The authors sincerely thank the Canadian Institutes of Health Research MOP 130258 and the Star Foundation for Children&#8217;s Health Research, along with the Molly Towell Foundation, for the provision of the research facility and financial support. The authors also sincerely thank Dr. Kinkead Richard for manuscript proofreading and advice.

Este método necessária a utilização de matérias de origem animal, permitidas pelo Comitê de Laval University animal Ética (protocolo nº 2012-170). 1. Configuração e Preparação <ol><li> Soluções <ol><li> Preparar soluções stock aCSF de acordo com as seguintes receitas 7,17. Outras receitas com variações de concentração estão disponíveis na literatura. Soluções de armazenar a 4 ° C durante até um mês. <ol><li> Solução de sal: adicionar 75,39 g de NaCl (129 mM final); 2,5 g de KCl (3,35 mM final); 0,81 g de NaH2PO4 (0,58 mM final); 2,33 g de MgCl2 (1,15 mM final); 1,85 g de CaCl2 (1,26 mM). Dissolver em 800 ml de água destilada, em seguida, enche-se a 1 L com água destilada. </li><li> Solução de bicarbonato de: adicionar 17,65 g de NaHCO3 (21 mM final). Dissolver em 900 ml de água destilada, em seguida, enche-se a 1 L com água destilada. Variações da concentração de bicarbonato irá resultar em variações do pH. </li><li> Solução de glicose: adicionar 54,06g de glucose (30 mM final). Dissolver em 400 ml de água destilada, em seguida, preencher a 500 ml com água destilada. </li></ol></li><li> Prepare aCSF, diluindo 100 ml de solução salina, 100 ml de solução de bicarbonato, e 50 ml de solução de glucose em 1 L de água destilada. Armazenar a 4 ° C até à sua utilização. </li><li> Prepare um eletrodo de vidro por aquecimento e alongamento um tubo de vidro até que quebre. Areia para baixo a ponta antes de usar. O eléctrodo pode ser reutilizado muitas tempo, desde que ele permaneça limpo. </li></ol></li><li> Setup Experimental Nota: Os detalhes de configuração são apresentados na Figura 2. <ol><li> Ligue o amplificador, averager, sistema de aquisição de dados, controlador de temperatura e bomba em movimento. Verifique a amplificação do sinal (ganho = 10.000), a filtração (baixo limiar, 10 Hz; limiar alto, 5 kHz), e a taxa de amostragem da conversão de analógico para digital do sinal bruto (2,5 kHz). </li><li> Encha uma garrafa com aCSF e bolha com carbogênio (95% O 2 </sub&gt;, 5% de CO 2). Induzir uma bubbled- e aqueceu-aCSF fluxo na câmara de gravação (volume de 5 ml) a 4 ml / min. Manter a temperatura na câmara de gravação em 26 (± 1) ° C. O pH deve ser 7,4 nestas condições padrão. </li><li> Mantenha 50 ml de RT e borbulhou-carbogénio aCSF em uma seringa de 50 ml, perto da câmara de dissecção. </li><li> Ligue o computador e iniciar o software de gravação. </li></ol></li></ol> 2. Dissecção <ol><li> Pesar e visualmente determinar o sexo do animal (machos têm um ano genital distância mais longa, enquanto as fêmeas têm uma distância ano-genital curto; machos genitais são muitas vezes escuro, enquanto órgãos genitais femininos são cor de rosa). </li><li> Anestesiar os roedores recém-nascidos por uma das seguintes opções: Crioanestesia (imergir completamente o animal em gelo durante 4 - 5 min 18), a injecção (equitensine a 4 ml / kg) 19 ou inalação de anestésicos voláteis (isoflurano 20 ou éter 21). Confirm um plano adequado de anestesia pela ausência de um reflexo de retirada da pata. </li><li> Colocar o animal no banco, cara ventral para baixo. Secção da parte da cabeça rostral coronalmente com um bisturi ao nível da bregma (visíveis através da pele e do crânio). Execute esta etapa imediatamente após o animal é anestesiado. </li><li> Seção do corpo coronalmente com um bisturi sob os membros anteriores. </li><li> Retire a pele, músculos, tecidos adiposos e víscera com tesouras cirúrgicas e finas e um alicate. Como os ossos são moles nesta idade, ser cauteloso para não danificar o sistema nervoso. Execute esta etapa no banco. </li><li> Coloque a preparação na câmara de dissecção. Utilizar a aCSF armazenado na seringa para oxigenar a preparação. A partir deste ponto para o fim da gravação, usar um microscópio. </li><li> Na face dorsal da preparação, cortar o crânio e vértebras do rostral à parte caudal ao longo do eixo médio com tesouras cirúrgicas finas e e um alicate. Abra o crânio cortee vértebras de modo a expor o tecido nervoso. Use a aCSF strored na seringa para oxigenar a preparação. </li><li> Com os alicates, remover a membrana aracnoideia, um tecido fino que cobre a superfície do tecido