Los autores desean agradecer a Ikiel Ptito por su apoyo técnico continuo. NSERC otorgar a MP.

Parte 1: Pre-procesamiento de tejidos <ol><li> Tejido debe ser buena perfusión con paraformaldehído, glutaraldehído, o formalina. Esto se puede lograr a través de la perfusión estándar transcardial normalmente se utiliza para la cosecha de otros órganos. En el presente estudio el tema fue profundamente sedado con clorhidrato de ketamina (10 mg / kg, im), sacrificados con una sobredosis de pentobarbital sódico (25 mg / kg, iv) y perfundidos transcardially con PBS 0,1 M hasta que esté completamente exsanguinated.This es seguido por una solución de paraformaldehído al 4% en PBS durante 5 minutos (aproximadamente 1 litro). </li></ol> Parte 2: estereotáxica bloqueo <ol><li> Antes de colocar la cabeza en el marco estereotáxico las coordenadas del plano cero Horsley-Clarke/interaural hay que tener. Este es el punto medio teórico entre las orejas. Para medir este plano, las barras del oído deben ser igualmente instalado en el aparato, coloque una hoja de bisturí en el brazo manipulador estereotáxica y medir el punto medio entre las barras de oído. Esto es importante para determinar dónde para bloquear el tejido sobre la base de las necesidades de investigación. Una vez completado el cráneo tiene que estar preparado para la fijación en el aparato estereotáxico. </li><li> Con el fin de colocar la cabeza en el marco estereotáxico la mandíbula inferior tendrá que ser removido con pinzas para los huesos y un bisturí. Además de la piel, músculo y tejido conjuntivo debe ser removido para exponer el cráneo. Una vez que el tejido conectivo es extraído del cráneo, exponer el cerebro por socavando la bóveda craneal (la parte de tabla del hueso occipital, así como el parietal, huesos anuncio frontal). En la parte experimento de la bóveda craneal tiene todo listo ha eliminado. Tenga cuidado de no eliminar por completo los huesos temporales, porque los canales del oído tendrá que estar intacto para colocar la cabeza en el marco estereotáxico. El resto de la materia dura debe ser removido desde el cerebro al descubierto. El cráneo está listo para ser colocado en el marco estereotáxico. </li><li> De la misma manera en la que se podría realizar una cirugía estereotáxica, ajustar el ojo, el paladar y bares del oído de tal manera que la cabeza esté bien fija en el aparato estereotáxico. Coloque el manipulador estereotáxica en las coordenadas predeterminadas anterior / posterior (A / P) y mover el manipulador de la cara lateral del cerebro. Baje lentamente la hoja en el cerebro, completamente levantar la hoja del cerebro, entonces se mueven en sentido medial e inferior de la hoja de nuevo. Repita estos dos pasos hasta que la hoja se ha llegado a la parte lateral del hemisferio opuesto. Con esto se completa el bloque de la corona en primer lugar. Para su posterior bloques coronal mover el manipulador de 1 cm en el eje A / P y repetir hasta que todo el cerebro ha sido bloqueado. </li></ol> Parte 3: Extracción del cerebro en el cráneo <ol><li> Retire el cabezal del marco estereotáxico y mantener el cerebro expuestos en la palma de su mano. Tratar de asegurar el cerebro ligeramente la colocación de mimos en el cerebro en la palma de su mano y coloque el dedo meñique a través de los lóbulos frontales, este movimiento reduce al mínimo del cerebro en el cráneo. A fin de evitar el secado de la superficie pial del cerebro, coloque un trozo de gasa humedecida PBS a través del cerebro. Mantenga la cabeza firme por el cráneo y socavar el resto de los huesos occipital y temporal, junto con la columna vertebral. Esto expone a los aspectos de base y laterales del cerebro. Por último eliminar cualquier resto de hueso frontal y el hueso nasal, lo que permitirá el acceso a los bulbos olfatorios. Es importante quitar el hueso frontal último porque, como se quita la base del cráneo, el cerebro se mueve un poco y los lóbulos frontales se pueden dañar en los bordes irregulares del hueso fontal. Cortar y retirar la restante materia dura. Levante con cuidado la parte frontal del cerebro, deslice el bisturí en el cerebro y el cerebro sin la del cráneo. </li></ol> Parte 4: Medidas <ol><li> Hay una serie de medidas útiles que se pueden hacer antes de la congelación. Por ejemplo, el eje A / P del cerebro con un calibrador. Además, la densidad específica puede ser medida por el peso del cerebro, la medición del volumen por el desplazamiento de agua en una probeta dividiendo el peso por el desplazamiento de volumen (Tabla 1). </li></ol> Parte 5: Termina bloqueo en el cerebro. <ol><li> Normalmente, cuando la hoja del bisturí se introduce en el cerebro no es lo suficientemente largo para penetrar por completo la totalidad de dorsal-ventral medida del cerebro. Una vez que se remueve el cerebro y las mediciones son brutos obtenidos, tomar un pañuelo de papel, corte la hoja y terminar bloqueando el cerebro a través de la parte ventral del cerebro (Figura 2). </li><li> Antes de la congelación de los bloques, es necesario cryoprotect el tejido de graduado soluciones de sacarosa PBS buffer (10, 20, y 30%) se mantiene a 4 ˚ C hasta que se hunde el cerebro. Normalmente se tarda una noche de incubación en el 10%, 2-3 días en el 20% y más de 3-5 días en el 30% de los bloques que se hunden hasta el fondo del recipiente. A cambio diario de the 30% de solución de sacarosa se recomienda. </li></ol> Parte 6: Resultados de Representante Hay una serie de graves medidas morfológicas que se puede hacer una vez que el cerebro ha sido extraído del cráneo. Estos incluyen una longitud / P, el peso y la densidad específica (Tabla 1). Por lo general, bloquear el cerebro en 6.7 medición de los bloques de 1 cm (Figura 1). Cada pieza se fotografiaron (Figura 2) y pueden ser más diseccionado en función de las necesidades de investigación o preparados para la congelación de soluciones de sacarosa calificado. <table border="1"><tbody><tr><td> Tema </td><td> A Longitud / P (Mm) </td><td> Peso (Gramos) </td><td> Desplazamiento de agua (Ml) </td><td> Densidad específica (G / ml) </td></tr><tr><td> O2303-2-1-1 </td><td> 64.3 </td><td> 28.1 </td><td> 24 </td><td> 1.171 </td></tr><tr><td> O5180-1 </td><td> 71.3 </td><td> 38.7 </td><td> 34 </td><td> 1.138 </td></tr><tr><td> O2708-3-1 </td><td> 62.8 </td><td> 28.7 </td><td> 26 </td><td> 1.104 </td></tr><tr><td> O9184-4-2 </td><td> 65.3 </td><td> 29.5 </td><td> 24 </td><td> 1.229 </td></tr><tr><td> N459-1-14-2 </td><td> 68.2 </td><td> 31.6 </td><td> 26 </td><td> 1.215 </td></tr><tr><td> PROMEDIO </td><td> 66.38 </td><td> 31.32 </td><td> 26.8 </td><td> 1.171 </td></tr><tr><td> ETS DEV </td><td> 3.38 </td><td> 4.33 </td><td> 4.17 </td><td> 0.052 </td></tr></tbody></table> Tabla 1. Bruto mediciones morfológicas del hemisferio derecho de 5 meses de edad monos verdes <img src="/files/ftp_upload/1259/1259fig1.jpg" alt="la figura 1" /> <img src="/files/ftp_upload/1259/1259fig2.jpg" alt="la figura 1.5" /> Figura 1. Esquemas de los planos coronal utilizado para bloquear el cerebro. Este es un ejemplo de un cerebro adulto vervet externalizados. Ejemplo del procedimiento de bloqueo. Las líneas verticales aquí están espaciados a 1 cm, por lo general producen siete bloques de la corona de cada hemisferio. <img src="/files/ftp_upload/1259/1259fig3.jpg" alt="la figura 2" /> Figura 2. Los bloques de tejido cerebral en el espacio estereotáxico. Cada bloque se producen aproximadamente 200 secciones tomadas a 50 micras. Con este esquema de muestreo de más de 1200 secciones a través de la corteza será tomado, y un adicional de 400 a 500 del cerebelo en rodajas en el plano coronal.

