Ámbar Southwell La instalación del equipo y entrenó a los autores en su uso. El financiamiento fue proporcionado por una beca del NINDS a Paul Patterson.

1. Este protocolo se basa en las de Southwell et. al. (2009) y Carter et. al. (2001). <ol><li> El aparato consiste en un haz de rayos metros con una superficie plana de 12 mm o 6 mm de ancho) en reposo 50 cm por encima de la mesa en dos polos. Un cuadro negro se coloca en el extremo de la viga en el punto de llegada. (Madera contrachapada y otros tipos de madera para las vigas, postes, y la caja se puede encontrar en cualquier ferretería). El material de nidificación de las jaulas en casa se coloca en la caja de negro para atraer el ratón al punto final. Una lámpara (con una bombilla de 60 vatios) se utiliza para brillar la luz por encima del punto de inicio y sirve como un estímulo aversivo. El tiempo para cruzar el centro de 80 cm se mide por dos detectores de movimiento (fabricado por Visopia): una a 0 cm que se inicia un temporizador y una de 80 cm que se detiene el cronómetro. Una hamaca de nylon (que se puede obtener de una tienda de telas local) se extiende por debajo de la viga, ~ 7.5 cm por encima de la mesa, para amortiguar cualquier caída. Una cámara de video se encuentra en un trípode para grabar el espectáculo. El aparato utilizado en esta demostración fue construido en el sitio. </li><li> Una cohorte de al menos 10 ratones para cada condición, se recomienda para esta prueba. Los ratones se encuentran en grupos, siempre que sea posible, en un 12 horas de luz (7am-7pm) / oscuridad ciclo y se aclimataron a las jaulas en las instalaciones de comportamiento por lo menos 2 semanas antes del comienzo de la prueba. Aproximadamente 10 minutos antes del entrenamiento / prueba, los ratones pueden ser transportados a la sala que contiene el aparato de la viga. </li><li> Los días de entrenamiento, cada ratón cruza el haz de 12 mm 3 veces y luego los tiempos de haz 6 mm 3. Vigas de mayor ancho (es decir, 28 mm) se puede utilizar para los ratones que no son capaces de atravesar los estrechos o grandes ratones. Los ratones se colocan en un extremo de una viga y el tiempo requerido para cruzar a la caja de escape en el otro extremo (80 cm) se mide por los detectores de movimiento. El temporizador se inicia por la nariz del ratón entre el centro de 80 cm, y se detuvo cuando el animal llega al final de los 80 cm. Los ratones reposar durante 10 min en sus jaulas en casa entre sesiones de entrenamiento en los dos haces. Durante este período, entre ensayos, otro 3-4 ratones pueden ser entrenados para hacer un uso eficiente del tiempo. Ratones C57BL / 6 suelen cruzar la viga con una cantidad mínima de parar o detener. Sin embargo, cuando lo hacen puesto, oler o mirar a su alrededor, sin proceder hacia adelante, el investigador debe fomentar el ratón para seguir avanzando por la insistencia, picar, o empujando desde atrás con los dedos enguantados. Una vez que los ratones se encuentran en la caja de seguridad, se les permite un tiempo (menos de 15 segundos) para descansar antes de la próxima prueba. Después de cada sesión de entrenamiento, los ratones son devueltos directamente a sus jaulas y llevado de vuelta a su habitación de la vivienda. Es de destacar que los ratones pueden detenerse más si tienen más capacitados y tener demasiada familiaridad con la tarea y el aparato. Por otro lado, la formación se puede continuar por días adicionales si los ratones todavía no atraviesan toda la viga con éxito después de dos días de entrenamiento. Si los tratamientos se aplican a los ratones, una medida de referencia sobre el rendimiento debe ser obtenido antes del tratamiento. </li><li> En el día de la prueba, los tiempos para cruzar cada haz se registran. Dos ensayos con éxito en el que el ratón no puesto en la viga se promedian. Las grabaciones de vídeo se puede utilizar para el análisis más fino de deslizamiento y otros déficits motores observables. Una forma alternativa de evaluar a pie del haz es de un 1,2 de puntuación el sistema neurológico, que las tasas de la imposibilidad de cruzar el rayo como una puntuación de &quot;1&quot; y la capacidad de atravesar el haz normalmente con las dos patas sobre la superficie de la viga y menos de dos se desliza el pie como un &quot;7&quot;. Un deslizamiento se define como el pie viene de la parte superior de la viga. En este sistema de indexación, una puntuación de &quot;3&quot; corresponden a cruzar la viga arrastrando extremidades. Un animal que los resultados de una en dos ensayos no una nueva prueba. Descripción del desempeño en la viga en relación con otros resultados se pueden encontrar en Feeney et al., (1982) y Carter et al., (2001). </li><li> Las vigas y el cuadro se limpian los excrementos de ratón y se limpió con una toalla empapada con el 70% de etanol y agua antes de la siguiente viga se coloca sobre el aparato. </li></ol> 2. Los resultados representativos: <img src="/files/ftp_upload/2376/2376fig1.jpg" alt="Figura 1" /> Figura 1. Tiempos de cruz de C57BL6 hombres y mujeres en las vigas de 6 mm y 12 mm. Una cohorte de 9 a 11 semanas de edad, los ratones C57BL/6-NCRL PESO fueron probados en los dos haces después de 2 días de entrenamiento. Los tiempos promedio para el cruce de la viga de 12 mm fueron 4,6 ± 0,4 segundos para los hombres (n = 14) y 3,3 ± 0,3 segundos para las mujeres (n = 11). Veces en la barra de 6 mm fueron 6,8 ± 0,7 segundos para los hombres (n = 13) y 5,9 ± 0,5 segundos para las mujeres (n = 10) (los errores se presentan como SEM).

