Los autores desean agradecer a los premios de sueldos, subsidios equipos y fondos de operación del Fonds de la Recherche en Santé Québec (FRSQ), los Institutos Canadienses de Investigación en Salud (CIHR), las Ciencias Naturales e Ingeniería de Investigación de Canadá (NSERC), y la Concordia University Research Chairs Program (CRUC), así como la retroalimentación reflexiva sobre este manuscrito de la Dra. Jane Stewart.

1. ALOJAMIENTO DE ANIMALES <ol><li> Jaula: Con el fin de registrar la actividad de rodaje de la rueda de un roedor individual, cada jaula debe albergar un roedor único, de rodadura de la rueda. Debido a que las ruedas para correr puede ser considerado una forma de enriquecimiento, todos los roedores en cualquier estudio deben tener el mismo acceso a una rueda para correr. </li><li> Cambios de ropa de cama: manejo de animales, así como cambios en las jaulas o ropa de cama todos podemos tener no photic efectos sobre los ritmos circadianos 21-23, por lo que, jaulas con piso de malla son ideales ya que minimizar el contacto con el animal. A pesar de la disponibilidad de este tipo de bandeja de sistema, cambios de ropa de cama se deben evitar durante las fases críticas de un experimento. Las alternativas incluyen el uso de las camas de larga duración, lo que permitiría una jaula más cambios frecuentes, o cambio de jaulas en un horario pseudo-aleatoria. </li><li> Cajas de aislamiento: Las jaulas deben ser guardados en cajas de aislamiento que son de sonido atenuado, light-controlled y bien ventilado. Dependiendo del tamaño y la configuración de las cajas de aislamiento, el número de jaulas dentro de cada cuadro típicamente estará en el intervalo de 1-8. Al albergar varias jaulas en una caja de aislamiento individual, se debe tener en cuenta que los olores y sonidos que vienen de otros animales pueden tener efectos de confusión sobre el comportamiento circadiano de los animales individuales. Para evitar estos problemas, se debería intentar alojar una jaula por caja de aislamiento. </li><li> Ventilación: el flujo de aire adecuado es imprescindible para hacer las cajas de un entorno familiar cómodo para los roedores. El ventilador en cada cuadro debe ser encapuchado, a fin de evitar que la luz desde fuera del área de alcanzar el interior. Además, los aficionados típicamente retirar el aire de la caja y golpe en la habitación. Pequeña luz-apretado rejillas de ventilación permiten que el aire entre las cajas de aislamiento de varios puntos, y ayudar a evitar brisas incómodas. Con el fin de comprobar que la ventilación es adecuada, la temperatura dentro de la caja de aislamiento (cuandocerrado durante varias horas, con las luces encendidas) debe ser prácticamente idéntica a la temperatura en la habitación donde se encuentra. </li><li> Iluminación: intensidad de la luz ambiental debería ser el mismo en todas las jaulas. Organice una sola luz en una ubicación similar por encima de cada jaula, y utilizar siempre la misma marca / tipo de bombilla. Utilice iluminación de intensidad moderada (100-300 lux) a nivel de la jaula. Evitar los niveles de iluminación excesivamente altas, que son más propensos a producir cambios directos en el comportamiento atribuibles a la luz en lugar de el sistema circadiano, per se (por