Este trabalho foi financiado pelo Wellcome Trust.

Antes de qualquer estudos podem ser realizados com a aprovação dos bebês éticos e informado por escrito o consentimento dos pais precisa ser procurado. Neste estudo aprovação ética foi obtida a partir do University College Hospital Comitê de Ética e informado por escrito o consentimento dos pais foi obtida antes de cada procedimento. Este estudo conformado com as normas estabelecidas pela Declaração de Helsinki e as diretrizes Boa Prática Clínica. 1. Coleta de dados - Configuração <ol><li> Coloque um mínimo de 16 individuais descartáveis ​​EEG eletrodos Ag / AgCl copo sobre a cabeça do bebê depois de preparar a pele de acordo com o sistema de colocação do eletrodo 10-20 (Figura 1A). Cobertura mais abrangente pode ser conseguido usando uma touca de EEG com eletrodos embutidos. Uso de uma tampa de EEG torna o processo mais rápido e menos disruptivo, particularmente como eletrodos são preparados com gel indutiva antes de colocar o boné na cabeça. A aplicação de eletrodos descartáveis ​​EEG requermais tempo e habilidade, mas geralmente resulta em uma melhor gravação. Considerar a redução do número de eletrodos se o acesso para a criança é limitada, mas sempre usar eletrodos da linha média (Cz, CPZ e FCz). Use FCz como eletrodo de referência para a gravação. </li><li> Use pasta condutora EEG para otimizar o eletrodo / pele de acoplamento elétrico. </li><li> Coloque uma rede elástica ao longo dos eletrodos para mantê-los no lugar. </li><li> Amarre a cabos-eletrodos em conjunto para minimizar a interferência elétrica. </li><li> Coloque um eletrodo terra colocada no peito ou na cabeça. </li><li> Colocação de eletrodos de EMG no bíceps femoral de ambas as pernas depois de preparar a pele (Figura 1B). </li><li> Para gravar a atividade ECG lugar levam uma eletrodos de ECG sobre o corpo, após preparar a pele (um eletrodo no lado esquerdo do peito, um na direita e use o eletrodo terra mesmo que para a EEG). </li><li> Coloque um transdutor de movimento sobre o abdome para medir a respiração. </li><li> Coloque uma sonda de oxímetro de pulso com o pépara medir a saturação de oxigênio e freqüência cardíaca. Certifique-se que a sonda está fixada no local e que um sinal contínuo é gravada sem interrupções. A sonda oxímetro precisa ser colocado com o pé que é contralateral ao pé que pretende estimular. </li><li> Configurar uma filmadora tripé montado para moldar a cara do bebê de modo que mudanças na expressão facial pode ser gravado. </li><li> Coloque um diodo emissor de luz (LED) no quadro da câmera. O LED está ligado ao circuito de tempo para que ela irá piscar quando a estimulação é apresentado para sincronizar o EEG, EMG e gravação de vídeo. </li></ol> 2. Coleta de dados - gravação <ol><li> Iniciar a gravação de vídeo. </li><li> Iniciar a gravação de oximetria de pulso. </li><li> Iniciar EEG / EMG de gravação. </li><li> Espere até que a criança seja resolvida. </li><li> Segure o pé como se realizar uma punção do calcanhar e marcar manualmente evento as gravações de EEG e EMG. Esta época vai ser usado para identificar uma seção de fundo do controle EEGe EMG. </li><li> Aplicar estimulação táctil batendo levemente uma rolha de borracha que está preso ao braço de um martelo contra a superfície do tendão do calcanhar. Estimular o pé que não está anexado ao oxímetro de pulso. Quando a criança é estimulada a EEG / EMG e gravação de vídeo deve ser um evento marcado para identificar o tempo na gravação quando o estímulo ocorreu. A estimulação táctil pode ser um evento marcado pelo acoplamento de um chefe de impedância para o martelo do tendão que liga o estimulador eletronicamente com o equipamento de gravação. A gravação de vídeo é um evento marcado pelo flash LED. Toques repetidos podem ser aplicados eo estímulo pode ser aplicado a diferentes regiões do corpo, ou seja, o ombro </li><li> Aplicar um estímulo controle não-nocivos ao girar a lanceta de 90 graus e colocando-a contra o pé de modo que quando a lâmina de mola é liberada, não entra em contato com a pele. Este evento pode ser tempo-locked usando um acelerômetro conectado à superfície superior do tele lança. O acelerômetro detecta a vibração que ocorre quando a lâmina é liberada. </li><li> Realizar a punção do calcanhar clinicamente essenciais, em conformidade com a prática clínica na unidade neonatal. Espere