<ol>
	<li>Feeney, D. M., Gonzalez, A., Law, W. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amphetamine,+haloperidol,+and+experience+interact+to+affect+rate+of+recovery+after+motor+cortex+injury.">Amphetamine, haloperidol, and experience interact to affect rate of recovery after motor cortex injury.</a> Science. 217, 855-857 (1982).</li><li>Carter, R. J., Morton, J., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+coordination+and+balance+in+rodents.">Motor coordination and balance in rodents.</a> Curr Protoc Neurosci. Chapter 8, (2001).</li><li>Wallace, J. E., Krauter, E. E., Campbell, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+and+reflexive+behavior+in+the+aging+rat.">Motor and reflexive behavior in the aging rat.</a> J Gerontol. 35, 364-370 (1980).</li><li>Brooks, S. P., Dunnett, S. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tests+to+assess+motor+phenotype+in+mice:+a+user's+guide.">Tests to assess motor phenotype in mice: a user's guide.</a> Nat Rev Neurosci. 10, 519-529 (2009).</li><li>Fox, G. B., Fan, L., Levasseur, R. A., Faden, A. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sustained+sensory/motor+and+cognitive+deficits+with+neuronal+apoptosis+following+controlled+cortical+impact+brain+injury+in+the+mouse.">Sustained sensory/motor and cognitive deficits with neuronal apoptosis following controlled cortical impact brain injury in the mouse.</a> J Neurotrauma. 15, 599-614 (1998).</li><li>Buccafusco, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Methods of behavioral analysis in neuroscience. , (2009).</li><li>Southwell, A. L., Ko, J., Patterson, P. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intrabody+gene+therapy+ameliorates+motor,+cognitive,+and+neuropathological+symptoms+in+multiple+mouse+models+of+Huntington's+disease.">Intrabody gene therapy ameliorates motor, cognitive, and neuropathological symptoms in multiple mouse models of Huntington's disease.</a> J Neurosci. 29, 13589-13602 (2009).</li><li>Carter, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+progressive+motor+deficits+in+mice+transgenic+for+the+human+Huntington's+disease+mutation.">Characterization of progressive motor deficits in mice transgenic for the human Huntington's disease mutation.</a> J Neurosci. 19, 3248-3257 (1999).</li><li>Gulinello, M., Chen, F., Dobrenis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+deficits+in+motor+coordination+and+cognitive+dysfunction+in+a+mouse+model+of+the+neurodegenerative+lysosomal+storage+disorder,+Sandhoff+disease.">Early deficits in motor coordination and cognitive dysfunction in a mouse model of the neurodegenerative lysosomal storage disorder, Sandhoff disease.</a> Behav Brain Res. 193, 315-319 (2008).</li><li>Stanley, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+mouse+beam+walking+assay+offers+improved+sensitivity+over+the+mouse+rotarod+in+determining+motor+coordination+deficits+induced+by+benzodiazepines.">The mouse beam walking assay offers improved sensitivity over the mouse rotarod in determining motor coordination deficits induced by benzodiazepines.</a> J Psychopharmacol. 19, 221-227 (2005).</li></ol>

Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por el Cuidado de Animales Caltech institucional y el empleo (IACUC).

