Este trabalho foi apoiado pelo SFB 604, DFG MO 1421/2-1 e Krebshilfe 107089 (para HM). AS é destinatário de um Prêmio Jovem Investigador de Tumor das Crianças Foundation (Nova York, EUA).

O presente estudo foi realizado de acordo com a Lei de Proteção dos Animais da República Federal da Alemanha (Bundesrepublik Deutschland Tierschutzgesetz der) e foi aprovada pela Secretaria de Estado da Turíngia para a Segurança Alimentar e Defesa do Consumidor (Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz). 1. Definir as Medidas <ol><li> Anestesiar os ratos por isoflurano / O 2 inalação - para a iniciação de anestesia 3%, para a manutenção de 2% de isoflurano em 100% de oxigénio (Figura 1). Confirme anestesia suficientes, testando os reflexos simples, como reflexos de movimento e teste de sensibilidade para a dor de baixo grau. O uso de pomada para os olhos para evitar o ressecamento durante a anestesia é recomendada, mas não indispensável, pois o procedimento normalmente leva apenas 30 min / animal no total. Em caso de experimentos de sobrevivência, administrar mais oportuna analgésicos de acção para controlar a dor pós-operatória. </li><li> Raspar a pele que cobre as patas traseiras com um barbeador elétrico e realizar depilação com um creme de depilação disponíveis no mercado enquanto que os animais já estão sob analgesia. A fim de manter o status de livre de patógenos durante todo o procedimento, use luvas de assepsia e sempre utilizar instrumentos cuidadosamente limpos com etanol 70%. </li><li> Controle de estabilidade de temperatura do corpo por uma placa de aquecimento controlado de feedback e sonda térmica retal (Figura 2). Se necessário, utilize um campo estéril para cobrir a placa de aquecimento entre os animais, a fim de manter um ambiente estéril experimental. Além disso, recomenda-se usar uma placa de aquecimento, o qual é controlado electronicamente por meio de um sensor integrado para limitar a temperatura de aquecimento para ≤ 40 ° C, a fim de evitar danos no tecido. <img alt="Figura 1" fo:content-width="4in" fo:src="/files/ftp_upload/51181/51181fig1highres.jpg" src="/files/ftp_upload/51181/51181fig1.jpg" /> Figura 1. Expconfiguração erimental mostrando um rato anestesiado com membros posteriores raspadas. </li><li> Tome eletrocardiograma (ECG) para monitorar a freqüência cardíaca como um parâmetro vital. Instalar três eléctrodos para ECG, como segue: um eléctrodo sob a pele de cada um dos membros anteriores e um eléctrodo sob a pele na área do pescoço. </li><li> Eletrodos coloque o anel usando contato gel para ótima resistência condutividade / transferência. O eléctrodo de detecção (rotulado de preto) é colocado na posição em que o músculo do gastrocnémio tem o seu diâmetro máximo. O eletrodo de referência (indicado em vermelho) é colocado logo abaixo do eletrodo de detecção. Nota: Por favor, veja &#39;table material&#39; para obter detalhes de equipamentos. </li><li> Marque as posições corretas de medição com pré-determinados, mas consistentes distâncias entre o proximal e distal a estimulação do nervo ciático eo eletrodo de chumbo. Proposta Experimental: A uma distância de 4 mm do eletrodo sensor, o estímulo distal ocorrerá.A uma distância de 16 mm e o eléctrodo de detecção, a estimulação proximal será realizado (Figura 2). </li></ol><img alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig2highres.jpg" src="/files/ftp_upload/51181/51181fig2.jpg" /> Figura 2. Imagem representativas mostrando a situação experimental apenas antes do inicio das medições. A seta branca indica a posição do meio de detecção (preto) e referência (vermelho) do eléctrodo no músculo gastrocnémio da pata posterior esquerda. A estimulação por eléctrodos de agulha vai ser realizada em posições definidas em relação ao eléctrodo de detecção preto. O ponto de estímulo distal (marca preta com &quot;DS&quot; no membro posterior esquerdo) tem uma distância de 4 mm do eletrodo sensor; o lugar da estimulação proximal (marca preta com &quot;ps&quot;) é 16 milímetros de distância. A linha vermelha na pata traseira direita mostra a aproximadamente<html> Imate curso anatômica do nervo ciático. Além disso, as posições aproximadas de músculos dos membros posteriores relevantes são mostrados como pontos de referência. O asterisco indica que a sonda térmica retal. 