Este trabajo fue apoyado por SFB 604, DFG MO 1421/2-1 y Krebshilfe 107089 (HM). AS es destinatario de un Premio Joven Investigador de Tumor Infantil Foundation (Nueva York, EE.UU.).

El presente estudio se realizó de acuerdo a la Ley de Protección de Animales de la República Federal de Alemania (Tierschutzgesetz der República Federal de Alemania) y fue aprobado por la Oficina Estatal de Turingia para la Inocuidad de los Alimentos y Protección del Consumidor (Thüringer Landesamt für Lebensmittelsicherheit und Verbraucherschutz). 1. Configuración de las mediciones <ol><li> Se anestesia a los ratones por isoflurano / O 2 inhalación - para la iniciación de la anestesia 3%, para el mantenimiento de 2% de isoflurano en oxígeno al 100% (Figura 1). Confirme la anestesia suficientes probando los reflejos simples, como los reflejos del movimiento y las pruebas de sensibilidad para el dolor leve. Se recomienda el uso de una pomada en los ojos para evitar la sequedad durante la anestesia, pero no es indispensable ya que el procedimiento suele durar sólo 30 min / animal en total. En el caso de los experimentos de supervivencia, administrar oportuna ya analgésicos de acción para el control del dolor postoperatorio. </li><li> Afeitarse la piel que cubre las extremidades posteriores con una maquinilla de afeitar eléctrica y realizar la depilación con una crema de depilación disponibles en el mercado, mientras que los animales están ya en la analgesia. Con el fin de mantener el estatus libre de patógenos durante todo el procedimiento, utilice guantes asépticos y utilizar siempre instrumentos cuidadosamente limpiados con etanol al 70%. </li><li> Control de estabilidad de la temperatura del cuerpo por una placa de calefacción controlada y retroalimentación (Figura 2) sonda termo rectal. Si es necesario, utilice un paño estéril para cubrir la placa de calentamiento entre los animales con el fin de mantener un entorno experimental estéril. Además, se recomienda utilizar una placa de calentamiento, que se controla electrónicamente a través de un sensor integrado para limitar la temperatura de calentamiento a ≤ 40 ° C con el fin de evitar daños en los tejidos. <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 Exp.configuración erimental mostrando un ratón anestesiado con extremidades posteriores afeitadas. </li><li> Tome electrocardiografía (ECG) grabaciones para monitorear la frecuencia cardiaca como un parámetro vital. Instalar tres electrodos para las grabaciones de ECG de la siguiente manera: un electrodo debajo de la piel de cada extremidad anterior y un electrodo debajo de la piel en la zona del cuello. </li><li> Electrodos Place anillo utilizando gel de contacto para la resistencia óptima conductividad / transferencia. El electrodo sensor (marcado en negro) se coloca en la posición en el músculo gastrocnemio tiene su diámetro máximo. El electrodo de referencia (indicado en rojo) se coloca justo debajo de la electrodo de detección. Nota: Por favor vea &#39;tabla de materiales &quot;para detalles del equipamiento. </li><li> Marque las posiciones de medida correctas con distancias predeterminadas pero consistentes entre los extremos proximal y estimulación del nervio ciático distal y el electrodo de plomo. Propuesta experimental: A una distancia de 4 mm del electrodo de detección, la estimulación distal se llevará a cabo.En una distancia de 16 mm hasta el electrodo de detección, la estimulación proximal se llevará a cabo (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. Imagen representativa que muestra la situación experimental justo antes del comienzo de las mediciones. La flecha blanca indica la posición de la detección (negro) y de referencia (rojo) de electrodo en el músculo gastrocnemio de la extremidad posterior izquierda. La estimulación por electrodos de aguja se realizará en las posiciones definidas en relación con el electrodo sensor negro. El punto de estimulación distal (punto negro con &quot;DS&quot; en la extremidad posterior izquierda) tiene una distancia de 4 mm del electrodo de detección; el lugar de la estimulación proximal (punto negro con &quot;ps&quot;) es de 16 mm de distancia. La línea roja en la extremidad posterior derecha muestra el ca<html> Imate curso anatómico del nervio ciático. Por otra parte, las posiciones aproximadas de los músculos de las extremidades traseras relevantes se muestran como puntos de referencia. El asterisco indica que la sonda térmica rectal. 