nervoso. Manter a pia-máter e vasos sanguíneos contra o tecido nervoso. Utilizar a aCSF armazenado na seringa para oxigenar a preparação. </li><li> Coloque a preparação com o dorsal viradas para baixo, e cuidadosamente derrubar o tronco cerebral ea medula espinhal, cortando os nervos e tecido conjuntivo com a tesoura, segurando a preparação no local. Uma abordagem dorsal também é possível. Manter as raízes dos nervos e o maior tempo possível. Remover os ossos para isolar o tronco cerebral e da medula espinhal. Utilizar a aCSF armazenado na seringa para oxigenar a preparação. </li><li> Remover o cerebelo e restantes estruturas rostral seccionando-los com o bisturi. Utilizar a aCSF armazenado no interior da agulha para oxigenar a preparação. </li><li> Opcionalmente, dependendo experimenprojeto tal, remova a ponte com o bisturi por uma seção coronal anterior à artéria cerebelar inferior 22. Note-se que manter os pons inteiras vai abrandar o ritmo em ratos e totalmente inibi-la em camundongos. Preparações que incluem apenas a parte caudal da ponte exibir uma atividade respiratória, como com uma frequência estável na raiz C4. </li></ol> 3. A gravação <ol><li> Coloque a preparação na câmara de gravação, ventral face para cima. Corrigir a preparação com os pinos na parte mais baixa da medula espinhal e rostral mais-parte do tronco cerebral. </li><li> Usando uma seringa ligada ao eléctrodo por sua agulha, induzir uma depressão no eléctrodo (diâmetro da ponta de vidro: 150 - 225 uM), puxando o êmbolo da seringa, a fim de preencher parcialmente o eléctrodo com aCSF. </li><li> Utilizando um micromanipulador, colocar cuidadosamente o eléctrodo próximo de uma das quarta raízes ventrais. Outras raízes ventrais também poderia apresentar um respiratório-like atividade (e.g., nervo craniano XII, C1 raiz ventral). </li><li> Induzir uma depressão no reservatório de eléctrodo através da seringa puxando o êmbolo para fora, a fim de aspirar suavemente a radícula nervo. Em seguida, mova cuidadosamente o eléctrodo de aplicá-la contra a medula espinhal. Nota: Idealmente, o tamanho das radículas corresponde ao tamanho da abertura do eléctrodo e, portanto, cria uma vedação entre os compartimentos internos e externos do eléctrodo. Desde amplificadores diferenciais são normalmente utilizados para tais gravações, qualquer abertura entre o interior do eléctrodo e a câmara de registo reduz a qualidade do sinal e faz com que seja difícil de eliminar o ruído de fundo. </li><li> Comece a gravação. </li><li> Grave o ritmo produzido pela preparação sob condições de normóxia (isto é, borbulhada com carbogénio aCSF: 95% de O2 e 5% de CO 2) durante pelo menos 20 minutos para determinar os parâmetros de linha de base de preparação. </li><li> Alterne a perfusão de borbulhou-carbogénio aCSF paraaCSF estímulo (isto é, feito borbulhar com 95% de N2 e 5% de CO 2 por estímulo hipoxia) durante 15 min. Alterar a duração da exposição, dependendo do protocolo experimental. Grave a duração da exposição sobre a gravação e animal folha de dados. Utilize tubos de aço inoxidável entre a garrafa e a câmara de aCSF sempre que possível para evitar a difusão de gás para o exterior do tubo. </li><li> Alterne a perfusão de volta para aCSF borbulhou-carbogénio padrão para, pelo menos, 15 min para uma gravação de recuperação. Grave este na gravação e animal folha de dados. </li><li> Fim da gravação. </li></ol> 4. Análise estatística <ol><li> A partir do sinal integrado, calcular a frequência como o número de rajadas por minuto (expressa em rajadas / min), a amplitude como a diferença entre a linha de base e o pico do sinal de sincronismo (expressa em mV), a duração do sincronismo como a duração de o início até ao fim do sinal de sincronismo (expresso em segundos), a área de explosão como a área sob a curve da explosão sobre o sinal integrado (expressa em mV · s) (Figura 3). </li><li> Calcular a frequência, amplitude, duração e área de estourar estourar sob normóxica (baseline), hipóxia (estímulo) e pós-condições de hipóxia (pós-estímulo) de recuperação como a média da última 5 min das gravações para cada condição. Não analisam a primeira porção de cada condição, porque existe um atraso entre a ligação da perfusão e aCSF homogeneização na câmara de gravação. </li><li> Expresse então estímulo (ou seja, a hipóxia) e pós-estímulo (ou seja, pós-hipóxia) valores de recuperação como uma percentagem dos valores de referência para a gravação correspondente. Exprimir os resultados em média ± SD </li></ol>