2. Medição <ol><li> Princípio: Realizar uma série de estímulos nervosos com repetitivamente gerados pulsos de 0,1 ms de duração por monopolar descartáveis ​​eletrodos de agulha 28 g de onda quadrada simples (taxa de repetição de 200 ms; ver Figura 3A). Para a análise de dados fora de linha, é recomendado para adquirir simultaneamente sinais de estimulação, juntamente com a curva de resposta da função neuromuscular (Figura 3B), devido à estimulação do nervo. Média, uma série de respostas máximas (&quot;representante resposta função neuromuscular&quot;) para posterior análise de dados. A fim de produzir dados confiáveis, ficha e posteriormente média pelo menos três curvas de resposta independentes, ótimo por local de estimulação e animal. p_upload/51181/51181fig3highres.jpg &quot;src =&quot; / files/ftp_upload/51181/51181fig3.jpg &quot;/&gt; Figura 3. Procedimento para a aquisição de dados e análise (representação esquemática). Impulsos repetitivos gerados são aplicados para o nervo isquiático através de eléctrodos em agulha (linha superior na Figura 3A). Simultaneamente, várias curvas de resposta neuromuscular correspondentes são registadas (linha inferior na Figura 3A). Quando a média e ampliada, as curvas de resposta neuromuscular devido a pulsos de estimulação (linha superior na Figura 3B) mostram as seguintes propriedades características de linha inferior (na Figura 3B): Latência da resposta do sinal, bem como a duração e a amplitude do sinal estão indicados e pode ser obtido para cálculos e estatísticas posteriores. Condução de sinal irregular e / ou gravações com qualidade inferior normalmente resulta em várias deformações de sinal com mais do que apenas um positivo e outro negativo deflexão ou deformação do sinal(Forma bimodal) com amplitude reduzida (Figura 3C). </li><li> Realizar estimulação proximal com um eléctrodo de agulha na posição determinada (&quot;MA&quot; na Figura 2). <ol><li> A fim de alcançar melhores condições de gravação com amplitudes máximas, visualizar as curvas de resposta reais em simultâneo com o processo de estimulação. Ao fazê-lo, o experimentador é capaz de avaliar imediatamente a forma das curvas de resposta, assim como o tamanho da amplitude. </li><li> Se necessário, ligeiramente e cuidadosamente manipular a posição e / ou o ângulo da agulha em relação à estimulação do nervo ciático. Esta optimização suave condições de estimulação permite atingir amplitudes constantes com o maior valor possível, e uma curva de resposta de forma típica bifásica (Figura 4). </li></ol></li><li> Realizar estimulação distal com um eléctrodo de agulha na posição determinada (&quot;DS&quot; na Figura 2). </li><li> Após completion da medição, transferir o mouse testado para uma gaiola separada até que ele recuperou a consciência suficiente para manter decúbito esternal. Não deixe um animal autônoma e na companhia de outros animais até que ele está totalmente recuperado da anestesia. Administrar oportuna mais analgésicos de acção para controlar a dor pós-operatória. A administração sistêmica de medicamentos anti-inflamatórios não esteróides (AINE) e opióides são recomendados para 1-3 dias. </li><li> Alternativamente, sacrificar o mouse ainda anestesiados de forma rápida e mais humanos, sem dor para o animal, por exemplo, por deslocamento cervical. </li></ol><img alt="Figura 4" fo:content-width="6in" fo:src="/files/ftp_upload/51181/51181fig4highres.jpg" src="/files/ftp_upload/51181/51181fig4.jpg" /> Figura 4. Ilustração para determinar as gravações CMAP com amplitudes máximas. Uma série de registo completo é apresentado. (Um </strong&gt;) Ponto de inserção com resposta mínima CMAP. (B) os resultados do movimento agulha estimulação leve em gravações CMAP com amplitudes máximas. (C) mudanças adicionais na colocação da agulha produzir gravações CMAP com diferentes amplitudes, incluindo quase máxima amplitudes. (D) Estimulação substituição da agulha com gravações CMAP série de amplitudes quase máximos. Nota: decremento típica em amplitudes CMAP pode ocorrer durante a estimulação repetitiva no local estimulação óptima 12, 13. Os asteriscos indicam gravações CMAP com amplitudes máximas representadas por média. 3. Análise <ol><li> Extraia parâmetros de condução nervosa com base no conjunto neuromuscular representante resposta função de dados usando um pacote de software adequado (por exemplo, AtisaPro). Nota: lidar com a determinação de dados relacionados com o tempo, com um cuidado especial, pois a determinação do ponto de inflexão do composto pot ação motoracial (CMAP) início e término pode ser difícil. Um procedimento com reprodutibilidade verificado é dado na Figura 3B, onde são usados ​​tangentes sobre desvios de sinal após o início e antes do término. </li><li> Calcular &#39;latência de sinal &quot;e valores&quot; CMAP&#39;. <ol><li> Latência &#39;representa o intervalo de tempo entre estímulo e CMAP início, ao passo que o intervalo de tempo entre o início da CAMP deflexão negativa inicial para rubricar retorno à linha de base é chamado de&#39; duração CMAP &#39;. Utilizar a diferença entre latências distais e proximais para calcular a velocidade de condução junto com a distância entre sítios de estimulação proximal e distal. A velocidade de condução = latência / distância entre sítios de estimulação </li><li> CMAP &#39;(composto potencial de ação motor) amplitude representa a magnitude entre o ponto de viragem máximo positivo e negativo do sinal de CMAP (dada em mV). CMAP = value &#39;positivo ponto de viragem &quot;- valor&quot; negativo po reviravoltaint &#39; </li></ol></li></ol>