El rendimiento en la barra de equilibrio es una medida útil de la coordinación fina y el equilibrio y ha sido validado por el trabajo anterior. Haz la prueba puede detectar déficits motores debido a la edad, lesiones nerviosas centrales Syst `, y la manipulación genética y farmacológica de los roedores jóvenes y mayores 2,3,4. Los ratones con lesiones corticales impacto a menudo exhiben contralateral caer en la viga de 5,6. Haz la prueba también se ha utilizado para diferenciar las habilida...

<ul><li> Vigas: 1-m listones de madera lisa con cuadrados pequeños, medianos y grandes (es decir, 6, 12 y 28 mm de ancho) secciones transversales </li><li> Soporte de pie para sostener la sección de inicio de la viga elevada (alrededor de 1,5 cm de sección y 50 cm de alto) </li><li> Caja de la meta (20 cm a cada lado), montado sobre un soporte (3 cm de sección y 50 cm de alto) </li><li> Hamaca-~ 7,5 cm por encima de mesa </li><li> De 60 vatios (W) lámpara de escritorio </li><li> Cámara de vídeo, trípode, y las cintas vírgenes de vídeo </li><li> Etanol, toallas de papel, materiales de limpieza </li></ul>

assessment of motor balance and coordination in mice using the balance beam

Lesión cerebral, las manipulaciones genéticas, y los tratamientos farmacológicos pueden resultar en alteraciones de las habilidades motoras en ratones. La coordinación motora fina y el equilibrio puede ser evaluado mediante el ensayo de caminar viga. El objetivo de esta prueba es para el ratón para mantenerse en pie y caminar a través de un haz estrecho elevado a una plataforma segura. Esta prueba se lleva a cabo durante 3 días consecutivos: 2 días de entrenamiento y un día de la prueba. Desempeño en la viga se cuantifica mediante la medición del tiempo que tarda el ratón para atravesar el haz y el número de hojas de la pata que se producen en el proceso. Aquí se presenta el protocolo utilizado en nuestro laboratorio, y los resultados de representante de un grupo de ratones C57BL / 6. Esta tarea es particularmente útil para la detección de los déficits en las habilidades motoras sutiles y el equilibrio que no puede ser detectado por las pruebas de motor, tales como la rotarod.

Watch this Scientific Journal Video about Assessment of Motor Balance and Coordination in Mice using the Balance Beam at JoVE.com

Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Los déficits en la coordinación motora fina puede ser evaluada con la prueba de barra de equilibrio. Desempeño en la viga se cuantifica mediante la velocidad a la que se recorre la viga y el número de veces que el ratón se desliza sobre la viga.

Evaluación del equilibrio del motor y la coordinación en ratones utilizando la barra de equilibrio

Brain injury, genetic manipulations, and pharmacological treatments can result in alterations of motor skills in mice.  Fine motor coordination and ...

assessment-motor-balance-coordination-mice-using-balance

Research

JoVE Journal

Neuroscience

46.3K vistas.  California Institute of Technology. Los déficits en la coordinación motora fina puede ser evaluada con la prueba de barra de equilibrio. Desempeño en la viga se cuantifica mediante la velocidad a la que se recorre la viga y el número de veces que el ratón se desliza sobre la viga.