2. Medición <ol><li> Principio: Realice una serie de estimulaciones nerviosas con repetitivamente generados pulsos de onda cuadrada única de 0,1 ms de duración por electrodos de aguja monopolar desechable 28 T (tasa de repetición 200 ms; ver Figura 3A). Para el análisis de datos fuera de línea, se recomienda para adquirir simultáneamente las señales de estimulación junto con la curva de respuesta de la función neuromuscular (Figura 3B) debido a la estimulación del nervio. Promedio, una serie de respuestas máximas (&quot;respuesta representativa de la función neuromuscular&quot;) para el análisis de datos posterior. Con el fin de obtener datos fiables, ficha y después promedio por lo menos 3, las curvas de respuesta óptimos independientes por zona de estimulación y de los animales. p_upload/51181/51181fig3highres.jpg &quot;src =&quot; / files/ftp_upload/51181/51181fig3.jpg &quot;/&gt; Figura 3. Procedimiento para la adquisición de datos y análisis (presentación esquemática). Repetitivamente impulsos generados son aplicados para el nervio ciático a través de electrodos de aguja (fila superior en la Figura 3A). Al mismo tiempo, se registran varias curvas de respuesta neuromuscular correspondientes (fila inferior en la Figura 3A). Cuando promediado y ampliada, curvas de respuesta neuromuscular debido a impulsos de estimulación (fila superior de la Figura 3B) muestran las propiedades siguientes características (fila inferior de la Figura 3B): Latencia de la respuesta de la señal, así como la duración y la amplitud de la señal se indican y pueden ser obtenido para los cálculos y las estadísticas posteriores. Conducción de la señal irregular y / o grabaciones subóptimas suelen dar lugar a diversas deformaciones de señal con algo más que una positiva y una desviación negativa o deformación de la señal(Forma bimodal) con una reducción de la amplitud (Figura 3C). </li><li> Realizar la estimulación proximal con un electrodo de aguja en la posición determinada (&quot;PS&quot; en la Figura 2). <ol><li> Con el fin de lograr mejores condiciones de grabación con amplitudes máximas, visualizar las curvas de respuesta reales simultáneas para el proceso de estimulación. Al hacerlo, el experimentador es capaz de evaluar inmediatamente la forma de las curvas de respuesta, así como el tamaño de la amplitud. </li><li> Si es necesario, ligeramente y cuidadosamente manipular la posición y / o el ángulo de la aguja con respecto a la estimulación del nervio ciático. Este suave optimización de las condiciones de estimulación permite alcanzar amplitudes constantes con el valor más grande posible y una curva de respuesta de forma típica bifásica (Figura 4). </li></ol></li><li> Realice la estimulación distal con un electrodo de aguja en la posición determinada (&quot;DS&quot; en la Figura 2). </li><li> Después completion de la medida, transferir el ratón a prueba a una jaula separada hasta que se haya recuperado el conocimiento suficiente para mantener decúbito esternal. No deje un animal desatendido y en la compañía de otros animales hasta que se haya recuperado totalmente de la anestesia. Administrar oportuna ya analgésicos de acción para el control del dolor postoperatorio. La administración sistémica de fármacos antiinflamatorios no esteroideos (AINE) y los opioides se recomiendan durante 1-3 días. </li><li> Alternativamente, sacrifica el ratón sigue anestesiado en una manera rápida y cruel y evitarle más dolor para el animal, por ejemplo, la dislocación 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. Ilustración para determinar las grabaciones CMAP con amplitudes máximas. Una serie de registro completo se presenta. (Un </strong&gt;) Punto de inserción con una respuesta mínima CMAP. (B) los resultados del movimiento de la aguja de estimulación leve en CMAP grabaciones con amplitudes máximas. (C) Los cambios adicionales en la colocación de agujas producen CMAP grabaciones con diferentes amplitudes entre ellos-cerca de la máxima amplitud. (D) La estimulación de reemplazo de la aguja con CMAP grabaciones de serie de amplitudes casi máximos. Nota: disminución típica en amplitudes de CMAP puede ocurrir durante la estimulación repetitiva en el sitio de estimulación óptima 12, 13. Los asteriscos indican las grabaciones CMAP con amplitudes máximas representados para el promediado. 3. Análisis <ol><li> Extraer parámetros de conducción nerviosa basadas en el conjunto de datos representativo respuesta de la función neuromuscular mediante un paquete de software apropiado (por ejemplo AtisaPro). Nota: Por favor, maneje la determinación de datos relacionada con el tiempo, con especial cuidado, porque la determinación del punto de inflexión de la acción motriz compuesto potrencial (CMAP) de inicio y terminación puede ser difícil. Un procedimiento con reproducibilidad verificado se da en la Figura 3B, donde se utilizan las tangentes en deflexiones de señal después del inicio y antes de rescisión. </li><li> Calcular los valores de los CMAP &#39;&#39; latencia de la señal &#39;y. <ol><li> Latencia &#39;representa el retardo de tiempo entre la estimulación y la CMAP inicio, mientras que el intervalo de tiempo entre el inicio de CAMP de deflexión inicial negativa a regresar a la línea de base se llama iniciales «duración CMAP&#39;. Utilice la diferencia entre latencias distales y proximales para calcular la velocidad de conducción junto con la distancia entre los sitios de estimulación distal y proximal. velocidad de conducción = latencia / distancia entre los sitios de estimulación </li><li> CMAP &#39;(compuesto potencial de acción motor) amplitud representa la magnitud entre el máximo punto de inflexión positivo y negativo de la señal CMAP (dado en mV). «Punto de inflexión positiva&quot; CMAP = valor - po de respuesta negativa de valor &quot;int &#39; </li></ol></li></ol>