<ol>
	<li>Gallagher, M., Rapp, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+animal+models+to+study+the+effects+of+aging+on+cognition.+Annu+Rev+Psychol+48.">The use of animal models to study the effects of aging on cognition. Annu Rev Psychol 48.</a> , 339-370 (1997).</li><li>Clancy, B., Darlington, R., Finlay, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translating+developmental+time+across+mammalian+species.">Translating developmental time across mammalian species.</a> Neuroscience. 105, 7-17 (2001).</li><li>Nowakowski, R. S., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+site+of+origin+and+route+and+rate+of+migration+of+neurons+to+the+hippocampal+region+of+the+rhesus+monkey.">The site of origin and route and rate of migration of neurons to the hippocampal region of the rhesus monkey.</a> J Comp Neurol. 196, 129-154 (1981).</li><li>Rakic, P., Bourgeois, J. P., Eckenhoff, M. F., Zecevic, N., Goldman-Rakic, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concurrent+overproduction+of+synapses+in+diverse+regions+of+the+primate+cerebral+cortex.">Concurrent overproduction of synapses in diverse regions of the primate cerebral cortex.</a> Science. 232, 232-235 (1986).</li><li>Granger, B., Tekaia, F., Le Sourd, A. M., Rakic, P., Bourgeois, J. 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N., Guzman-Flores, C., Juarez, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Voluntary+consumption+of+beverage+alcohol+by+vervet+monkeys:+population+screening,+descriptive+behavior+and+biochemical+measures.">Voluntary consumption of beverage alcohol by vervet monkeys: population screening, descriptive behavior and biochemical measures.</a> Pharmacol Biochem Behav. 36, 367-373 (1990).</li><li>Palmour, R. M., Mulligan, J., Howbert, J. 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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+neural+stem+cells+migrate+along+the+nigrostriatal+pathway+in+a+primate+model+of+Parkinson's+disease.">Human neural stem cells migrate along the nigrostriatal pathway in a primate model of Parkinson's disease.</a> Exp Neurol. 211, 362-369 (2008).</li><li>Lemere, C. A., Beierschmitt, A., Iglesias, M., Spooner, E. T., Bloom, J. K., Leverone, J. F., Zheng, J. B., Seabrook, T. J., Louard, D., Li, D., Selkoe, D. J., Palmour, R. M., Ervin, F. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease+abeta+vaccine+reduces+central+nervous+system+abeta+levels+in+a+non-human+primate,+the+Caribbean+vervet.">Alzheimer's disease abeta vaccine reduces central nervous system abeta levels in a non-human primate, the Caribbean vervet.</a> Am J Pathol. 165, 283-297 (2004).</li><li>Mash, D. C., Staley, J. K., Doepel, F. M., Young, S. N., Ervin, F. R., Palmour, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Altered+dopamine+transporter+densities+in+alcohol-preferring+vervet+monkeys.">Altered dopamine transporter densities in alcohol-preferring vervet monkeys.</a> Neuroreport. 7, 457-462 (1996).</li><li>Burke, M. W., Palmour, R. M., Ervin, F. R., Ptito, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+reduction+in+frontal+cortex+of+primates+after+prenatal+alcohol+exposure.">Neuronal reduction in frontal cortex of primates after prenatal alcohol exposure.</a> Neuroreport. 20, 13-17 (2009).</li></ol>