Video: Assessment of Motor Balance and Coordination in Mice using the Balance Beam

Measuring Motor Coordination in Mice

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such as emotionality or cognition can be assessed, however, it is vital to determine whether the motor capabilities of e.g. a mutant or lesioned mouse allow such an assessment. Performance on a maze task requiring strength and coordination, such as the Morris water maze, might well be impaired in a mouse by motor, rather than cognitive, impairments, so it is essential to selectively dissect the latter from the former. For example, sensorimotor impairments caused by NMDA antagonists have been shown to impair water maze performance2. 
Motor coordination has traditionally been assessed in mice and rats by the rotarod test, in which the animal is placed on a horizontal rod that rotates about its long axis; the animal must walk forwards to remain upright and not fall off. Both set speed and accelerating versions of the rotarod are available.
The other three tests described in this article (horizontal bar, static rods and parallel bars) all measure coordination on static apparatus. The horizontal bar also requires strength for adequate performance, particularly of the forelimbs as the mouse initially grips the bar just with the front paws. 
Adult rats do not perform well on tests such as the static rods and parallel bars (personal observations); they appear less well coordinated than mice. I have only tested male rats, however, and male mice seem generally less well coordinated than females. Mice appear to have a higher strength:weight ratio than rats; the Latin name, Mus musculus, seems entirely appropriate. The rotarod, the variations of the foot fault test12 or the Catwalk (Noldus)15 apparatus are generally used to assess motor coordination in rats.

Protocols are presented for two established motor coordination tasks, the accelerating rotarod and horizontal bar, also two tests developed in Oxford recently, the static rods and parallel bars. These tests can detect motor impairments potentially of interest in their own right, as well as being possible variables in tests of other areas of behavior.

Medición de Coordinación motora en ratones

Mice are increasingly being used in behavioral neuroscience, largely replacing rats as the behaviorist's animal of choice. Before aspects of behavior such ...

Investigación

Neurociencias

Assessment of Morphine-induced Hyperalgesia and Analgesic Tolerance in Mice Using Thermal and Mechanical Nociceptive Modalities

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular mechanisms underlying both phenomena are not well understood and their elucidation should benefit from the study of animal models and from the design of appropriate experimental protocols. 
We describe here a methodological approach for inducing, recording and quantifying morphine-induced hyperalgesia as well as for evidencing analgesic tolerance, using the tail-immersion and tail pressure tests in wild-type mice. As shown in the video, the protocol is divided into five sequential steps. Handling and habituation phases allow a safe determination of the basal nociceptive response of the animals. Chronic morphine administration induces significant hyperalgesia as shown by an increase in both thermal and mechanical sensitivity, whereas the comparison of analgesia time-courses after acute or repeated morphine treatment clearly indicates the development of tolerance manifested by a decline in analgesic response amplitude. This protocol may be similarly adapted to genetically modified mice in order to evaluate the role of individual genes in the modulation of nociception and morphine analgesia. It also provides a model system to investigate the effectiveness of potential therapeutic agents to improve opiate analgesic efficacy.

We describe a protocol to examine the development of opioid-induced hyperalgesia and tolerance in mice. Based on the measurement of thermal and mechanical nociceptive responses of na&#239;ve and morphine-treated animals, it allows to quantify the increase in pain sensitivity (hyperalgesia) and decrease in analgesia (tolerance) associated with chronic opiate administration.

Evaluación de la hiperalgesia inducida por la morfina y analgésicos Tolerancia en ratones utilizando térmicas y mecánicas Modalidades nociceptivos

Opioid-induced hyperalgesia and tolerance severely impact the clinical efficacy of opiates as pain relievers in animals and humans. The molecular ...

Assessment of Vascular Regeneration in the CNS Using the Mouse Retina

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). It is increasingly being recognized that several neurodegenerative diseases such as Alzheimer&#8217;s, multiple sclerosis, and amyotrophic lateral sclerosis present elements of vascular compromise. In addition, the most prominent causes of blindness in pediatric and working age populations (retinopathy of prematurity and diabetic retinopathy, respectively) are characterized by vascular degeneration and failure of physiological vascular regrowth. The aim of this technical paper is to provide a detailed protocol to study CNS vascular regeneration in the retina. The method can be employed to elucidate molecular mechanisms that lead to failure of vascular growth after ischemic injury. In addition, potential therapeutic modalities to accelerate and restore healthy vascular plexuses can be explored. Findings obtained using the described approach may provide therapeutic avenues for ischemic retinopathies such as that of diabetes or prematurity and possibly benefit other vascular disorders of the CNS.

