn can be influenced by preceding sounds depending on the perceptibility of this preceding stimulus: Sounds well above hearing threshold will completely inhibit the amplitude of the ASR; sounds close to threshold will only slightly inhibit the ASR. This phenomenon is called pre-pulse inhibition (PPI) 3,4, and the amount of PPI on the ASR gradually depends on the perceptibility of the pre-pulse. PPI of the ASR is therefore well suited to determine behavioral audiograms in na&iuml;ve, non-trained animals, to determine hearing impairments or even to detect possible subjective tinnitus percepts in these animals. In this paper we demonstrate the use of this method in a rodent model (cf. also ref. 5), the Mongolian gerbil (Meriones unguiculatus), which is a well know model species for startle response research within the normal human hearing range (e.g. 6).

A fast and inexpensive method for the behavioral determination of hearing parameters like hearing thresholds, hearing impairments or phantom perceptions (subjective tinnitus) is described. It uses pre-pulse inhibition of the acoustic startle response and can be easily implemented in a personal computer using a programmable AD/DA-converter and a piezo sensor.

A configuração de Baixo Custo para audiometria comportamental em roedores

In auditory animal research it is crucial to have precise information about basic hearing parameters of the animal subjects that are involved in the ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A-máquina interface humana Integrando sensores de baixo custo com uma estimulação do sistema neuromuscular eléctrico para pós-AVC Reabilitação Balance

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

A Simple and Inexpensive Method for Determining Cold Sensitivity and Adaptation in Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life. The cold plantar assay allows the objective and inexpensive assessment of cold sensitivity in mice, and can quantify both analgesia and hypersensitivity. Mice are acclimated on a glass plate, and a compressed dry ice pellet is held against the glass surface underneath the hindpaw. The latency to withdrawal from the cooling glass is used as a measure of cold sensitivity.
Cold sensation is also important for survival in regions with seasonal temperature shifts, and in order to maintain sensitivity animals must be able to adjust their thermal response thresholds to match the ambient temperature. The Cold Plantar Assay (CPA) also allows the study of adaptation to changes in ambient temperature by testing the cold sensitivity of mice at temperatures ranging from 30 &#176;C to 5 &#176;C. Mice are acclimated as described above, but the glass plate is cooled to the desired starting temperature using aluminum boxes (or aluminum foil packets) filled with hot water, wet ice, or dry ice. The temperature of the plate is measured at the center using a filament T-type thermocouple probe. Once the plate has reached the desired starting temperature, the animals are tested as described above.
This assay allows testing of mice at temperatures ranging from innocuous to noxious. The CPA yields unambiguous and consistent behavioral responses in uninjured mice and can be used to quantify both hypersensitivity and analgesia. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

The Cold Plantar Assay (CPA) measures cold responsiveness between 30 &#176;C and 5 &#176;C, and can also measure cold adaptation. This protocol describes how to use the CPA to measure cold hypersensitivity, analgesia, and adaptation in mice.

Um método simples e barato para a determinação da sensibilidade Fria e Adaptação no Mice

Cold hypersensitivity is a serious clinical problem, affecting a broad subset of patients and causing significant decreases in quality of life.  The cold ...

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies using Drosophila have contributed important breakthroughs in understanding neurological processes. Thus, with the goal of understanding the cellular and molecular basis of TBI pathologies in humans, we developed the High Impact Trauma (HIT) device to inflict closed head TBI in flies. Flies subjected to the HIT device display phenotypes consistent with human TBI such as temporary incapacitation and progressive neurodegeneration. The HIT device uses a spring-based mechanism to propel flies against the wall of a vial, causing mechanical damage to the fly brain. The device is inexpensive and easy to construct, its operation is simple and rapid, and it produces reproducible results. Consequently, the HIT device can be combined with existing experimental tools and techniques for flies to address fundamental questions about TBI that can lead to the development of diagnostics and treatments for TBI. In particular, the HIT device can be used to perform large-scale genetic screens to understand the genetic basis of TBI pathologies.

A Method to Inflict Closed Head Traumatic Brain Injury in Drosophila

Here we describe a method to inflict closed head traumatic brain injury (TBI) in Drosophila. This method provides a gateway to investigate the cellular and molecular mechanisms that underlie TBI pathologies using the vast array of experimental tools and techniques available for flies.