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Os autores não têm nada a revelar.

O protocolo descrito fornece um método fácil e confiável para determinar propriedades de condução do nervo ciático em ratos anestesiados, sem a necessidade de expor o nervo de interesse. No entanto, este procedimento experimental provoca lesão tecidual por punção da agulha. Por isso, é uma opção razoável para sacrificar os animais depois de terminar as gravações. No entanto, em comparação com outros procedimentos invasivos que exigem a exposição do nervo antes da gravação, o dano tecidual é compara...

Medidas eletrofisiológicas são uma ferramenta indispensável para a investigação da integridade funcional dos nervos periféricos, tanto em ambientes clínicos e laboratoriais. Em seres humanos, um grande número de desordens neuromusculares e neuropatias diagnóstico dependem de medições electrofisiológicas. Ao medir as propriedades como velocidade de condução do nervo ou potenciais amplitudes do sinal, é possível caracterizar a origem áspero de doenças dos nervos periféricos. A velocidade de condução nervosa é altamente dependente de propagação do sinal rápida ativada por mielinização. Portanto, os processos desmielinizantes geralmente apresentam diminuição velocidades de condução 4. O composto potencial de ação motor (CMAP) - correlacionando-se com o número de axônios funcionais - é um indicador de dano axonal quando reduziu significativamente 5. Assim, por meio de métodos electrofisiológicos da etiologia da lesão do nervo periféricopode ser discriminado, tal como para as neuropatias hereditárias, neuropatia diabética 6,7 8,9, polineuropatias inflamatórias crónicas desmielinizantes (PDIC) 10, ou neuropatias metabólicas 11. Normalmente, na aplicação humana são os preferidos gravações não-invasivos no nervo sural ou ulnar. Em camundongos, é muito simples para analisar propriedades nervosas do nervo ciático, o maior nervo do sistema nervoso periférico (SNP), contendo tanto grandes - e axônios de pequeno calibre do sistema motor e sensorial. O procedimento como demonstrado aqui é um método rápido, fácil e confiável para medir todos os valores padrão relevantes para eletrofisiologia em nervos periféricos no rato intacto. Ao tomar gravações de um organismo preservados, são garantidas condições fisiológicas do ambiente do nervo.