The authors have nothing to disclose.

El San Cristóbal mono verde (Chlorocebus aethiops sabeus) es un primate del viejo mundo con patrones similares y las tasas de desarrollo del cerebro cortical y subcortical a la de los seres humanos. Esta especie ha sido utilizada para modelar complejos trastornos de la conducta humana como un comportamiento ansioso, hipertensión 8, 9 hemisferectomía, la enfermedad de Parkinson 10, la enfermedad de Alzhemier de 11, y el abuso del alcohol 12. Más recientemente, esta...

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<td>Scalpel</td>
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dissecting the non-human primate brain in stereotaxic space

El uso de primates no humanos proporciona un excelente modelo de traslación para nuestra comprensión de los procesos de desarrollo y el envejecimiento en los seres humanos<sup&gt; 1-6</sup&gt;. Además, el uso de primates no humanos, recientemente ha dado la oportunidad de modelo natural complejo trastornos psiquiátricos como el abuso de alcohol<sup&gt; 7</sup&gt;. A continuación se describe una técnica para bloquear el cerebro en el plano coronal del mono verde (<em&gt; Chlorocebus aethiops sabeus</em&gt;) En el cráneo intacto en el espacio estereotáxico. El método descrito aquí proporciona un plano estándar de la sección entre los bloques y los temas y minimiza las secciones parciales entre los bloques. Seccionar un bloque de tejido en el plano coronal también facilita la delimitación de un área de interés. Este método proporciona bloques de tamaño manejable desde un solo hemisferio de los monos verdes se obtienen más de 1200 secciones en corte a 50 micras. Además mediante el bloqueo del cerebro en bloques de 1 cm, que facilita la penetración de la sacarosa para cyroprotection y permite que el bloque a cortar en un criostato estándar.