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The authors have no competing financial interests or conflicts of interests to disclose.

Quantificação exata da atividade respiratória pode ser um desafio. Na verdade, a respiração é uma função que pode ser tanto automática e voluntária, e que é modulada de acordo com o ambiente, as necessidades do corpo, o estado emocional eo comportamento. A vantagem desta técnica é o isolamento de elementos neurais responsáveis ​​por produzir o comando respiratória. Assim, as gravações electrofisiológicas de preparações da medula espinal e do tronco cerebral-pletismografia são técnicas complemen...

A respiração é uma actividade fundamental complexo e controlado pelo cérebro, permitindo dioxigénio (O2) e a absorção de dióxido de carbono (CO 2) eliminação. O comando central da respiração é gerado por uma complexa rede localizado no tronco cerebral em ambos os mamíferos 1, 2 anfíbios, répteis, pássaros 3 4 5 e peixes. Mesmo se o estudo de respiração pode ser processada in vivo, investigações mecanicistas precisos requerem acesso directo à rede de controlo respiratório. Para este fim, Adrian e Buytendijk desenvolveu uma preparação peixinho reduzida, no qual os eletrodos posicionados na superfície do tronco cerebral registro do ritmo gerado associada a ventilação branquial 5. Esta abordagem foi subsequentemente adaptado por Suzue em 1984 6 para utilização em roedores recém-nascidos. O advento desta preparação tem levado a avanços significativos na neurobiologia respiratória. Uma vez que é relativamente simples, a técnica apresentada here é passível de uma ampla gama de investigações básicas de comportamentos motores rítmicas e as suas origens em roedores recém-nascidos. O objetivo geral deste método consiste em gravar o correlato neural da atividade inspiratória, um ritmo respiratório, chamado respiração fictício, produzido pela rede respiratória. Este método pode ser utilizado em uma ampla gama de objetivos de pesquisa, visando respostas inspiratória a variações respiratórias ou farmacologia em ambos os selvagens tipo 7 e 8 animais transgênicos. Dado que as experiências são realizadas a uma temperatura baixa, sem aferentes sensoriais, e sob condições nas quais as concentrações de glicose e O 2 no interior da aCSF são elevados, foram levantadas questões sobre a relevância fisiológica do sinal gravado. Embora existam diferenças claras entre in vivo e in vitro condições (por exemplo., A freqüência de rajadas inspiratória) permanece o fato de que a presença deos principais elementos da rede respiratória 6 torná-lo possível estudar um ritmo robusto associado a uma função homeostática vital 9,10. A lógica por trás do desenvolvimento e da utilização dessa técnica é facilitar o acesso direto aos elementos do tronco cerebral da rede respiratória, que são de difícil acesso in vivo, especialmente em recém-nascidos. O tronco cerebral é colocado sob condições estritamente controladas: o ritmo gravada não é modulada por entradas aferentes periféricas dos pulmões ou dos órgãos carótidas, permitindo o estudo para se concentrar no comando central da respiração em si 11. Assim, este acesso é utilizada para aplicar estímulos e gravar o sinal de saída. Em contraste com pletismografia gravações, o ritmo respiratório é modulada por todos os seus componentes ao longo do corpo (por ex., A distensão do pulmão, sensores químicos periféricas), tornando-o difícil de aplicar estímulos precisos. Numarato ewborn, o protocolo consiste em gravar o quarto sinal ventral raiz em um tronco cerebral isolado e uma medula espinhal truncado, mantida no líquido cefalorraquidiano artificial (aCSF). O ritmo gerada pelos preparativos da medula do tronco cerebral-espinhal é composta de explosões lentas individuais que estão ligadas ao sinal inspiratória 9. Isolado preparações da medula do tronco cerebral-espinhal são facilmente graváveis ​​em ratos