<table border="0" cellpadding="0" cellspacing="0" width="917">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:321px;">Concentric Needle Electrodes (Stimulation)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td style="width:119px;">9013S0901</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Digital Ring Electrodes (Recording)</td>
			<td style="width:311px;">Natus Medical Incorporated 
			San Carlos, CA 94070, USA</td>
			<td>9013S0302</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">ToM - Tower of Measurement (A/D converter)</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">AtisaPro, Data acquisition &#38; analysis software</td>
			<td>GJB Datentechnik GmbH, Langewiesen, Germany</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HSE-Stimulator T</td>
			<td colspan="2">Hugo Sachs Elektronik, Hugstetten, Germany</td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo electrophysiological measurements on mouse sciatic nerves

Estudos electrofisiológicos permitir uma classificação racional de várias doenças neuromusculares e são de ajuda, em conjunto com técnicas de neuropatológicos, na compreensão da patofisiologia subjacente 1. Aqui nós descrevemos um método para realizar estudos eletrofisiológicos no nervo ciático de rato in vivo. Os animais são anestesiados com isoflurano, a fim de assegurar a analgesia para os ratinhos testados e não perturbada ambiente de trabalho durante as medições que levam cerca de 30 min / animal. A temperatura corporal constante de 37 ° C é mantida por uma placa de aquecimento e medida continuamente por uma sonda térmica rectal 2. Além disso, um eletrocardiograma (ECG) é rotineiramente registrada durante as medições, a fim de monitorar continuamente o estado fisiológico dos animais investigados. Registros eletrofisiológicos são executadas no nervo ciático, o maior nervo do the sistema nervoso periférico (SNP), fornecendo o membro posterior do rato com feixes de fibras tanto motoras e sensoriais. No nosso protocolo, os nervos ciáticos permanecer in situ e, portanto, não têm de ser extraídos ou exposto, permitindo medições sem qualquer irritação nervosas adversas juntamente com gravações reais. Usando eletrodos de agulha apropriadas 3 realizamos tanto proximal e distal estímulos nervosos, registrar os potenciais de transmissão com eletrodos de sensoriamento no músculo gastrocnêmio. Após o processamento de dados, de confiança e valores altamente consistentes para a velocidade de condução nervosa (NCV) e do composto potencial de ação motor (CMAP), os principais parâmetros para a quantificação do funcionamento dos nervos periféricos bruto, pode ser alcançado.

<html> Foi realizada uma série de medições in vivo eletrofisiológicos no nervo ciático de 12 ratos no total para este estudo: 6 animais de cada sexo. As medições foram realizadas com o protocolo apresentado e entregues os seguintes resultados: Ambos os ratos machos e fêmeas exibir uma velocidade de condução do nervo ciático média de cerca de 20 m / s (Figura 5). Isto é consistente com outras medidas na literatura. Além disso, mostra que não há diferen...

Watch this Scientific Journal Video about In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves at JoVE.com

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Medições das propriedades de condução nervosa em vivo exemplificar uma ferramenta poderosa para caracterizar vários modelos animais de doenças neuromusculares. Aqui, apresentamos um protocolo de fácil e confiável pelo qual análise eletrofisiológica em nervo ciático de ratos anestesiados pode ser realizada.

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 ...

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In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

33.0K visualizações.  Fritz Lipmann Institute.  Medições das propriedades de condução nervosa em vivo exemplificar uma ferramenta poderosa para caracterizar vários modelos animais de doenças neuromusculares. Aqui, apresentamos um protocolo de fácil e confiável pelo qual análise eletrofisiológica em nervo ciático de ratos anestesiados pode ser realizada. 