Watch this Scientific Journal Video about Dissecting the Non-human Primate Brain in Stereotaxic Space at JoVE.com

Dissecting the Non-human Primate Brain in Stereotaxic Space

Los primates no humanos es una importante especie de traslación para nuestra comprensión del proceso normal del cerebro. La organización anatómica del cerebro de los primates puede proporcionar información importante sobre las condiciones normales y patológicas en los seres humanos.

La disección de cerebro de los primates no humanos en el espacio estereotáxica

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In ...

dissecting-the-non-human-primate-brain-in-stereotaxic-space

Research

JoVE Journal

Biology

10.0K vistas.  University of Montreal. Los primates no humanos es una importante especie de traslación para nuestra comprensión del proceso normal del cerebro. La organización anatómica del cerebro de los primates puede proporcionar información importante sobre las condiciones normales y patológicas en los seres humanos.

Video: Dissecting the Non-human Primate Brain in Stereotaxic Space

Survivable Stereotaxic Surgery in Rodents 

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different systemic challenges (pharmacological or physiological) to the CNS. For example, one can directly measure how the animal's midbrain dopamine projections respond to dopamine-releasing drugs like d-amphetamine or natural stimuli like food. In this video, we show you how to implant guide cannulas targeting specific sites in the rat brain, how to insert and implant a microdialysis probe and how to use high performance liquid chromatography coupled with electrochemical detection (HPLC-EC) to measure extracellular levels of oxidizable neurotransmitters and metabolites. Local precise introduction of drugs through the microdialysis probe allows for refined work on site specificity in a compound s mechanism of action. This technique has excellent anatomical and chemical resolution but only modest time resolution as microdialysis samples are usually processed every 20-30 minutes to ensure detectable neurotransmitter levels. Complementary ex vivo tools (i.e., slice and cell culture electrophysiology) can assist with monitoring real-time neurotransmission.

Survivable Stereotaxic Surgery in Rodents

The monitoring of extracellular neurotransmitter levels in distinct brain regions of freely moving animals offers insights on the link between neurotransmitter release and behavior. In vivo microdialysis coupled with electrochemical detection provides excellent anatomical and chemical resolution; and information on how basal neurotransmission is altered by pharmacological or physiological manipulations.

La cirugía estereotáxica de supervivencia en los roedores

The ability to measure extracellular basal levels of neurotransmitters in the brain of awake animals allows for the determination of effects of different ...

Investigación

Biología

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

Disección y de grabación de la unión neuromuscular C. Elegans

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

The Gateway to the Brain: Dissecting the Primate Eye

The visual system in humans is considered the gateway to the world and plays a principal role in the plethora of sensory, perceptual and cognitive processes. It is therefore not surprising that quality of vision is tied to quality of life . Despite widespread clinical and basic research surrounding the causes of visual disorders, many forms of visual impairments, such as retinitis pigmentosa and macular degeneration, lack effective treatments. Non-human primates have the closest general features of eye development to that of humans. Not only do they have a similar vascular anatomy, but amongst other mammals, primates have the unique characteristic of having a region in the temporal retina specialized for high visual acuity, the fovea1. Here we describe a general technique for dissecting the primate retina to provide tissue for retinal histology, immunohistochemistry, laser capture microdissection, as well as light and electron microscopy. With the extended use of the non-human primate as a translational model, our hope is that improved understanding of the retina will provide insights into effective approaches towards attenuating or reversing the negative impact of visual disorders on the quality of life of affected individuals.

The non-human primate is an important translational species for our understanding of development and aging. The anatomical organization of the primate retina may provide important insights into normal and pathological conditions in humans.

La puerta de enlace para el cerebro: la disección de los ojos de Primates

The visual system in humans is considered the  gateway  to the world and plays a principal role in the plethora of sensory, perceptual and cognitive ...