até que a atividade EEG é liquidado antes de fazer a punção. Tempo de travamento do lance do calcanhar pode ser realizada da mesma forma como para a estimulação de controle. Após a punção do calcanhar, não espremer o pé por pelo menos 30 segundos para garantir que as respostas gravadas são causados ​​exclusivamente por lance. </li><li> Garantir que a quantidade necessária de sangue é coletado e preparar as amostras para análises clínicas. </li><li> Salvar os dados e parar todas as gravações. </li><li> Registro de informações demográficas da criança e os detalhes experimentais e introduzi-los em um banco de dados para armazenamento seguro e referência futura. </li><li> Repita este procedimento na amostra necessário de crianças. Neste exemplo, o número de crianças = 23. </li></ol> 3. Análise de dados EEG <ol><li> Criar épocas EEG de 1,7 segundos que correspondem a cada estímulo controle de toque, e lança fundo e EEG. Épocas deve começar a 0,6 segundo antes de cada evento. O número de épocas correspondentes a cada modalidade deve ser o mesmo. </li><li> Linha de base correta das épocas subtraindo-se o sinal de linha de base média e alta filtro passa-los a 0,1 Hz. </li><li> Considere as épocas gravado em CPZ ou Cz para posterior análise e excluir as épocas, que foram contaminados por artefato movimento. Artefacto movimento é definido como uma mudança na amplitude maior do que 50μV em menos de 50ms. </li><li> Alinhar os traços para corrigir a latência jitter entre 50 e 300 ms pós-estímulo e conduta Análise de Componentes Principais (PCA) neste intervalo de tempo para identificar o potencial tátil (atividade EEG ou seja, relacionados com a estimulação tátil). Considere as épocas para ser as variáveis ​​eo tempo de pontos das observações. PCA decompõe a EEG em épocas básicas formas de onda, denominadas componentes principais (PCs) e representam variação sistemática na amplitude do sinal através de pontos de tempo. </li><li> Executar one-way análise de variância (ANOVA) sobre os pesos de cada um dos 2 primeiros PCs para determinar que PC representa o potencial tátil. Este será o PC, cujo peso estava significativamente maior estimulação tátil seguinte em relação ao fundo EEG. </li><li> Alinhar os traços para corrigir jitter de latência entre 300 e 700 ms pós-estímulo e conduta PCA neste intervalo de tempo. </li><li> Executar one-way ANOVA sobre os pesos de cada um dos 2 primeiros PCs para determinar que PC representa o potencial nociceptivo específico. Este será o PC cujos pesos foram significativamente maiores após estimulação nociva em relação ao estímulo tátil e de base no EEG. </li></ol> 4. EMG análise de dados <ol><li> Calcular a raiz quadrada média (RMS) do sinal EMG na primeira estimulação pós 1000 ms para o controle e estímulos lance. </li><li> Realizar um teste t-ono RMS valores para determinar o nociceptivos específicos retirada espinhal reflexo. </li></ol> 5. Resultados representante <img alt="Figura 1" src="/files/ftp_upload/3118/3118fig1.jpg" /> <strong&gt; Figura 1 (A) Diagrama (i) e (ii) foto da colocação de eletrodos para gravações de EEG (10/20 modificada internacionais sistema de colocação de eletrodos);. (B) Diagrama das colocações de eletrodos para gravações EMG no bíceps femoral . <img alt="Figura 2" src="/files/ftp_upload/3118/3118fig2.jpg" /> . Figura 2 (A) Exemplos do potencial sensorial em Cz evocado pelo toque em 3 crianças; (B) exemplos do potencial nociceptivos-específicos em Cz evocada pelo lance nocivos em 3 crianças. <img alt="Figura 3" src="/files/ftp_upload/3118/3118fig3.jpg" /> Figura 3. Dependência dos pesos PC na modalidade de estímulo em Cz (média ± SEM). O PC obtida entre 50-300 ms após o início do estímulo tátil representa um potencial eo PC obtidos entre 300-700 ms após o início do estímulo representa um potencial nociceptivos-específicos. Os PCs (linhas em negrito) são sobrepostos no médias grandobtidos em todos os tipos de estímulo (EEG de fundo, lança, toque nocivos), após os traços individuais foram alinhados no intervalo de tempo indicado. <img alt="Figura 4" src="/files/ftp_upload/3118/3118fig4.jpg" /> Figura 4. (A) Exemplo de EMG atividade em uma criança após (i) a punção do calcanhar nociva e (ii) toque de calcanhar. (B) Média (± SE) EMG raiz quadrada média da atividade EMG em recém-nascidos após o calcanhar nociva e estímulos não-nocivos toque.