Vídeo: In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

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

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

The targeting and refinement of RGC projections to the midbrain is a popular and powerful model system for studying how precise patterns of neural connectivity form during development. In mice, retinofugal projections are arranged in a topographic manner and form eye-specific layers in the Lateral Geniculate Nucleus (dLGN) of the thalamus and the Superior Colliculus (SC). The development of these precise patterns of retinofugal projections has typically been studied by labeling populations of RGCs with fluorescent dyes and tracers, such as horseradish peroxidase1-4. However, these methods are too coarse to provide insight into developmental changes in individual RGC axonal arbor morphology that are the basis of retinotopic map formation. They also do not allow for the genetic manipulation of RGCs.
Recently, electroporation has become an effective method for providing precise spatial and temporal control for delivery of charged molecules into the retina5-11. Current retinal electroporation protocols do not allow for genetic manipulation and tracing of retinofugal projections of a single or small cluster of RGCs in postnatal mice. It has been argued that postnatal in vivo electroporation is not a viable method for transfecting RGCs since the labeling efficiency is extremely low and hence requires targeting at embryonic ages when RGC progenitors are undergoing differentiation and proliferation6. 
In this video we describe an in vivo electroporation protocol for targeted delivery of genes, shRNA, and fluorescent dextrans to murine RGCs postnatally. This technique provides a cost effective, fast and relatively easy platform for efficient screening of candidate genes involved in several aspects of neural development including axon retraction, branching, lamination, regeneration and synapse formation at various stages of circuit development. In summary we describe here a valuable tool which will provide further insights into the molecular mechanisms underlying sensory map development.

Transfection of Mouse Retinal Ganglion Cells by in vivo Electroporation

We demonstrate an in vivo electroporation protocol for transfecting single or small clusters of retinal ganglion cells (RGCs) and other retinal cell types in postnatal mice over a wide range of ages. The ability to label and genetically manipulate postnatal RGCs in vivo is a powerful tool for developmental studies.

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

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

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 ...

Functional Neuroimaging Using Ultrasonic Blood-brain Barrier Disruption and Manganese-enhanced MRI

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains technically challenging. One approach, Activation-Induced Manganese-enhanced MRI (AIM MRI), has been used successfully to map neuronal activity in rodents 1-5. In AIM MRI, Mn2+ acts a calcium analog and accumulates in depolarized neurons 6,7. Because Mn2+ shortens the T1 tissue property, regions of elevated neuronal activity will enhance in MRI. Furthermore, Mn2+ clears slowly from the activated regions; therefore, stimulation can be performed outside the magnet prior to imaging, enabling greater experimental flexibility. However, because Mn2+ does not readily cross the blood-brain barrier (BBB), the need to open the BBB has limited the use of AIM MRI, especially in mice.
One tool for opening the BBB is ultrasound. Though potentially damaging, if ultrasound is administered in combination with gas-filled microbubbles (i.e., ultrasound contrast agents), the acoustic pressure required for BBB opening is considerably lower. This combination of ultrasound and microbubbles can be used to reliably open the BBB without causing tissue damage 8-11.
Here, a method is presented for performing AIM MRI by using microbubbles and ultrasound to open the BBB. After an intravenous injection of perflutren microbubbles, an unfocused pulsed ultrasound beam is applied to the shaved mouse head for 3 minutes. For simplicity, we refer to this technique of BBB Opening with Microbubbles and UltraSound as BOMUS 12. Using BOMUS to open the BBB throughout both cerebral hemispheres, manganese is administered to the whole mouse brain. After experimental stimulation of the lightly sedated mice, AIM MRI is used to map the neuronal response.
To demonstrate this approach, herein BOMUS and AIM MRI are used to map unilateral mechanical stimulation of the vibrissae in lightly sedated mice 13. Because BOMUS can open the BBB throughout both hemispheres, the unstimulated side of the brain is used to control for nonspecific background stimulation. The resultant 3D activation map agrees well with published representations of the vibrissae regions of the barrel field cortex 14. The ultrasonic opening of the BBB is fast, noninvasive, and reversible; and thus this approach is suitable for high-throughput and/or longitudinal studies in awake mice.