The rodent retina has long been recognized as an accessible window to the brain. In this technical paper we provide a protocol that employs the mouse model of oxygen-induced retinopathy to study the mechanisms that lead to failure of vascular regeneration within the central nervous system after ischemic injury. The described system can also be harnessed to explore strategies to promote regrowth of functional blood vessels within the retina and CNS.

Evaluación de la Vascular regeneración en el SNC Utilizando la retina del ratón

The rodent retina is perhaps the most accessible mammalian system in which to investigate neurovascular interplay within the central nervous system (CNS). ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

La unión neuromuscular: Medición Synapse Tamaño, fragmentación y los cambios en la densidad de proteína sináptica Uso confocal microscopía de fluorescencia

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Assessment of Dendritic Arborization in the Dentate Gyrus of the Hippocampal Region in Mice

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity and extent of dendritic arborization correlates well with the synaptic plasticity in these cells. A reliable method for assessment of dendritic arborization is needed to characterize the deleterious effects of neurological disorders on these structures and to determine the effects of therapeutic interventions. However, quantification of these structures has proven to be a formidable task given their complex and dynamic nature. Fortunately, sophisticated imaging techniques can be paired with conventional staining methods to assess the state of dendritic arborization, providing a more reliable and expeditious means of assessment. Below is an example of how these imaging techniques were paired with staining methods to characterize the dendritic arborization in wild type mice. These complementary imaging methods can be used to qualitatively and quantitatively assess dendritic arborization that span a rather wide area within the hippocampal region.

We describe two methods for visualization and quantification of dendritic arborization in the hippocampus of mouse models: real-time and extended depth of field imaging. While the former method allows sophisticated topographical tracing and quantification of the extent of branching, the latter allows speedy visualization of the dendritic tree.

Evaluación de la arborización dendrítica en el giro dentado de la región del hipocampo en ratones

Dendritic arborization has been shown to be a reliable marker for examination of structural and functional integrity of neurons. Indeed, the complexity ...

A Human-machine-interface Integrating Low-cost Sensors with a Neuromuscular Electrical Stimulation System for Post-stroke Balance Rehabilitation

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing delivery of oxygen and nutrients. About half of the stroke survivors are left with some degree of disability. Innovative methodologies for restorative neurorehabilitation are urgently required to reduce long-term disability. The ability of the nervous system to reorganize its structure, function and connections as a response to intrinsic or extrinsic stimuli is called neuroplasticity. Neuroplasticity is involved in post-stroke functional disturbances, but also in rehabilitation. Beneficial neuroplastic changes may be facilitated with non-invasive electrotherapy, such as neuromuscular electrical stimulation (NMES) and sensory electrical stimulation (SES). NMES involves coordinated electrical stimulation of motor nerves and muscles to activate them with continuous short pulses of electrical current while SES involves stimulation of sensory nerves with electrical current resulting in sensations that vary from barely perceivable to highly unpleasant. Here, active cortical participation in rehabilitation procedures may be facilitated by driving the non-invasive electrotherapy with biosignals (electromyogram (EMG), electroencephalogram (EEG), electrooculogram (EOG)) that represent simultaneous active perception and volitional effort. To achieve this in a resource-poor setting, e.g., in low- and middle-income countries, we present a low-cost human-machine-interface (HMI) by leveraging recent advances in off-the-shelf video game sensor technology. In this paper, we discuss the open-source software interface that integrates low-cost off-the-shelf sensors for visual-auditory biofeedback with non-invasive electrotherapy to assist postural control during balance rehabilitation. We demonstrate the proof-of-concept on healthy volunteers.

A novel low-cost human-machine interface for interactive post-stroke balance rehabilitation system is presented in this article. The system integrates off-the-shelf low-cost sensors towards volitionally driven electrotherapy paradigm. The proof-of-concept software interface is demonstrated on healthy volunteers.

A-máquina humana integración de sensores de bajo coste con un sistema de estimulación eléctrica neuromuscular para la Rehabilitación de Balance post-accidente cerebrovascular

A stroke is caused when an artery carrying blood from heart to an area in the brain bursts or a clot obstructs the blood flow to brain thereby preventing ...