de pós-natal dia 0-4 (P0 - P4) 7. Esta abordagem é vulgarmente usado para avaliar a resposta hipóxica da rede respiratória, e também a resposta a hipercapnia, acidose ou drogas. Um protocolo de hipóxia aguda é apresentado aqui. Esta estimulação é obtido por retirada de O 2 no aCSF; esta abordagem é utilizada para avaliar a tolerância e a capacidade de resposta a insultos hipóxicos. O protocolo induz uma depressão do ritmo a partir do primeiro minuto até ao final da exposição a hipoxia (Figura 1) 12. Esta depressão é invertidadurante a pós-hipóxica 12 recuperação. No que respeita ao delineamento experimental, é importante notar que a ponte, localizada na parte rostral do tronco cerebral, tem uma ação inibitória sobre o gerador de ritmo 8. Assim, as preparações de tronco cerebral e da medula espinal integral rostral exibir um ritmo inferior. Inclusão da ponte na amostra isolada para a gravação é determinado de acordo com o objetivo do experimento 13; o estudo da influência pontina na rede medula oblongata exigiria gravações com e sem a ponte para comparar os resultados 14. Além disso, uma das vantagens desta técnica é a possibilidade de estender a porção rostral do preparado para incluir mesencefálico e / ou regiões diencefálica 15,16, tornando-se possível avaliar o efeito destas regiões na rede ponto-medular respiratória.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sylgard</td>
			<td>Sigma Aldrich</td>
			<td>761036-5EA</td>
			<td>Use under hood</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Bioshop</td>
			<td>SOD002</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Bioshop</td>
			<td>POC888</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Bioshop</td>
			<td>CCL444</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Bioshop</td>
			<td>MAG510</td>
			<td></td>
		</tr>
		<tr>
			<td>NaHCO3</td>
			<td>Bioshop</td>
			<td>SOB999</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4</td>
			<td>Bioshop</td>
			<td>SPM306</td>
			<td></td>
		</tr>
		<tr>
			<td>D-glucose</td>
			<td>Bioshop</td>
			<td>GLU501</td>
			<td></td>
		</tr>
		<tr>
			<td>Carbogen</td>
			<td>Linde</td>
			<td>343-02-0006&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Temperature Controller</td>
			<td>Warner Instruments, Hamden, CT, USA</td>
			<td>TC-324B</td>
			<td></td>
		</tr>
		<tr>
			<td>Suction electrode</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 573000</td>
			<td></td>
		</tr>
		<tr>
			<td>Differential AC amplifier</td>
			<td>A-M Systems, Everett, WA, USA</td>
			<td>model 1700</td>
			<td></td>
		</tr>
		<tr>
			<td>Moving averager</td>
			<td>CWE, Ardmore, PA, USA</td>
			<td>model MA-821</td>
			<td></td>
		</tr>
		<tr>
			<td>Data acquisition system</td>
			<td>Dataq Instruments, Akron, OH, USA</td>
			<td>model DI-720</td>
			<td></td>
		</tr>
		<tr>
			<td>LabChart software</td>
			<td>ADInstruments, Colorado Springs, CO, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Prism sofware</td>
			<td>Graphpad, La Jolla, CA, USA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection chamber</td>
			<td>Plastic box (e.g. petri box) will do</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Recording chamber</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Micromanipulator</td>
			<td>World Precision Instrument Inc, Sarasota, FL, USA</td>
			<td>KITE-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Base</td>
			<td>Kanetec, Bensenville, IL, USA</td>
			<td>MB</td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson, Middleton, WI, USA</td>
			<td>MINIPULS 3</td>
			<td></td>
		</tr>
		<tr>
			<td>Faraday Cage</td>
			<td>Home made</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Computer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

electrophysiology on isolated brainstem-spinal cord preparations from newborn rodents allows neural respiratory network output recording