A technique is described for broadly opening the blood-brain barrier in the mouse using microbubbles and ultrasound. Using this technique, manganese can be administered to the mouse brain. Because manganese is an MRI contrast agent that accumulates in depolarized neurons, this approach enables imaging of neuronal activity.

Neuroimagem Funcional Usando rompimento da barreira hemato-encefálica ultra-sônico e Manganês-RM

Although mice are the dominant model system for studying the genetic and molecular underpinnings of neuroscience, functional neuroimaging in mice remains ...

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety of brain disorders1,2. To better understand the mechanisms by which brain circuits react to an animal's experience requires the ability to monitor the experience-dependent molecular changes in a given set of neurons, over a prolonged period of time, in the live animal. While experience and associated neural activity is known to trigger gene expression changes in neurons1,2, most of the methods to detect such changes do not allow repeated observation of the same neurons over multiple days or do not have sufficient resolution to observe individual neurons3,4. Here, we describe a method that combines in vivo two-photon microscopy with a genetically encoded fluorescent reporter to track experience-dependent gene expression changes in individual cortical neurons over the course of day-to-day experience. 
One of the well-established experience-dependent genes is Activity-regulated cytoskeletal associated protein (Arc)5,6. The transcription of Arc is rapidly and highly induced by intensified neuronal activity3, and its protein product regulates the endocytosis of glutamate receptors and long-term synaptic plasticity7. The expression of Arc has been widely used as a molecular marker to map neuronal circuits involved in specific behaviors3. In most of those studies, Arc expression was detected by in situ hybridization or immunohistochemistry in fixed brain sections. Although those methods revealed that the expression of Arc was localized to a subset of excitatory neurons after behavioral experience, how the cellular patterns of Arc expression might change with multiple episodes of repeated or distinctive experiences over days was not investigated. 
In vivo two-photon microscopy offers a powerful way to examine experience-dependent cellular changes in the living brain8,9. To enable the examination of Arc expression in live neurons by two-photon microscopy, we previously generated a knock-in mouse line in which a GFP reporter is placed under the control of the endogenous Arc promoter10. This protocol describes the surgical preparations and imaging procedures for tracking experience-dependent Arc-GFP expression patterns in neuronal ensembles in the live animal. In this method, chronic cranial windows were first implanted in Arc-GFP mice over the cortical regions of interest. Those animals were then repeatedly imaged by two-photon microscopy after desired behavioral paradigms over the course of several days. This method may be generally applicable to animals carrying other fluorescent reporters of experience-dependent molecular changes4.

In Vivo Two-photon Imaging Of Experience-dependent Molecular Changes In Cortical Neurons

Experience-dependent molecular changes in neurons are essential for the brain's ability to adapt in response to behavioral challenges. An in vivo two-photon imaging method is described here that allows the tracking of such molecular changes in individual cortical neurons through genetically encoded reporters.

Vivo em dois fótons de imagem de experiência dependentes de alterações moleculares em neurônios corticais

The brain's ability to change in response to experience is essential for healthy brain function, and abnormalities in this process contribute to a variety ...

Simultaneous Electrophysiological Recording and Calcium Imaging of Suprachiasmatic Nucleus Neurons

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in regulating the intracellular calcium concentration. Changing environmental conditions, such as the light-dark cycle, can modify neuronal and neural network activity and the expression of a family of circadian clock genes within the suprachiasmatic nucleus (SCN), the location of the master circadian clock in the mammalian brain. Excitatory synaptic transmission leads to an increase in the postsynaptic Ca2+ concentration that is believed to activate the signaling pathways that shifts the rhythmic expression of circadian clock genes. Hypothalamic slices containing the SCN were patch clamped using microelectrodes filled with an internal solution containing the calcium indicator bis-fura-2. After a seal was formed between the microelectrode and the SCN neuronal membrane, the membrane was ruptured using gentle suction and the calcium probe diffused into the neuron filling both the soma and dendrites. Quantitative ratiometric measurements of the intracellular calcium concentration were recorded simultaneously with membrane potential or current. Using these methods it is possible to study the role of changes of the intracellular calcium concentration produced by synaptic activity and action potential firing of individual neurons. In this presentation we demonstrate the methods to simultaneously record electrophysiological activity along with intracellular calcium from individual SCN neurons maintained in brain slices.