Um Método para infligir Cabeça Fechada Lesão cerebral traumático em<em&gt; Drosophila</em

Traumatic brain injury (TBI) affects millions of people each year, causing impairment of physical, cognitive, and behavioral functions and death. Studies ...

Direct-current Stimulation and Multi-electrode Array Recording of Seizure-like Activity in Mice Brain Slice Preparation

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the stimulation parameters (e.g., orientation, field strength, and stimulation duration) need to be examined in mice brain slice preparations. Testing and arranging the orientation of the electrode relative to the position of the mice brain slice are feasible. The present method preserves the thalamocingulate pathway to evaluate the effect of DCS on anterior cingulate cortex seizure-like activities. The results of the multichannel array recordings indicated that cathodal DCS significantly decreased the amplitude of the stimulation-evoked responses and duration of 4-aminopyridine and bicuculline-induced seizure-like activity. This study also found that cathodal DCS applications at 15 min caused long-term depression in the thalamocingulate pathway. The present study investigates the effects of DCS on thalamocingulate synaptic plasticity and acute seizure-like activities. The current procedure can test the optimal stimulation parameters including orientation, field strength, and stimulation duration in an in vitro mouse model. Also, the method can evaluate the effects of DCS on cortical seizure-like activities at both the cellular and network levels.

Studies have shown that cathodal transcranial direct-current stimulation can produce suppressive effects on drug-resistant seizures. In this study, an in vitro experimental setup was devised in which the direct-current stimulation and multielectrode array recording of seizure-like activity were evaluated in mice brain slice preparation. The direct-current stimulation parameters were evaluated.

A estimulação de corrente contínua e Multi-eletrodo de gravação matriz de perda de consciência em Ratos Cérebro Slice Preparação

Cathodal transcranial direct-current stimulation (tDCS) induces suppressive effects on drug-resistant seizures. To perform effective actions, the ...

High-density Electroencephalographic Acquisition in a Rodent Model Using Low-cost and Open-source Resources

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network connectivity, are applicable to an expanding variety of questions in neuroscience. Performing these kinds of analyses in a rodent model requires higher electrode density than traditional screw electrodes can accomplish. While higher-density electroencephalographic montages for rodents exist, they are of limited availability to most researchers, are not robust enough for repeated experiments over an extended period of time, or are limited to use in anesthetized rodents.1-3 A proposed low-cost method for constructing a durable, high-count, transcranial electrode array, consisting of bilaterally implantable headpieces is investigated as a means to perform advanced electroencephalogram analyses in mice or rats.

Procedures for headpiece fabrication and surgical implantation necessary to produce high signal to noise, low-impedance electroencephalographic and electromyographic signals are presented. While the methodology is useful in both rats and mice, this manuscript focuses on the more challenging implementation for the smaller mouse skull. Freely moving mice are only tethered to cables via a common adapter during recording. One version of this electrode system that includes 26 electroencephalographic channels and 4 electromyographic channels is described below.

Instructions for the low-cost construction and surgical implantation of a chronic transcranial high-density electroencephalographic montage into mice are provided. Signal recording, extraction, and processing techniques are also described.

De alta densidade Electroencefalografia Aquisição em um modelo de roedor Usando de baixo custo e de código aberto Recursos

Advanced electroencephalographic analysis techniques requiring high spatial resolution, including electrical source imaging and measures of network ...

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at the synaptic level. The Active Zone (AZ) is the region at the presynaptic membrane that mediates neurotransmitter release and is composed of a dense collection of scaffold proteins. AZs of Drosophila melanogaster (Drosophila) photoreceptors undergo molecular remodeling after prolonged exposure to natural ambient light. Thus the level of neuronal activity can rearrange the molecular composition of the AZ and contribute to the regulation of the functional output.
Starting from the light exposure set-up preparation to the immunohistochemistry, this protocol details how to quantify the number, the spatial distribution, and the delocalization level of synaptic molecules at AZs in Drosophila photoreceptors. Using image analysis software, clusters of the GFP-fused AZ component Bruchpilot were identified for each R8 photoreceptor (R8) axon terminal. Detected Bruchpilot spots were automatically assigned to individual R8 axons. To calculate the distribution of spot frequency along the axon, we implemented a customized software plugin. Each axon&#39;s start-point and end-point were manually defined and the position of each Bruchpilot spot was projected onto the connecting line between start and end-point. Besides the number of Bruchpilot clusters, we also quantified the delocalization level of Bruchpilot-GFP within the clusters. These measurements reflect in detail the spatially resolved synaptic dynamics in a single neuron under different environmental conditions to stimuli.