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Los autores no tienen nada que revelar.

El protocolo descrito proporciona un método sencillo y fiable para determinar las propiedades de conducción del nervio ciático en ratones anestesiados y sin la necesidad de exponer el nervio de interés. Sin embargo, este procedimiento experimental provoca lesión tisular por punción con aguja. Por tanto, es una opción razonable para sacrificar a los animales después de terminar las grabaciones. Sin embargo, en comparación con otros procedimientos más invasivos, que requieren la exposición del nervio antes de g...

Las medidas electrofisiológicas son una herramienta indispensable para la investigación de la integridad funcional de los nervios periféricos, tanto en entornos clínicos y de laboratorio. En los seres humanos, un gran número de trastornos neuromusculares y neuropatías diagnóstico se basan en mediciones electrofisiológicas. Mediante la medición de propiedades nerviosas como la velocidad de conducción o potenciales amplitudes de la señal, es posible caracterizar el origen áspera de las enfermedades nerviosas periféricas. La velocidad de conducción nerviosa es altamente dependiente de la rápida propagación de la señal habilitada por la mielinización. Por lo tanto, los procesos desmielinizantes generalmente muestran una disminución de la velocidad de conducción 4. El potencial de acción compuesto de motor (CMAP) - se correlaciona con el número de axones funcionales - es un indicador de daño axonal cuando redujo significativamente 5. Por lo tanto, por medio de métodos electrofisiológicos la etiología de daño nervioso periféricopuede ser discriminado, tal como, por neuropatías hereditarias 6,7, neuropatía diabética 8,9, polineuropatías desmielinizantes inflamatorias crónicas (PDIC) 10, o neuropatías metabólicas 11. Normalmente, en la aplicación en seres humanos se prefieren las grabaciones no invasivas en el nervio sural o cubital. En los ratones, es sencillo para analizar propiedades nerviosas de los nervios ciáticos, el nervio más grande del sistema nervioso periférico (SNP) que contiene tanto grandes - y axones de pequeño calibre del sistema motriz y sensorial. El procedimiento como se ha demostrado aquí es un método rápido, sencillo y fiable para medir todos los valores estándar pertinentes para electrofisiología en los nervios periféricos en el ratón intacto. Al tomar grabaciones de un organismo conservado, se garantizan las condiciones fisiológicas del medio ambiente nervio.

<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

Los estudios electrofisiológicos permiten una clasificación racional de diversas enfermedades neuromusculares y son de ayuda, junto con técnicas de neuropatológicos, en el entendimiento de la fisiopatología subyacente 1. Aquí se describe un método para realizar estudios electrofisiológicos en nervios ciáticos de ratones in vivo. Los animales se anestesiaron con isoflurano con el fin de garantizar la analgesia para los ratones probados y sin molestias entorno de trabajo durante las mediciones que se llevan alrededor de 30 min / animal. Una temperatura corporal constante de 37 ° C es mantenida por una placa de calentamiento y mide de forma continua por una sonda termo rectal 2. Además, un electrocardiograma (ECG) se registra habitualmente durante las mediciones con el fin de vigilar continuamente el estado fisiológico de los animales investigados. Los registros electrofisiológicos se realizan en el nervio ciático, el nervio más grande de the sistema nervioso periférico (SNP), el suministro de la cierva extremidad ratón con tractos de fibras, tanto motoras y sensoriales. En nuestro protocolo, nervios ciáticos permanecer in situ y por lo tanto no tienen que ser extraídos o expuestos, lo que permite mediciones sin ningún irritaciones nerviosas adversos junto con grabaciones reales. El uso de electrodos de aguja apropiadas 3 que realizamos tanto proximal y estimulaciones nerviosas distales, el registro de los potenciales de transmisión con electrodos sensores en músculos gemelos. Después del procesamiento de datos, fiable y valores altamente consistentes para la velocidad de conducción nerviosa (NCV) y el compuesto potencial de acción motor (CMAP), los parámetros clave para la cuantificación de funcionamiento de los nervios periféricos bruto, se puede lograr.