ejemplo, enmascaramiento). </li><li> La oscuridad / luz roja Dim: Si es necesario manipular o tratar a los animales en la oscuridad (por ejemplo, en constante oscuridad o la noche), gafas de visión nocturna se debe utilizar. Alternativamente, debido a que el sistema circadiano es relativamente insensible al rojo de onda, tenue iluminación roja puede utilizarse. La luz roja específico que utilice debe ser probado para asegurar que no se alter rodaje rueda de actividad (enmascaramiento, por ejemplo) o ajustar el reloj circadiano (por ejemplo, producir un cambio de fase). </li></ol> 2. Recopilación de datos (Ver Figura 1 - Configuración VitalView Hardware) <ol><li> Ejecución de ruedas: El diámetro y la ergonomía de la rueda de funcionamiento cambiará la cantidad de uso 24. Por lo tanto, utilizar ruedas más pequeñas y ligeras para los ratones, y las ruedas más grandes más pesadas para las ratas. Al lavarse y volver a instalar las ruedas, asegúrese de que las ruedas puedan girar sin obstáculos, no se &quot;tambalea&quot;, y que la grabación de micro-interruptores son activados por cada vuelta de la rueda. </li><li> Micro-interruptores: cada revolución de la rueda de funcionamiento debe activar un campo magnético o mecánico micro-interruptor. La información de la micro-interruptor se transmite a través de un solo canal y registrada por un ordenador que puede bin los datos en el tiempo (por ejemplo, cada 2, 5, 6, o min 10). </li><li> Hardware: Nuestra Runnirueda ng-grabaciones se realizan con VitalView, una plataforma de hardware y software desarrollado por Mini Mitter ( <a href="http://www.minimitter.com/vitalview_software.cfm" target="_blank">http://www.minimitter.com/vitalview_software.cfm</a> ). Sin embargo, hay otras plataformas de grabación tales como ClockLab, desarrollado por Actimetrics ( <a href="http://www.actimetrics.com/ClockLab/" target="_blank">http://www.actimetrics.com/ClockLab/</a> ). Ambas plataformas reunir datos de muchas fuentes de un solo canal (por ejemplo, un único micro-interruptor activado por una sola rueda en marcha) en un archivo único equipo. Los datos de canales individuales se pueden representar gráficamente y se analizaron por separado en una fecha posterior. </li></ol> 3. Grabaciones de datos <ol><li> Archivos: Las plataformas de software mencionados anteriormente se pueden utilizar para separar los canales individuales de manera que se crean los archivos individuales para cada registro de rodaje de la rueda. Estos datos se visualizan mejor y se representan con especial designed programas como ActiView (Minimitter, Bend, OR), Circadia o Clocklab (Actimetrics, Wilmette, IL) que todos pueden producir periodograms y actograms. Sin embargo, un solo canal archivos también se pueden abrir y analizados mediante programas generales de hoja de cálculo como Excel (Microsoft, Redmond, WA). </li><li> Cálculo del tiempo circadiano (CT): CT 12 es, por definición, el inicio de la actividad de rodaje de la rueda en roedores nocturnos. Paralelamente a la jornada de 24 horas, por convención, un día circadiano se divide en 24 horas circadiano. En consecuencia, si el circadiano de funcionamiento libre período es 24 hr 30 min, medido por el reloj de pared, CT 0 se producirá aproximadamente 12 hr 15 min después de CT12. </li></ol>