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to stimuli remain to be fully understood. To overcome the difficulty of accessing the brainstem in the whole animal, isolation of the brainstem and part of the spinal cord is performed. This preparation is maintained in artificial cerebro-spinal fluid where gases, concentrations, and temperature are controlled and monitored. The output signal from the respiratory network is recorded by a suction electrode placed on the fourth ventral root. In this manner, stimuli can be directly applied onto the brainstem, and the effect can be recorded directly. The signal recorded is linked to the inspiratory signal sent to the diaphragm via the phrenic nerve, and can be described as bursts (around 8 bursts per minute). Analysis of these bursts (frequency, amplitude, length, and area under the curve) allows precise characterization of the stimulus effect on the respiratory network. The main limitation of this method is the viability of the preparation beyond the early post-natal stages. Thus, this method greatly focuses on the study of the whole network without the peripheral inputs in the newborn rat.

Como mencionado na introdução, uma das vantagens mais importantes desta técnica é o acesso directo ao tronco cerebral para aplicar vários estímulos. Como um exemplo, a hipoxia foi aplicado aqui. Figura 1. AB exibe uma gravação protocolo completo, com ambas as condições de normóxia e de hipóxia. A Figura 1.CE exibe o ritmo registada em condições de normóxia (isto é, aCSF borbulhar com 95% de O2 e 5% CO2 a 26 ° C). Como anteriormente demonst...

Watch this Scientific Journal Video about Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording at JoVE.com

Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Eletrofisiologia no isolado do tronco encefálico-espinal Preparações do cordão umbilical de recém-nascidos Roedores permite a gravação de saída Neural Rede Respiratory

While it is well known that the central respiratory drive is located in the brainstem, several aspects of its basic function, development, and response to ...

electrophysiology-on-isolated-brainstem-spinal-cord-preparations-from

Research

JoVE Journal

Neuroscience

8.5K visualizações.  Laval University. The central respiratory drive is located in the brainstem. Spontaneous respiratory motor output from an isolated brainstem-spinal cord is recorded by placing an electrode on the fourth ventral root. This experimental approach is valuable for pharmacological investigations or the assessment of respiratory challenges and genetic manipulations on rhythmic motor behavior.

Vídeo: Electrophysiology on Isolated Brainstem-spinal Cord Preparations from Newborn Rodents Allows Neural Respiratory Network Output Recording

Dissection and Culture of Commissural Neurons from Embryonic Spinal Cord

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies of these neurons are located in the dorsal spinal cord and their axons follow stereotyped trajectories during embryonic development. Commissural axons initially project ventrally towards the floorplate. After crossing the midline, these axons turn anteriorly and project towards the brain. Each of these steps is regulated by the action of several guidance cues. Cultures highly enriched in commissural neurons are ideally suited for many experiments addressing the mechanisms of axon pathfinding, including turning assays, immunochemistry and biochemistry. Here, we describe a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. First, the spinal cord is isolated and dorsal strips are dissected out. The dorsal tissue is then dissociated into a cell suspension by trypsinization and mechanical disruption. Neurons are plated onto poly-L-lysine-coated glass coverslips or tissue-culture dishes. After 30 hours in vitro, most neurons have extended an axon. The purity of the culture (Yam et al. 2009), typically over 90%, can be assessed by immunolabeling with the commissural neuron markers DCC, LH2 and TAG1 (Helms and Johnson, 1998). This neuronal preparation is a useful tool for in vitro studies of the cellular and molecular mechanisms of commissural axon growth and guidance during spinal cord development.

This video demonstrates a method to dissect and culture commissural neurons from E13 rat dorsal spinal cord. Dissociated commissural neurons are useful to study the cellular and molecular mechanisms of axon growth and guidance.

Dissecção e Cultura de Neurônios da Medula Espinhal comissural embrionárias

Commissural neurons have been widely used to investigate the mechanisms underlying axon guidance during embryonic spinal cord development. The cell bodies ...