<html> Hemos llevado a cabo una serie de in vivo mediciones electrofisiológicas sobre los nervios ciáticos de 12 ratones en total para este estudio: 6 animales de cada sexo. Las mediciones se realizaron con el protocolo presentado y se entregan los siguientes resultados: Ambos ratones machos y hembras muestran una velocidad de conducción del nervio ciático media de aproximadamente 20 m / seg (Figura 5). Esto es consistente con otras mediciones en la literatura. A...

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

Las mediciones de las propiedades de conducción nerviosa in vivo ejemplo de una poderosa herramienta para caracterizar diversos modelos animales de enfermedades neuromusculares. A continuación, presentamos un protocolo sencillo y fiable por lo cual el análisis electrofisiológico en los nervios ciáticos de ratones anestesiados se puede realizar.

En Vivo electrofisiológicos mediciones sobre los nervios ciáticos de ratones

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

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Research

JoVE Journal

Neuroscience

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

32.8K vistas.  Fritz Lipmann Institute.  Las mediciones de las propiedades de conducción nerviosa in vivo ejemplo de una poderosa herramienta para caracterizar diversos modelos animales de enfermedades neuromusculares. A continuación, presentamos un protocolo sencillo y fiable por lo cual el análisis electrofisiológico en los nervios ciáticos de ratones anestesiados se puede realizar. 

Video: 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.

Las mediciones electrofisiológicas de un sistema olfativo Polilla

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

Investigación

Neurociencias

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.

La transfección de células de ratón del ganglio de la retina por In vivo La electroporación

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.

Las mediciones electrofisiológicas y Análisis de la nocicepción en los bebés humanos

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

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.

In vivo de dos fotones de imágenes de dependientes Experience-Los cambios moleculares en las neuronas corticales

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.

En Vivo Transferencia de genes a células de Schwann en el nervio ciático de roedores mediante electroporación

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.

Análisis de las células inmunes en solo de los nervios ciático y de ganglio de raíz Dorsal de un ratón mediante citometría de flujo

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.

En Vivo Medición electrofisiológica de potencial de acción compuesto del músculo de las extremidades anteriores en modelos de ratón de la degeneración de la motoneurona

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.

En proyección de imagen del calcio Vivo de las células de pelo de la línea Lateral en el pez cebra larvas

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 ...

In Vitro Wedge Slice Preparation for Mimicking In Vivo Neuronal Circuit Connectivity

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively thin to properly visualize and access neurons for patch-clamping or imaging, and in vitro examination of brain circuitry is limited to only what is physically present in the acute slice. To maintain the benefits of in vitro slice experimentation while preserving a larger portion of presynaptic nuclei, we developed a novel slice preparation. This &#8220;wedge slice&#8221; was designed for patch-clamp electrophysiology recordings to characterize the diverse monaural, sound-driven inputs to medial olivocochlear (MOC) neurons in the brainstem. These neurons receive their primary afferent excitatory and inhibitory inputs from neurons activated by stimuli in the contralateral ear and corresponding cochlear nucleus (CN). An asymmetrical brain slice was designed which is thickest in the rostro-caudal domain at the lateral edge of one hemisphere and then thins towards the lateral edge of the opposite hemisphere. This slice contains, on the thick side, the auditory nerve root conveying information about auditory stimuli to the brain, the intrinsic CN circuitry, and both the disynaptic excitatory and trisynaptic inhibitory afferent pathways that converge on contralateral MOC neurons. Recording is performed from MOC neurons on the thin side of the slice, where they are visualized using DIC optics for typical patch-clamp experiments. Direct stimulation of the auditory nerve is performed as it enters the auditory brainstem, allowing for intrinsic CN circuit activity and synaptic plasticity to occur at synapses upstream of MOC neurons. With this technique, one can mimic in vivo circuit activation as closely as possible within the slice. This wedge slice preparation is applicable to other brain circuits where circuit analyses would benefit from preservation of upstream connectivity and long-range inputs, in combination with the technical advantages of in vitro slice physiology.

Integration of diverse synaptic inputs to neurons is best measured in a preparation that preserves all pre-synaptic nuclei for natural timing and circuit plasticity, but brain slices typically sever many connections. We developed a modified brain slice to mimic in vivo circuit activity while maintaining in vitro experimentation capability.

In Vitro Wedge Slice Preparación para imitar la conectividad del circuito neuronal In Vivo

In vitro slice electrophysiology techniques measure single-cell activity with precise electrical and temporal resolution. Brain slices must be relatively ...