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U.S.A. 101, 5339-5346 (2004).</li><li>Guilding, C., Piggins, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Challenging+the+omnipotence+of+the+suprachiasmatic+timekeeper:+are+circadian+oscillators+present+throughout+the+mammalian+brain?.">Challenging the omnipotence of the suprachiasmatic timekeeper: are circadian oscillators present throughout the mammalian brain?.</a> Eur. J. Neurosci. 25, 3195-3216 (2007).</li><li>Boulos, Z., Terman, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Food+availability+and+daily+biological+rhythms.">Food availability and daily biological rhythms.</a> Neurosci. Biobehav. Rev. 4, 119-131 (1980).</li><li>Boulos, Z., Rosenwasser, A. 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Seguimiento de los ritmos de actividad diaria con ruedas para correr es el método más utilizado y fiable para evaluar la salida del reloj circadiano maestro en roedores nocturnos. Rueda de marcha la actividad, sin embargo, es sólo uno de los muchos aspectos del comportamiento y la fisiología que pueden ser monitoreados continuamente. Aunque la gran mayoría de la actividad de rodaje de la rueda se produce durante la noche, más del 30% de la vigilia total se produce durante el día 25,26. Otras variables ...

<table border="1"&gt;<tbody&gt;<tr&gt;<td<strong&gt; Nombre del reactivo</strong</td&gt;<td<strong&gt; Empresa</strong</td&gt;<td<strong&gt; Número de catálogo</strong</td&gt;<td<strong&gt; Comentarios (opcional)</strong</td&gt;</tr&gt;<tr&gt;<td&gt; VitalView Cartas y Software</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 855-0030-00</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; DP24 Dataport</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 840-0024-00</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; QA4-Módulo</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 130-0050-00</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; Interruptor magnético</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 130-0015-00</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; Montaje C-50 Cable</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 060-0045-10</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; Rat funcionamiento conjunto de la rueda</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 640-0700-00</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; Cage y bandeja de soporte</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 640-0400-00</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; Utilizable cortar jaula</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 664-2154-00</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; Cuadrícula piso de la jaula</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 676-2154-00</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; Residuos bandeja</td&gt;<td&gt; Mini Mitter</td&gt;<td&gt; # 684-2154-00</td&gt;<td&gt; (Bend, Oregón, EE.UU.)</td&gt;</tr&gt;<tr&gt;<td&gt; Caja de lámpara</td&gt;<td&gt; Microlitos Científico</td&gt;<td&gt; # R-101</td&gt;<td&gt; (Toronto, ON, Canadá)</td&gt;</tr&gt;<tr&gt;<td&gt; 4W Las lámparas fluorescentes</td&gt;<td&gt; Microlitos Científico</td&gt;<td&gt; # F4T5/CW</td&gt;<td&gt; (Toronto, ON, Canadá)</td&gt;</tr&gt;<tr&gt;<td&gt; Aislamiento de cámaras</td&gt;<td&gt; Custom construido</td&gt;<td</td&gt;<td&gt; 28 &quot;H x 20&quot; W x 28 &quot;D ½&quot; Negro melamina.</td&gt;</tr&gt;</tbody&gt;</table

recording and analysis of circadian rhythms in running-wheel activity in rodents

Cuando los roedores tienen libre acceso a una rueda de funcionamiento en su jaula, el uso voluntario de esta rueda dependerá del tiempo de 1-5 días. Roedores nocturnos, incluyendo ratas, hámsteres y ratones, son activos durante la noche y relativamente inactivos durante el día. Muchas otras medidas fisiológicas y de comportamiento también exhiben ritmos diarios, pero en roedores, corriendo ruedas actividad sirve como una medida particularmente fiable y conveniente de la salida del reloj circadiano maestro, el núcleo supraquiasmático (SCN) del hipotálamo. En general, a través de un proceso llamado arrastre, el patrón de actividad diario de rodaje de ruedas naturalmente se alinearán con el medio ambiente de luz-oscuridad ciclo (ciclo LD, por ejemplo, 12 horas de luz: 12 hr-oscuro). Ritmos circadianos Sin embargo se endógenamente generado patrones de comportamiento que exhiben un período de ~ 24 h, y persisten en la oscuridad constante. Por lo tanto, en ausencia de un ciclo LD, el registro y el análisis de la actividad de rodaje rueda puedeser usado para determinar la subjetiva de tiempo de días. Debido a que estos ritmos son dirigidos por el reloj circadiano del subjetiva de tiempo de día se conoce como el tiempo circadiano (CT). En contraste, cuando un ciclo LD está presente, el tiempo del día que se determina por el medio ambiente ciclo LD se denomina tiempo de zeitgeber (ZT). A pesar de los ritmos circadianos en la actividad de rodaje de ruedas generalmente están vinculados al reloj del SCN 6-8, osciladores circadianos en muchas otras regiones del cerebro y del cuerpo 9-14 también podrían estar involucrados en la regulación de los ritmos de actividad diaria. Por ejemplo, ritmos diarios en alimentos anticipatoria actividad no requieren el SCN 15,16 y en su lugar, se correlacionan con los cambios en la actividad de los osciladores extra-SCN 17-20. Así, ejecutando ruedas grabaciones actividad puede proporcionar importante información de comportamiento no sólo sobre la salida del reloj maestro SCN, sino también de la actividad de los osciladores extra-SCN. A continuación se descrOIE equipo y los métodos utilizados para registrar, analizar y mostrar los ritmos circadianos de actividad locomotora en ratones de laboratorio.