Neurociência

Spinal Cord Electrophysiology II: Extracellular Suction Electrode Fabrication

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate a method to fabricate suction electrodes that are used to examine CPG activity, or fictive locomotion, in dissected rodent spinal cords. The rodent spinal cords are placed in artificial cerebrospinal fluid and the ventral roots are drawn into the suction electrode. The electrode is constructed by modifying a commercially available suction electrode. A heavier silver wire is used instead of the standard wire given by the commercially available electrode. The glass tip on the commercial electrode is replaced with a plastic tip for increased durability. We prepare hand drawn electrodes and electrodes made from specific sizes of tubing, allowing consistency and reproducibility. Data is collected using an amplifier and neurogram acquisition software. Recordings are performed on an air table within a Faraday cage to prevent mechanical and electrical interference, respectively.

A demonstration of the fabrication and use of an extracellular suction electrode used to measure electrophysiological recordings of neonatal rodent spinal cords in vitro

Spinal Cord Eletrofisiologia II: Fabricação Eletrodo de Sucção Extracelular

Development of neural circuitries and locomotion can be studied using neonatal rodent spinal cord central pattern generator (CPG) behavior. We demonstrate ...

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from individual neurons are critical for understanding how neural circuits function under natural conditions. Traditionally, these recordings have been performed 'blind', meaning the identity of the recorded cell is unknown at the start of the recording. Cellular identity can be subsequently determined via intracellular1, juxtacellular2 or loose-patch3 iontophoresis of dye, but these recordings cannot be pre-targeted to specific neurons in regions with functionally heterogeneous cell types. Fluorescent proteins can be expressed in a cell-type specific manner permitting visually-guided single-cell electrophysiology4-6. However, there are many model systems for which these genetic tools are not available. Even in genetically accessible model systems, the desired promoter may be unknown or genetically homogenous neurons may have varying projection patterns. Similarly, viral vectors have been used to label specific subgroups of projection neurons7, but use of this method is limited by toxicity and lack of trans-synaptic specificity. Thus, additional techniques that offer specific pre-visualization to record from identified single neurons in vivo are needed. Pre-visualization of the target neuron is particularly useful for challenging recording conditions, for which classical single-cell recordings are often prohibitively difficult8-11. The novel technique described in this paper uses retrograde transport of a fluorescent dye applied using tungsten needles to rapidly and selectively label a specific subset of cells within a particular brain region based on their unique axonal projections, thereby providing a visual cue to obtain targeted electrophysiological recordings from identified neurons in an intact circuit within a vertebrate CNS.
The most significant novel advancement of our method is the use of fluorescent labeling to target specific cell types in a non-genetically accessible model system. Weakly electric fish are an excellent model system for studying neural circuits in awake, behaving animals12. We utilized this technique to study sensory processing by "small cells" in the anterior exterolateral nucleus (ELa) of weakly electric mormyrid fish. "Small cells" are hypothesized to be time comparator neurons important for detecting submillisecond differences in the arrival times of presynaptic spikes13. However, anatomical features such as dense myelin, engulfing synapses, and small cell bodies have made it extremely difficult to record from these cells using traditional methods11, 14. Here we demonstrate that our novel method selectively labels these cells in 28% of preparations, allowing for reliable, robust recordings and characterization of responses to electrosensory stimulation.

Retrograde Fluorescent Labeling Allows for Targeted Extracellular Single-unit Recording from Identified Neurons In vivo

Retrograde transport of fluorescent dye labels a sub-population of neurons based on anatomical projection. Labeled axons can be visually targeted in vivo, permitting extracellular recording from identified axons. This technique facilitates recording when neurons cannot be labeled through genetic manipulation or are difficult to isolate using 'blind' in vivo approaches.

Labeling fluorescentes retrógrada Permite alvejado Extracelular Gravação Single-unidade dos neurônios identificados<em&gt; In vivo</em

The overall goal of this method is to record single-unit responses from an identified population of neurons. In vivo electrophysiological recordings from ...