Functional and Morphological Assessment of Diaphragm Innervation by Phrenic Motor Neurons

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and morphological levels. Compound muscle action potential (CMAP) recording following phrenic nerve stimulation can be used to quantitatively assess functional diaphragm innervation by PhMNs of the cervical spinal cord in vivo in anesthetized rats and mice. Because CMAPs represent simultaneous recording of all myofibers of the whole hemi-diaphragm, it is useful to also examine the phenotypes of individual motor axons and myofibers at the diaphragm NMJ in order to track disease- and therapy-relevant morphological changes such as partial and complete denervation, regenerative sprouting and reinnervation. This can be accomplished via whole-mount immunohistochemistry (IHC) of the diaphragm, followed by detailed morphological assessment of individual NMJs throughout the muscle. Combining CMAPs and NMJ analysis provides a powerful approach for quantitatively studying diaphragmatic innervation in rodent models of CNS and PNS disease.

Compound muscle action potential recording quantitatively assesses functional diaphragm innervation by phrenic motor neurons. Whole-mount diaphragm immunohistochemistry assesses morphological innervation at individual neuromuscular junctions. The goal of this protocol is to demonstrate how these two powerful methodologies can be used in various rodent models of spinal cord disease.

Evaluación funcional y morfológica de Diafragma Inervación por frénico neuronas motoras

This protocol specifically focuses on tools for assessing phrenic motor neuron (PhMN) innervation of the diaphragm at both the electrophysiological and ...

Assessment of Hippocampal Dendritic Complexity in Aged Mice Using the Golgi-Cox Method

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching variations of the neuronal dendrites within the hippocampus are implicated in cognition and memory formation. There are several approaches to Golgi staining, all of which have been useful for determining the morphological characteristics of dendritic arbors and produce a clear background. The present Golgi-Cox method, (a slight variation of the protocol that is provided with a commercial Golgi staining kit), was designed to assess how a relatively low dose of the chemotherapeutic drug 5-flurouracil (5-Fu) would affect dendritic morphology, the number of spines, and the complexity of arborization within the hippocampus. The 5-Fu significantly modulated the dendritic complexity and decreased the spine density throughout the hippocampus in a region-specific manner. The data presented show that the Golgi staining method effectively stained the mature neurons in the CA1, the CA3, and the dentate gyrus (DG) of the hippocampus. This protocol reports the details for each step so that other researchers can reliably stain tissue throughout the brain with high quality results and minimal troubleshooting.

Here we present a Golgi-Cox protocol in extensive detail. This reliable tissue stain method allows for a high-quality assessment of the cytoarchitecture in the hippocampus, and throughout the entire brain, with minimal troubleshooting.

Evaluación de la complejidad dendrítica del hipocampo en ratones envejecidos utilizando el método de Golgi-Cox

Dendritic spines are the protuberances from the neuronal dendritic shafts that contain&#160; excitatory synapses. The morphological and branching ...

Retrograde Neuroanatomical Tracing of Phrenic Motor Neurons in Mice

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic nerves innervating the respiratory diaphragm. In spinal cord slices, phrenic motor neurons cannot be identified from other motor neurons on morphological or biochemical criteria. We provide the description of procedures for visualizing phrenic motor neuron cell bodies in mice, following intrapleural injections of cholera toxin subunit beta (CTB) conjugated to a fluorophore. This fluorescent neuroanatomical tracer has the ability to be caught up at the diaphragm neuromuscular junction, be carried retrogradely along the phrenic axons and reach the phrenic cell bodies. Two methodological approaches of intrapleural CTB delivery are compared: transdiaphragmatic versus transthoracic injections. Both approaches are successful and result in similar number of CTB-labeled phrenic motor neurons. In conclusion, these techniques can be applied to visualize or quantify the phrenic motor neurons in various experimental studies such as those focused on the diaphragm-phrenic circuitry.

Here, we describe a protocol for identifying phrenic motor neurons in mice after intrapleural delivery of fluorophore conjugated cholera toxin subunit beta. Two techniques are compared to inject the pleural cavity: transdiaphragmatic versus transthoracic approaches.

Seguimiento Neuroanatomical retrógrada frénico de neuronas de Motor en ratones

Phrenic motor neurons are cervical motor neurons originating from C3 to C6 levels in most mammalian species. Axonal projections converge into phrenic ...

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

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

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

Análisis cinemático 3D para la evaluación funcional en el modelo de rata de lesión de aplastamiento de nervios ciáticos

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