<ol><li> Los programas de ordenador: programas informáticos especializados se utilizan normalmente en la generación de actograms y el cálculo del período circadiano. Estos programas incluyen, pero no se limitan a, ActiView (Minimitter, Bend, OR) y Circadia. </li><li> Actograms: Actograms proporcionar una ilustración gráfica de los patrones diarios de actividad de rodaje de la rueda. Hay un solo trazado (eje x = 24 hr) y doble traza-(eje x = 48 hr) actograms. Ambos métodos trama secuencial días desde arr...

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Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

Los ritmos circadianos en voluntaria rueda de funcionamiento en mamíferos están fuertemente acoplados a las oscilaciones moleculares de un reloj maestro en el cerebro. Como tales, estos ritmos diarios en el comportamiento puede ser usado para estudiar la influencia de los factores genéticos, farmacológicos, y ambiental sobre el funcionamiento de este reloj circadiano.

Registro y análisis de los ritmos circadianos en funcionamiento las ruedas Actividad en roedores

When rodents have free access to a running wheel in their home cage, voluntary use of this wheel will depend on the time of day1-5. Nocturnal rodents, ...

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Neuroscience

21.2K vistas.  McGill University. Los ritmos circadianos en voluntaria rueda de funcionamiento en mamíferos están fuertemente acoplados a las oscilaciones moleculares de un reloj maestro en el cerebro. Como tales, estos ritmos diarios en el comportamiento puede ser usado para estudiar la influencia de los factores genéticos, farmacológicos, y ambiental sobre el funcionamiento de este reloj circadiano.

Video: Recording and Analysis of Circadian Rhythms in Running-wheel Activity in Rodents

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

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

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

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

Ensayo Actividad locomotora para el Estudio de los ritmos circadianos y del sueño en los parámetros Drosophila</em

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

Investigación

Neurociencias

Measuring Circadian and Acute Light Responses in Mice using Wheel Running Activity

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2. They are responsible for timing our sleep/wake cycles and hormone secretion. Since this timing is not precisely 24-hours, it is synchronized to the solar day by light input. This is accomplished via photic input from the retina to the suprachiasmatic nucleus (SCN) which serves as the master pacemaker synchronizing peripheral clocks in other regions of the brain and peripheral tissues to the environmental light dark cycle3-7. The alignment of rhythms to this environmental light dark cycle organizes particular physiological events to the correct temporal niche, which is crucial for survival8. For example, mice sleep during the day and are active at night. This ability to consolidate activity to either the light or dark portion of the day is referred to as circadian photoentrainment and requires light input to the circadian clock9. Activity of mice at night is robust particularly in the presence of a running wheel. Measuring this behavior is a minimally invasive method that can be used to evaluate the functionality of the circadian system as well as light input to this system. Methods that will covered here are used to examine the circadian clock, light input to this system, as well as the direct influence of light on wheel running behavior.

This article will review methods that can be used to determine circadian function and light responsiveness in mice.

Medición de las respuestas de luz circadianos y aguda en ratones con actividad rueda para correr

Circadian rhythms are physiological functions that cycle over a period of approximately 24 hours (circadian- circa: approximate and diem: day)1, 2.  They ...