<ol>
	<li>Fitzgerald, M., Walker, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infant+pain+management:+a+developmental+neurobiological+approach.">Infant pain management: a developmental neurobiological approach.</a> Nat. Clin. Pract. Neurol. 5 (1), 35-35 (2009).</li><li>Slater, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Oral+sucrose+as+an+analgesic+drug+for+procedural+pain+in+newborn+infants:+a+randomised+controlled+trial.">Oral sucrose as an analgesic drug for procedural pain in newborn infants: a randomised controlled trial.</a> Lancet. 376 (9748), 1225-1225 (2010).</li><li>Slater, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evoked+potentials+generated+by+noxious+stimulation+in+the+human+infant+brain.">Evoked potentials generated by noxious stimulation in the human infant brain.</a> Eur. J. Pain. 14 (3), 321-321 (2010).</li><li>Slater, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Premature+infants+display+increased+noxious-evoked+neuronal+activity+in+the+brain+compared+to+healthy+age-matched+term-born+infants.">Premature infants display increased noxious-evoked neuronal activity in the brain compared to healthy age-matched term-born infants.</a> Neuroimage. 52 (2), 583-583 (2010).</li></ol>

Os autores declaram que não têm nada a revelar.

Este vídeo mostra como respostas eletrofisiológicas, evocado por estimulação tátil e nocivas, pode ser caracterizada no bebê humano usando gravações de EEG e EMG. Estudos deste tipo ajudará a entender o desenvolvimento e plasticidade do processamento da dor humana, e levar a melhor avaliação clínica e tratamento da dor infantil 1, 2. O sucesso dessas experiências exige a estreita colaboração de uma equipe multidisciplinar. Perícia é necessária no campo da neonatologia, neurofisiologia clínica, bioengenharia e neurociência do desenvolvimento. É essencial que o cuidado da criança tem prioridade mais alta quando essas experiências são realizadas. A enfermeira de pesquisa ou médico que realiza a punção do calcanhar tem a responsabilidade de bem-estar da criança e as necessidades para garantir que o experimento é feito em conformidade com a prática clínica. Aprovação do comitê de ética do hospital e written o consentimento dos pais são necessários para realizar esses estudos. O lance do calcanhar é um evento clinicamente necessário e não pode ser repetido para o propósito do estudo. Portanto, é essencial que a gravação é recolhida de forma confiável e robusto que não interfere com a prática clínica 3, 4. Em particular, é crucial que o método utilizado para o tempo de bloqueio da estimulação é confiável e não impedir a coleta de sangue clínico. Todas as medidas de dor infantil são necessariamente indiretos, como tal, é importante certificar-se que a atividade registrada é especificamente evocada pelo evento nocivo e aspectos não outros cuidados do bebê 2. Isto pode ser conseguido através de projeto técnico cuidadoso e pós-processamento dos dados.

electrophysiological measurements and analysis of nociception in human infants

A dor é uma experiência sensorial e emocional desagradável. Uma vez que as crianças não podem verbalmente relatam suas experiências, os métodos atuais de avaliação da dor são baseados em reações comportamentais e fisiológicas do corpo, tais como movimentos chorando, corpo ou mudanças na expressão facial. Enquanto estas medidas demonstram que as crianças montar uma resposta após a estimulação nociva, eles são limitados: eles são baseados em ativação de somáticas subcortical e vias motoras autônomo que não pode ser confiavelmente ligados à central de processamento sensorial no cérebro. Conhecimento de como o sistema nervoso central responde a eventos nocivos poderiam fornecer uma visão de como a informação nociceptiva e dor é processada em recém-nascidos. O calcanhar de procedimento de punção utilizado para extrair o sangue de crianças hospitalizadas oferece uma oportunidade única para estudar a dor na infância. Neste vídeo podemos descrever como eletroencefalografia (EEG) e eletromiografia (EMG) em tempo bloqueado para este procedimento can ser usado para investigar a atividade nociceptiva no cérebro e medula espinhal. Esta abordagem integrativa para a medição da dor do bebê tem o potencial de abrir caminho para uma ferramenta de medição eficaz e sensível clínicos.

Watch this Scientific Journal Video about Electrophysiological Measurements and Analysis of Nociception in Human Infants at JoVE.com

Electrophysiological Measurements and Analysis of Nociception in Human Infants

A avaliação e tratamento da dor em crianças é difícil porque as crianças não podem verbalmente relatam sua experiência. Neste vídeo podemos descrever quantitativa métodos eletrofisiológicos e técnicas de análise que pode ser usado para medir a resposta a eventos nocivos do sistema nervoso infantil.

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 ...