Analyzing Synaptic Modulation of Drosophila melanogaster Photoreceptors after Exposure to Prolonged Light

Here we show how to quantify the number and spatial distribution of synaptic active zones in Drosophila melanogaster&#160;photoreceptors, highlighted with a genetically encoded molecular marker, and their modulation after prolonged exposure to light.

Analisando Synaptic Modulação da<em&gt; Drosophila melanogaster</em&gt; Fotorreceptores após exposição à luz prolongada

The nervous system has the remarkable ability to adapt and respond to various stimuli. This neural adjustment is largely achieved through plasticity at ...

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the pathophysiological basis of these diseases, it is necessary to precisely characterize in which way the processing of information is disturbed between the different neuronal parts of the circuit. Using extracellular in vivo electrophysiological recordings, it is possible to accurately delineate neuronal activity within a neuronal network. The application of this method has several advantages over alternative techniques, e.g., functional magnetic resonance imaging and calcium imaging, as it allows a unique temporal and spatial resolution and does not rely on genetically engineered organisms. However, the use of extracellular in vivo recordings is limited since it is an invasive technique that cannot be universally applied. In this article, a simple and easy to use method is presented with which it is possible to simultaneously record extracellular potentials such as local field potentials and multiunit activity at multiple sites of a network. It is detailed how a precise targeting of subcortical nuclei can be achieved using a combination of stereotactic surgery and online analysis of multi-unit recordings. Thus, it is demonstrated, how a complete network such as the hyperdirect cortico-basal ganglia loop can be studied in anesthetized animals in vivo.

Acute In Vivo Electrophysiological Recordings of Local Field Potentials and Multi-unit Activity from the Hyperdirect Pathway in Anesthetized Rats

In this study, the methodology is presented on how to perform multi-site in vivo electrophysiological recordings from the hyperdirect pathway under urethane anesthesia.

Agudo<em&gt; In Vivo</em&gt; Gravações eletrofisiológicas de Potenciais de campo locais e atividade de múltiplas unidades a partir do caminho hiperdiretivo em ratos anestesiados

Converging evidence shows that many neuropsychiatric diseases should be understood as disorders of large-scale neuronal networks. To better understand the ...

In vivo Calcium Imaging in Mouse Inferior Olive

Inferior olive (IO), a nucleus in the ventral medulla, is the only source of climbing fibers that form one of the two input pathways entering the cerebellum. IO has long been proposed to be crucial for motor control and its activity is currently considered to be at the center of many hypotheses of both motor and cognitive functions of the cerebellum. While its physiology and function have been relatively well studied on single-cell level in vitro, presently there are no reports on the organization of the IO network activity in living animals. This is largely due to the extremely challenging anatomical location of the IO, making it difficult to subject to conventional fluorescent imaging methods, where an optic path must be created through the entire brain located dorsally to the region of interest.
Here we describe an alternative method for obtaining state-of-the-art -level calcium imaging data from the IO network. The method takes advantage of the extreme ventral location of the IO and involves a surgical procedure for inserting a gradient-refractive index (GRIN) lens through the neck viscera to come into contact with the ventral surface of the calcium sensor GCaMP6s-expressing IO in anesthetized mice. A representative calcium imaging recording is shown to demonstrate the feasibility to record IO neuron activity after the surgery. While this is a non-survival surgery and the recordings must be conducted under anesthesia, it avoids damage to life-critical brainstem nuclei and allows conducting large variety of experiments investigating spatiotemporal activity patterns and input integration in the IO. This procedure with modifications could be used for recordings in other, adjacent regions of the ventral brainstem.

In vivo Calcium Imaging in Mouse Inferior Olive

We present a protocol to expose the brainstem of adult mouse from the ventral side. By using a gradient-refractive index lens with a miniature microscope, calcium imaging can be used to examine the activity of inferior olive neural somata in vivo.