Large-scale Recording of Neurons by Movable Silicon Probes in Behaving Rodents

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of neurons and circuits requires methods with the spatial selectivity and temporal resolution appropriate for mechanistic analysis of neural ensembles in the behaving animal, i.e. recording of representatively large samples of isolated single neurons. Ensemble monitoring of neuronal activity has progressed remarkably in the past decade in both small and large-brained animals, including human subjects1-11. Multiple-site recording with silicon-based devices are particularly effective because of their scalability, small volume and geometric design.
Here, we describe methods for recording multiple single neurons and local field potential in behaving rodents, using commercially available micro-machined silicon probes with custom-made accessory components. There are two basic options for interfacing silicon probes to preamplifiers: printed circuit boards and flexible cables. Probe supplying companies (<a href="http://www.neuronexustech.com/" >http://www.neuronexustech.com/</a>; <a href="http://www.sbmicrosystems.com/">http://www.sbmicrosystems.com/</a>; <a href="http://www.acreo.se/">http://www.acreo.se/</a>) usually provide the bonding service and deliver probes bonded to printed circuit boards or flexible cables. Here, we describe the implantation of a 4-shank, 32-site probe attached to flexible polyimide cable, and mounted on a movable microdrive. Each step of the probe preparation, microdrive construction and surgery is illustrated so that the end user can easily replicate the process.

We describe methods for large-scale recording of multiple single units and local field potential in behaving rodents with silicon probes. Drive fabrication, probe attachment to the drive and probe implantation processes are illustrated in sufficient details for easy replication.

A gran escala de grabación de las neuronas por medio de sondas de silicio móviles en comportarse roedores

A major challenge in neuroscience is linking behavior to the collective activity of neural assemblies. Understanding of input-output relationships of ...

A Fully Automated and Highly Versatile System for Testing Multi-cognitive Functions and Recording Neuronal Activities in Rodents

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be investigated in a single task sequence. The unique feature of this system is a custom-made, acoustically transparent chamber that eliminates many of the issues associated with auditory cue control in most commercially available chambers. The ease with which operant devices can be added or replaced makes this system quite versatile, allowing for the implementation of a variety of auditory, visual, and olfactory behavioral tasks. Automation of the system allows fine temporal (10 ms) control and precise time-stamping of each event in a predesigned behavioral sequence. When combined with a multi-channel electrophysiology recording system, multiple cognitive brain functions, such as motivation, attention, decision-making, patience, and rewards, can be examined sequentially or independently.

In this report, we present a fully automated and highly versatile system capable of simultaneously testing multi-cognitive behaviors and recording neuronal activities for rodents.

Un sistema totalmente automatizado y altamente versátil para las pruebas múltiples funciones cognitivas y el registro de las actividades neuronales en los roedores

We have developed a fully automated system for operant behavior testing and neuronal activity recording by which multiple cognitive brain functions can be ...

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.

Marcaje fluorescente retrógrada Permite dirigida extracelular sola unidad de grabación de neuronas identificadas<em&gt; En 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 ...

Multi-unit Recording Methods to Characterize Neural Activity in the Locust (Schistocerca Americana) Olfactory Circuits

Detection and interpretation of olfactory cues are critical for the survival of many organisms. Remarkably, species across phyla have strikingly similar olfactory systems suggesting that the biological approach to chemical sensing has been optimized over evolutionary time1. In the insect olfactory system, odorants are transduced by olfactory receptor neurons (ORN) in the antenna, which convert chemical stimuli into trains of action potentials. Sensory input from the ORNs is then relayed to the antennal lobe (AL; a structure analogous to the vertebrate olfactory bulb). In the AL, neural representations for odors take the form of spatiotemporal firing patterns distributed across ensembles of principal neurons (PNs; also referred to as projection neurons)2,3. The AL output is subsequently processed by Kenyon cells (KCs) in the downstream mushroom body (MB), a structure associated with olfactory memory and learning4,5. Here, we present electrophysiological recording techniques to monitor odor-evoked neural responses in these olfactory circuits.
First, we present a single sensillum recording method to study odor-evoked responses at the level of populations of ORNs6,7. We discuss the use of saline filled sharpened glass pipettes as electrodes to extracellularly monitor ORN responses. Next, we present a method to extracellularly monitor PN responses using a commercial 16-channel electrode3. A similar approach using a custom-made 8-channel twisted wire tetrode is demonstrated for Kenyon cell recordings8. We provide details of our experimental setup and present representative recording traces for each of these techniques.