Procedures are described to perform simultaneous recordings of membrane potential or current and changes of intracellular calcium concentration. Suprachiasmatic nucleus neurons are filled with the calcium indicator bis-fura-2 using a patch clamp electrode in the whole cell patch clamp configuration.

Simultaneous electrophysiological and fluorescent imaging recording methods were used to study the role of changes of membrane potential or current in ...

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous system. SC-selective genetic manipulation in living animals is a powerful technique for studying the molecular and cellular mechanisms of SC myelination and demyelination in vivo. While knockout/knockin and transgenic mice are powerful tools for studying SC biology, these methods are costly and time consuming. Viral vector-mediated transgene introduction into the sciatic nerve is a simpler and less laborious method. However, viral methods have limitations, such as toxicity, transgene size constraints, and infectivity restricted to certain developmental stages. Here, we describe a new method that allows selective transfection of myelinating SCs in the rodent sciatic nerve using electroporation. By applying electric pulses to the sciatic nerve at the site of plasmid DNA injection, genes of interest can be easily silenced or overexpressed in SCs in both neonatal and more mature animals. Furthermore, this in vivo electroporation method allows for highly efficient simultaneous expression of multiple transgenes. Our novel technique should enable researchers to efficiently manipulate SC gene expression, and facilitate studies on SC development and function.

In Vivo Gene Transfer to Schwann Cells in the Rodent Sciatic Nerve by Electroporation

Here, we present an in vivo technique for gene transfer to Schwann cells (SCs) in the rodent sciatic nerve. This simple technique is useful for investigating signaling mechanisms involved in the development and maintenance of myelinating SCs.

Em Gene Vivo Transferência de células de Schwann no nervo ciático de roedores por Eletroporação

The formation of the myelin sheath by Schwann cells (SCs) is essential for rapid conduction of nerve impulses along axons in the peripheral nervous ...

Analysis of Immune Cells in Single Sciatic Nerves and Dorsal Root Ganglion from a Single Mouse Using Flow Cytometry

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of the PNS are affected by injury and disease, affecting the nerve function and the capacity for regeneration. Neuronal immune cells are commonly analyzed by immunofluorescence (IF). While IF is essential for determining the location of the immune cells in the nerve, IF is only semi-quantitative and the method is limited to the number of markers that can be analyzed simultaneously and the degree of surface expression. In this study, flow cytometry was used for quantitative analysis of leukocyte infiltration into sciatic nerves or dorsal root ganglions (DRGs) of individual mice. Single cell analysis was performed using DAPI and several proteins were analyzed simultaneously for either surface or intracellular expression. Both sciatic nerves from one mouse that were treated according to this protocol generated &#8805; 30,000 single nucleated events. The proportion of leukocytes in the sciatic nerves, determined by expression of CD45, was approximately 5% of total cell content in the sciatic nerve and approximately 5-10% in the DRG. Although this protocol focuses primarily on the immune cell population within the PNS, the flexibility of flow cytometry to measure a number of markers simultaneously means that the other cells populations present within the nerve, such as Schwann cells, pericytes, fibroblasts, and endothelial cells, can also be analyzed using this method. This method therefore provides a new means for studying systemic effects on the PNS, such as neurotoxicology and genetic models of neuropathy or in chronic diseases, such as diabetes.

Quantitative analysis of cell content within the murine sciatic nerve is difficult due to the scarcity of the tissue. This protocol describes a method for tissue digestion and preparation that provides sufficient cells for flow cytometry analysis of immune cell populations from nerves of individual mice.