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Research

JoVE Journal

Neuroscience

17.1K visualizações.  University College London. A avaliação e tratamento da dor em crianças é difícil porque as crianças não podem verbalmente relatam sua experiência. Neste vídeo podemos descrever quantitativa métodos eletrofisiológicos e técnicas de análise que pode ser usado para medir a resposta a eventos nocivos do sistema nervoso infantil.

Vídeo: Electrophysiological Measurements and Analysis of Nociception in Human Infants

Electrophysiological Measurements from a Moth Olfactory System

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively. These approaches have been exploited for getting a better understanding of insect chemical communication. The identified stimuli can then be used as either attractants or repellents in management strategies for insect pests.

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single sensillum recordings (SSR), which are summed responses from all odorant receptor neurons (ORNs) located on the antenna and from those housed in individual sensilla, respectively.

Medidas eletrofisiológicas partir de um sistema olfativo Traça

Insect olfactory systems provide unique opportunities for recording odorant-induced responses in the forms of electroantennograms (EAG) and single ...

Neurociência

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Lapso de tempo de imagens ao vivo de relação clonal células progenitoras neurais no desenvolvimento Zebrafish Encéfalo anterior

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography (dEEG)

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any other current brain imaging technology1,2, is being used increasingly in the study of social cognitive functioning in infants and adults. While dEEG is enabling researchers to examine brain activity patterns with unprecedented levels of sensitivity, conventional EEG recording systems continue to face certain limitations, including 1) poor spatial resolution and source localization3,4,2) the physical discomfort for test subjects of enduring the individual application of numerous electrodes to the surface of the scalp, and 3) the complexity for researchers of learning to use multiple software packages to collect and process data. Here we present an overview of an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults. Although several analytical software techniques can be used to establish indirect indices of source localization to improve the spatial resolution of dEEG, the HydroCel Geodesic Sensor Net (HCGSN) by Electrical Geodesics, Inc. (EGI), a dense sensory array that maintains equal distances among adjacent recording electrodes on all surfaces of the scalp, further enhances spatial resolution4,5,6 compared to standard dEEG systems. The sponge-based HCGSN can be applied rapidly and without scalp abrasion, making it ideal for use with adults7,8, children9,10,11, and infants12, in both research and clinical4,5,6,13,14,15 settings. This feature allows for considerable cost and time savings by decreasing the average net application time compared to other dEEG systems. Moreover, the HCGSN includes unified, seamless software applications for all phases of data, greatly simplifying the collection, processing, and analysis of dEEG data.
The HCGSN features a low-profile electrode pedestal, which, when filled with electrolyte solution, creates a sealed microenvironment and an electrode-scalp interface. In all Geodesic dEEG systems, EEG sensors detect changes in voltage originating from the participant's scalp, along with a small amount of electrical noise originating from the room environment. Electrical signals from all sensors of the Geodesic sensor net are received simultaneously by the amplifier, where they are automatically processed, packaged, and sent to the data-acquisition computer (DAC). Once received by the DAC, scalp electrical activity can be isolated from artifacts for analysis using the filtering and artifact detection tools included in the EGI software. Typically, the HCGSN can be used continuously for only up to two hours because the electrolyte solution dries out over time, gradually decreasing the quality of the scalp-electrode interface.
In the Parent-Infant Research Lab at the University of Toronto, we are using dEEG to study social cognitive processes including memory, emotion, goals, intentionality, anticipation, and executive functioning in both adult and infant participants.

Investigating Social Cognition in Infants and Adults Using Dense Array Electroencephalography dEEG

Dense array electroencephalography is being used increasingly to study social cognitive functions in infants and adults. Here we present an established methodology that represents a significant improvement on conventional methodologies for studying EEG in infants and adults.

Investigando Cognição Social em Crianças e Adultos usando eletroencefalografia Matriz Densa ( D EEG)

Dense array electroencephalography (dEEG), which provides a non-invasive window for measuring brain activity and a temporal resolution unsurpassed by any ...