Multi-unit Recording Methods to Characterize Neural Activity in the Locust Schistocerca Americana Olfactory Circuits

We demonstrate variations of the extracellular multi-unit recording technique to characterize odor-evoked responses in the first three stages of the invertebrate olfactory pathway. These techniques can easily be adapted to examine ensemble activity in other neural systems as well.

Varias unidades Métodos de grabación para caracterizar la actividad neuronal en el Locust (<em&gt; Schistocerca americana</em&gt;) Circuitos olfativas

Detection and interpretation of olfactory cues are critical for the survival  of many organisms. Remarkably, species across phyla have strikingly similar ...

Deep Brain Stimulation with Simultaneous fMRI in Rodents

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for using blood oxygen level dependent (BOLD) functional magnetic resonance imaging (fMRI) to image rodents with simultaneous DBS. DBS fMRI presents a number of technical challenges, including accuracy of electrode implantation, MR artifacts created by the electrode, choice of anesthesia and paralytic to minimize any neuronal effects while simultaneously eliminating animal motion, and maintenance of physiological parameters, deviation from which can confound the BOLD signal.&#160;Our laboratory has developed a set of procedures that are capable of overcoming most of these possible issues. For electrical stimulation, a homemade tungsten bipolar microelectrode is used, inserted stereotactically at the stimulation site in the anesthetized subject. In preparation for imaging, rodents are fixed on a plastic headpiece and transferred to the magnet bore. For sedation and paralysis during scanning, a cocktail of dexmedetomidine and pancuronium is continuously infused, along with a minimal dose of isoflurane; this preparation minimizes the BOLD ceiling effect of volatile anesthetics. In this example experiment, stimulation of the subthalamic nucleus (STN) produces BOLD responses which are observed primarily in ipsilateral cortical regions, centered in motor cortex. Simultaneous DBS and fMRI allows the unambiguous modulation of neural circuits dependent on stimulation location and stimulation parameters, and permits observation of neuronal modulations free of regional bias. This technique may be used to explore the downstream effects of modulating neural circuitry at nearly any brain region, with implications for both experimental and clinical DBS.

This protocol describes a standard method for simultaneous functional magnetic resonance imaging and deep brain stimulation in the rodent. The combined use of these experimental tools allows for the exploration of global downstream activity in response to electrical stimulation at virtually any brain target.

La estimulación cerebral profunda con simultánea fMRI en roedores

In order to visualize the global and downstream neuronal responses to deep brain stimulation (DBS) at various targets, we have developed a protocol for ...

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the different organ systems from the periphery towards the central nervous system. As such, the increased activity or &#39;sensitization&#39; of mesenteric afferent nerves has been allocated an important role in the pathophysiology of visceral hypersensitivity and abdominal pain syndromes.
Mesenteric afferent nerve activity can be measured in vitro in an isolated intestinal segment that is mounted in a purpose-built organ bath and from which the splanchnic nerve is isolated, allowing researchers to directly assess nerve activity adjacent to the gastrointestinal segment. Activity can be recorded at baseline in standardized conditions, during distension of the segment or following the addition of pharmacological compounds delivered intraluminally or serosally. This technique allows the researcher to easily study the effect of drugs targeting the peripheral nervous system in control specimens; besides, it provides crucial information on how neuronal activity is altered during disease. It should be noted however that measuring afferent neuronal firing activity only constitutes one relay station in the complex neuronal signaling cascade, and researchers should bear in mind not to overlook neuronal activity at other levels (e.g., dorsal root ganglia, spinal cord or central nervous system) in order to fully elucidate the complex neuronal physiology in health and disease.
Commonly used applications include the study of neuronal activity in response to the administration of lipopolysaccharide, and the study of afferent nerve activity in animal models of irritable bowel syndrome. In a more translational approach, the isolated mouse intestinal segment can be exposed to colonic supernatants from IBS patients. Furthermore, a modification of this technique has been recently shown to be applicable in human colonic specimens.