Neural Circuit Recording from an Intact Cockroach Nervous System

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological effects of insecticides. This article describes the scope of cockroach sensory modalities that can be used to assay how an insect nervous system responds to environmental perturbations. Emphasis here is on the escape behavior mediated by cerci to giant fiber transmission in Periplaneta americana. This in situ preparation requires only moderate dissecting skill and electrophysiological expertise to generate reproducible recordings of neuronal activity. Peptides or other chemical reagents can then be applied directly to the nervous system in solution with the physiological saline. Insecticides could also be administered prior to dissection and the escape circuit can serve as a proxy for the excitable state of the central nervous system. In this context the assays described herein would also be useful to researchers interested in limb regeneration and the evolution of nervous system development for which P. americana is an established model organism.

This article describes the cockroach ventral nerve cord dissection and extracellular recordings from the cercal nerve and connectives. Evoked responses are generated by electrical stimulation of the cercal nerve or direct mechanical stimulation of the cerci.

Neural Circuit gravação de um Sistema Nervoso Barata Intacta

The cockroach ventral nerve cord preparation is a tractable system for neuroethology experiments, neural network modeling, and testing the physiological ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Aumento Time-dependente na resposta da rede à estimulação de culturas de pilha neuronais em matrizes do micro-elétrodo

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Preparation of Rhythmically-active In Vitro Neonatal Rodent Brainstem-spinal Cord and Thin Slice

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a signal driving the rhythmic contraction of inspiratory muscles. Rhythmic neural activity generated in the pBC and carried to other neuronal pools to drive the musculature of breathing may be studied using various approaches, including en bloc nerve recordings and transverse slice recordings. However, previously published methods have not extensively described the brainstem-spinal cord dissection process in a transparent and reproducible manner for future studies. Here, we present a comprehensive overview of a method used to reproducibly cut rhythmically-active brainstem slices containing the necessary and sufficient neuronal circuitry for generating and transmitting inspiratory drive. This work builds upon previous brainstem-spinal cord electrophysiology protocols to enhance the likelihood of reliably obtaining viable and rhythmically-active slices for recording neuronal output from the pBC, hypoglossal premotor neurons (XII pMN), and hypoglossal motor neurons (XII MN). The work presented expands upon previous published methods by providing detailed, step-by-step illustrations of the dissection, from whole rat pup, to in vitro slice containing the XII rootlets.

This protocol both visually communicates the brainstem-spinal cord preparation and clarifies the preparation of brainstem transverse slices in a comprehensive step-by-step manner. It was designed to increase reproducibility and enhance the likelihood of obtaining viable, long lasting, rhythmically-active slices for recording neural output from the respiratory regions of the brainstem.

Preparação de ritmicamente ativo em Vitro Neonatal roedores tronco cerebral-medular e fatia fina

Mammalian inspiratory rhythm is generated from a neuronal network in a region of the medulla called the preB&#246;tzinger complex (pBC), which produces a ...

Wide-field Single-photon Optical Recording in Brain Slices Using Voltage-sensitive Dye

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural circuits. Due to the fractional change in the light signal, it has been difficult to use this method as a quantitative assay. This article describes special optics and slice handling systems, which render this technique stable and reliable. The present article demonstrates the slice handling, staining, and recording of the VSD-stained hippocampal slices in detail. The system maintains physiological conditions for a long time, with good staining, and prevents mechanical movements of the slice during the recordings. Moreover, it enables staining of slices with a small amount of the dye. The optics achieve high numerical aperture at low magnification, which allows recording of the VSD signal at the maximum frame rate of 10 kHz, with 100 pixel x 100-pixel spatial resolution. Due to the high frame rate and spatial resolution, this technique allows application of the post-recording filters that provide sufficient signal-to-noise ratio to assess the changes in neural circuits.

We introduce a reproducible and stable optical recording method for brain slices using voltage-sensitive dye. The article describes voltage-sensitive dye staining and recording of optical signals using conventional hippocampal slice preparations.

Gravação ótica do único-fóton do largo-campo em fatias do cérebro usando a tintura tensão-sensível

Wide-field single photon voltage-sensitive dye (VSD) imaging of brain slice preparations is a useful tool to assess the functional connectivity in neural ...