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.
The Drosophila larva is an established model system to study thermal nociception, a sensory response to potentially harmful temperatures that is evolutionarily conserved across species1,2. The advantages of Drosophila for such studies are the relative simplicity of its nervous system and the sophistication of the genetic techniques that can be used to dissect the molecular basis of the underlying biology4-6 In Drosophila, as in all metazoans, the response to noxious thermal stimuli generally involves a "nocifensive" aversive withdrawal to the presented stimulus7. Such stimuli are detected through free nerve endings or nociceptors and the amplitude of the organismal response depends on the number of nociceptors receiving the noxious stimulus8. In Drosophila, it is the class IV dendritic arborization sensory neurons that detect noxious thermal and mechanical stimuli9 in addition to their recently discovered role as photoreceptors10. These neurons, which have been very well studied at the developmental level, arborize over the barrier epidermal sheet and make contacts with nearly all epidermal cells11,12. The single axon of each class IV neuron projects into the ventral nerve cord of the central nervous system11 where they may connect to second-order neurons that project to the brain.
Under baseline conditions, nociceptive sensory neurons will not fire until a relatively high threshold is reached. The assays described here allow the investigator to quantify baseline behavioral responses or, presumably, the sensitization that ensues following tissue damage. Each assay provokes distinct but related locomotory behavioral responses to noxious thermal stimuli and permits the researcher to visualize and quantify various aspects of thermal nociception in Drosophila larvae. The assays can be applied to larvae of desired genotypes or to larvae raised under different environmental conditions that might impact nociception. Since thermal nociception is conserved across species, the findings gleaned from genetic dissection in Drosophila will likely inform our understanding of thermal nociception in other species, including vertebrates.

Local and Global Methods of Assessing Thermal Nociception in Drosophila Larvae

In this article, we demonstrate assays to study thermal nociception in Drosophila larvae. One assay involves spatially-restricted (local) stimulation of thermal nociceptors1,2 while the second involves a wholesale (global) activation of most or all such neurons3. Together, these techniques allow visualization and quantification of the behavioral functions of Drosophila nociceptive sensory neurons.

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 ...

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Dois registros eletrofisiológicos de sinapticamente evocadas Respostas astroglial e neuronal em fatias hipocampais agudas

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

Stereotaxic Injection of a Viral Vector for Conditional Gene Manipulation in the Mouse Spinal Cord

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central nervous system. We demonstrate a stereotaxic injection technique that allows targeted gene expression or silencing in the dorsal horn of the mouse spinal cord. The surgical procedure is brief. It requires laminectomy of a single vertebra, providing for quick recovery of the animal and unimpaired motility of the spine. Controlled injection of a small vector suspension volume at low speed and use of a microsyringe with beveled glass cannula minimize the tissue lesion. The local immune response to the vector depends on the intrinsic properties of the virus employed; in our experience, it is minor and short-lived when a recombinant adeno-associated virus is used. A reporter gene such as enhanced green fluorescent protein facilitates monitoring spatial distribution of the vector, and the efficacy and cellular specificity of the transfection.

Viral vectors allow for targeted gene manipulation. We demonstrate a method for conditional gene expression or ablation in the mouse spinal cord, using stereotaxic injection of a viral vector into the dorsal horn, a prominent site of synaptic contact between primary somatosensory afferents and neurons of the central nervous system.

Injeção estereotáxica de um vetor viral para manipulação genética condicional no Cabo de Rato Spinal

Intraparenchymal injection of a viral vector enables conditional gene manipulation in distinct populations of neurons or particular regions of the central ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Em Medidas Vivo eletrofisiológicos nos nervos ciático Rato

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

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 ...

3D Kinematic Analysis for the Functional Evaluation in the Rat Model of Sciatic Nerve Crush Injury

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of sciatic nerve injury rodent models. In this protocol, we describe a novel kinematic analysis method that uses a three-dimensional (3D) motion capture apparatus for functional evaluations using a rat sciatic nerve crush injury model. First, the rat is familiarized with treadmill walking. Markers are then attached to the designated bone landmarks and the rat is made to walk on the treadmill at the desired speed. Meanwhile, the posterior limb movements of the rat are recorded using four cameras. Depending on the software used, marker tracings are created using both automatic and manual modes and the desired data are produced after subtle adjustments. This method of kinematic analysis, which uses a 3D motion capture apparatus, offers numerous advantages, including superior precision and accuracy. Many more parameters can be investigated during the comprehensive functional evaluations. This method has several shortcomings that require consideration: The system is expensive, can be complicated to operate, and may produce data deviations due to skin shifting. Nevertheless, kinematic analysis using a 3D motion capture apparatus is useful for performing functional anterior and posterior limb evaluations. In the future, this method may become increasingly useful for generating accurate assessments of various traumas and diseases.

We introduce a kinematic analysis method that uses a three-dimensional motion capture apparatus containing four cameras and data processing software for performing functional evaluations during fundamental research involving rodent models.

Análise Cinética 3D para a Avaliação Funcional no modelo de rat da lesão ciática de esmagamento nervoso

Compared to the Sciatic Functional Index (SFI), kinematic analysis is a more reliable and sensitive method for performing functional evaluations of ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Preparação de fatias hipocampais humanas agudas para gravações eletrofisiológicas

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...