In Vitro Recording of Mesenteric Afferent Nerve Activity in Mouse Jejunal and Colonic Segments

Mesenteric afferent nerves convey information from the gastrointestinal tract towards the brain regarding normal homeostasis as well as pathophysiology. Gastrointestinal afferent nerve activity can be assessed by mounting isolated intestinal segments with attached afferent nerves into an organ bath, isolating the nerve, and assessing basal as well as stimulated activity.

In Vitro de grabación de la actividad nerviosa aferente mesentérica en ratón yeyunal y segmentos del colon

Afferent nerves not only convey information concerning normal physiology, but also signal disturbed homeostasis and pathophysiological processes of the ...

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic tectum, is a popular model to study how neural circuits self-assemble. The ability to carry out whole cell patch clamp recordings from tectal neurons and to record RGC-evoked responses, either in vivo or using a whole brain preparation, has generated a large body of high-resolution data about the mechanisms underlying normal, and abnormal, circuit formation and function. Here we describe how to perform the in vivo preparation, the original whole brain preparation, and a more recently developed horizontal brain slice preparation for obtaining whole cell patch clamp recordings from tectal neurons. Each preparation has unique experimental advantages. The in vivo preparation enables the recording of the direct response of tectal neurons to visual stimuli projected onto the eye. The whole brain preparation allows for the RGC axons to be activated in a highly controlled manner, and the horizontal brain slice preparation allows recording from across all layers of the tectum.

Preparations and Protocols for Whole Cell Patch Clamp Recording of Xenopus laevis Tectal Neurons

In this paper, we discuss three brain preparations used for whole cell patch clamp recording to study the retinotectal circuit of Xenopus laevis tadpoles. Each preparation, with its own specific advantages, contributes to the experimental tractability of the Xenopus tadpole as a model to study neural circuit function.

Preparaciones y protocolos para todo celular parche abrazadera de neuronas Tectal de Xenopus laevis

The Xenopus tadpole retinotectal circuit, comprised of the retinal ganglion cells (RGCs) in the eye which form synapses directly onto neurons in the optic ...

The Power of Interstimulus Interval for the Assessment of Temporal Processing in Rodents

Temporal processing deficits have been implicated as a potential elemental dimension of higher-level cognitive processes, commonly observed in neurocognitive disorders. Despite the popularization of prepulse inhibition (PPI) in recent years, many current protocols promote using a percent of control measure, thereby precluding the assessment of temporal processing. The present study used cross-modal PPI and gap prepulse inhibition (gap-PPI) to demonstrate the benefits of employing a range of interstimulus intervals (ISIs) to delineate effects of sensory modality, psychostimulant exposure, and age. Assessment of sensory modality, psychostimulant exposure, and age reveals the utility of an approach varying the interstimulus interval (ISI) to establish the shape of the ISI function, including increases (sharper curve inflections) or decreases (flattening of the response amplitude curve) in startle amplitude. Additionally, shifts in peak response inhibition, suggestive of a differential sensitivity to the manipulation of ISI, are often revealed. Thus, the systematic manipulation of ISI affords a critical opportunity to evaluate temporal processing, which may reveal the underlying neural mechanisms involved in neurocognitive disorders.

Temporal processing, a preattentive process, may underlie deficits in higher-level cognitive processes, including attention, commonly observed in neurocognitive disorders. Using prepulse inhibition as an exemplar paradigm, we present a protocol for manipulating interstimulus interval (ISI) to establish the shape of the ISI function to provide an assessment of temporal processing.

Methods Article

Registro y análisis de los ritmos circadianos en funcionamiento las ruedas Actividad en roedores