Recording Network Activity in Spinal Nociceptive Circuits Using Microelectrode Arrays

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an increasingly detailed view of the circuits underpinning spinal pain processing. However, the effects of these connections for broader network activity in the DH remain less well understood because most studies focus on the activity of single neurons and small microcircuits. Alternatively, the use of microelectrode arrays (MEAs), which can monitor electrical activity across many cells, provides high spatial and temporal resolution of neural activity. Here, the use of MEAs with mouse spinal cord slices to study DH activity induced by chemically stimulating DH circuits with 4-aminopyridine (4-AP) is described. The resulting rhythmic activity is restricted to the superficial DH, stable over time, blocked by tetrodotoxin, and can be investigated in different slice orientations. Together, this preparation provides a platform to investigate DH circuit activity in tissue from na&#239;ve animals, animal models of chronic pain, and mice with genetically altered nociceptive function. Furthermore, MEA recordings in 4-AP-stimulated spinal cord slices can be used as a rapid screening tool to assess the capacity of novel antinociceptive compounds to disrupt activity in the spinal cord DH.

The combined use of microelectrode array technology and 4-aminopyridine-induced chemical stimulation for investigating network-level nociceptive activity in the spinal cord dorsal horn is outlined.

Atividade de rede de gravação em circuitos nociceptivos da coluna vertebral usando matrizes de microeletrodos

The roles and connectivity of specific types of neurons within the spinal cord dorsal horn (DH) are being delineated at a rapid rate to provide an ...

Electromagnetic Controlled Closed-Head Model of Mild Traumatic Brain Injury in Mice

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and understanding the mechanisms of how a TBI alters brain function. The availability of multiple animal models of TBI is necessary to model the different aspects and severities of TBI seen in people. This manuscript describes the use of a midline closed head injury (CHI) to develop a mouse model of mild TBI. The model is considered mild because it does not produce structural brain lesions based on neuroimaging or gross neuronal loss. However, a single impact creates enough pathology that cognitive impairment is measurable at least 1 month after injury. A step-by-step protocol to induce a CHI in mice using a stereotaxically guided electromagnetic impactor is defined in the paper. The benefits of the mild midline CHI model include the reproducibility of the injury-induced changes with low mortality. The model has been temporally characterized up to 1 year after the injury for neuroimaging, neurochemical, neuropathological, and behavioral changes. The model is complementary to open skull models of controlled cortical impact using the same impactor device. Thus, labs can model both mild diffuse TBI and focal moderate-to-severe TBI with the same impactor.

The protocol describes mild traumatic brain injury in a mouse model. In particular, a step-by-step protocol to induce a mild midline closed head injury and the characterization of the animal model is fully explained.

Modelo de Lesão Cerebral Traumática Leve em Camundongos com Controle Eletromagnético

Highly reproducible animal models of traumatic brain injury (TBI), with well-defined pathologies, are needed for testing therapeutic interventions and ...

Compreendendo as respostas do neurônio motor à estimulação da medula espinhal

The Ex vivo Preparation of Spinal Cord Slice for the Whole-Cell Patch-Clamp Recording in Motor Neurons During Spinal Cord Stimulation

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to execute sensorimotor behaviors, directly studying the electrical responses of motor neurons with SCS can help us understand the underlying logic of spinal motor modulation. To simultaneously record diverse stimulus characteristics and cellular responses, a patch-clamp is a good method to study the electrophysiological characteristics at a single-cell scale. However, there are still some complex difficulties in achieving this goal, including maintaining cell viability, quickly separating the spinal cord from the bony structure, and using the SCS to successfully induce action potentials. Here, we present a detailed protocol using patch-clamp to study the electrical responses of motor neurons to SCS with high spatiotemporal resolution, which can help researcher improve their skills in separating the spinal cord and maintaining the cell viability at the same time to smoothly study the electrical mechanism of SCS on motor neuron and avoid unnecessary trial and mistake.

Autor em destaque: Avançando a estimulação da medula espinhal - explorando as respostas celulares dos neurônios motores por meio da eletrofisiologia do patch-clamp 

This protocol describes a method using a patch-clamp to study the electrical responses of motor neurons to spinal cord stimulation (SCS) with high spatiotemporal resolution, which can help researchers improve their skills in separating the spinal cord and maintaining cell viability simultaneously.

A Preparação Ex Vivo do Corte da Medula Espinhal para o Registro de Patch-Clamp de Células Inteiras em Neurônios Motores Durante a Estimulação da Medula Espinhal

Spinal cord stimulation (SCS) can effectively restore locomotor function after spinal cord injury (SCI). Because the motor neurons are the final unit to ...