Análise de células do sistema imunológico em único nervos ciático e gânglio da raiz Dorsal de um único rato usando citometria de fluxo

Nerve-resident immune cells in the peripheral nervous system (PNS) are essential to maintaining neuronal integrity in a healthy nerve. The immune cells of ...

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings provide a sensitive approach to measure nerve conduction in humans and rodent models. To broaden the technical possibilities for electromyography in mice, the measurement of compound muscle action potentials (CMAPs) from the brachial plexus nerve in the forelimb using needle electrodes is described here. CMAP recordings after stimulating the sciatic nerve in hindlimbs have been previously described. The newly introduced method here allows for the evaluation of the nerve conductivity at an additional site, and thus provides a more profound overview of the neuromuscular functionality. The technique provides information on both the relative number of functional axons and the myelination level. Thereby, this method can be applied to assess both axonal diseases as well as demyelinating conditions. This minimally invasive method does not require extraction of the nerve and therefore it is suitable for repeated measurements for longitudinal follow-up in the same animal. Similar recordings are performed in clinical setups to emphasize the translational relevance of the method.

In Vivo Electrophysiological Measurement of Compound Muscle Action Potential from the Forelimbs in Mouse Models of Motor Neuron Degeneration

The measurement of nerve conduction is a useful tool to assess mouse models of neurodegeneration but it is frequently only applied to stimulate the sciatic nerve in hindlimbs. Here, we describe a technique to measure compound muscle action potential (CMAP) in vivo in the mouse forelimb muscles innervated by the brachial plexus.

Na Vivo Eletrofisiológica medição do potencial de ação muscular composto de membros dianteiros em modelos de Mouse de degeneração do neurônio Motor

Assessing the functionality of the nerve axon provides detailed information on the progression of neuromuscular disorders. Electrophysiological recordings ...

In Vivo Calcium Imaging of Lateral-line Hair Cells in Larval Zebrafish

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory stimuli that mechanically deflect apical protrusions called hair bundles. Deflection opens mechanotransduction (MET) channels in hair bundles, leading to an influx of cations, including calcium. This cation influx depolarizes the cell and opens voltage-gated calcium channels located basally at the hair-cell presynapse. In mammals, hair cells are encased in bone, and it is challenging to functionally assess these activities in vivo. In contrast, larval zebrafish are transparent and possess an externally located lateral-line organ that contains hair cells. These hair cells are functionally and structurally similar to mammalian hair cells and can be functionally assessed in vivo. This article outlines a technique that utilizes a genetically encoded calcium indicator (GECI), GCaMP6s, to measure stimulus-evoked calcium signals in zebrafish lateral-line hair cells. GCaMP6s can be used, along with confocal imaging, to measure in vivo calcium signals at the apex and base of lateral-line hair cells. These signals provide a real-time, quantifiable readout of both mechanosensation- and presynapse-dependent calcium activities within these hair cells. These calcium signals also provide important functional information regarding how hair cells detect and transmit sensory stimuli. Overall, this technique generates useful data about relative changes in calcium activity in vivo. It is less well-suited for quantification of the absolute magnitude of calcium changes. This in vivo technique is sensitive to motion artifacts. A reasonable amount of practice and skill are required for proper positioning, immobilization, and stimulation of larvae. Ultimately, when properly executed, the protocol outlined in this article provides a powerful way to collect valuable information about the activity of hair-cells in their natural, fully integrated states within a live animal.

The zebrafish is a model system that has many valuable features including optical clarity, rapid external development, and, of particular importance to the field of hearing and balance, externally located sensory hair cells. This article outlines how transgenic zebrafish can be used to assay both hair-cell mechanosensation and presynaptic function in toto.

Na imagem latente de cálcio Vivo de células de cabelo linha Lateral no Zebrafish Larval

Sensory hair cells are mechanoreceptors found in the inner ear that are required for hearing and balance. Hair cells are activated in response to sensory ...

Methods Article

Em Medidas Vivo eletrofisiológicos nos nervos ciático Rato