Knowing What Counts: Unbiased Stereology in the Non-human Primate Brain

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased stereology, the method accepted as state-of-the-art for quantification of biological objects in tissue sections2, generates reliable structural data for biological features in the mammalian brain3. The key components of the approach are unbiased (systematic-random) sampling of anatomically defined structures (reference spaces), combined with quantification of cell numbers and size, fiber and capillary lengths, surface areas, regional volumes and spatial distributions of biological objects within the reference space4. Among the advantages of these stereological approaches over previous methods is the avoidance of all known sources of systematic (non-random) error arising from faulty assumptions and non-verifiable models. This study documents a biological application of computerized stereology to estimate the total neuronal population in the frontal cortex of the vervet monkey brain (Chlorocebus aethiops sabeus), with assistance from two commercially available stereology programs, BioQuant Life Sciences and Stereologer (Figure 1). In addition to contrast and comparison of results from both the BioQuant and Stereologer systems, this study provides a detailed protocol for the Stereologer system.

The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans. Unbiased stereology is a method for accurately and efficiently estimating the total neuron number (or other cell type) in a given reference space1.

Sabiendo lo que cuenta: Estereología imparcial en el cerebro de los primates no humanos

The non-human primate is an important translational species for understanding the normal function and disease processes of the human brain. Unbiased ...

Trajectory Data Analyses for Pedestrian Space-time Activity Study

It is well recognized that human movement in the spatial and temporal dimensions has direct influence on disease transmission1-3. An infectious disease typically spreads via contact between infected and susceptible individuals in their overlapped activity spaces. Therefore, daily mobility-activity information can be used as an indicator to measure exposures to risk factors of infection. However, a major difficulty and thus the reason for paucity of studies of infectious disease transmission at the micro scale arise from the lack of detailed individual mobility data. Previously in transportation and tourism research detailed space-time activity data often relied on the time-space diary technique, which requires subjects to actively record their activities in time and space. This is highly demanding for the participants and collaboration from the participants greatly affects the quality of data4.
Modern technologies such as GPS and mobile communications have made possible the automatic collection of trajectory data. The data collected, however, is not ideal for modeling human space-time activities, limited by the accuracies of existing devices. There is also no readily available tool for efficient processing of the data for human behavior study. We present here a suite of methods and an integrated ArcGIS desktop-based visual interface for the pre-processing and spatiotemporal analyses of trajectory data. We provide examples of how such processing may be used to model human space-time activities, especially with error-rich pedestrian trajectory data, that could be useful in public health studies such as infectious disease transmission modeling.
The procedure presented includes pre-processing, trajectory segmentation, activity space characterization, density estimation and visualization, and a few other exploratory analysis methods. Pre-processing is the cleaning of noisy raw trajectory data. We introduce an interactive visual pre-processing interface as well as an automatic module. Trajectory segmentation5 involves the identification of indoor and outdoor parts from pre-processed space-time tracks. Again, both interactive visual segmentation and automatic segmentation are supported. Segmented space-time tracks are then analyzed to derive characteristics of one's activity space such as activity radius etc. Density estimation and visualization are used to examine large amount of trajectory data to model hot spots and interactions. We demonstrate both density surface mapping6 and density volume rendering7. We also include a couple of other exploratory data analyses (EDA) and visualizations tools, such as Google Earth animation support and connection analysis. The suite of analytical as well as visual methods presented in this paper may be applied to any trajectory data for space-time activity studies.

A suite of spatiotemporal processing methods are presented to analyze human trajectory data, such as that collected using a GPS device, for the purpose of modeling pedestrian space-time activities.

Los análisis de trayectoria de datos para el estudio de peatones Actividad espacio-tiempo

It is well recognized that human movement in the  spatial and temporal dimensions has direct influence on disease transmission1-3.  An infectious disease ...

Sampling Blood from the Lateral Tail Vein of the Rat

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, proteins, and glucose, yet the composition of the blood is dynamically regulated and easily perturbed. One factor that can change the blood composition is the stress response triggered by the sampling procedure, which can contribute to variability in the measures of interest. Here we describe a procedure for blood sampling from the lateral tail vein in the rat. This procedure offers significant advantages over other more commonly used techniques. It permits rapid sampling with minimal pain or invasiveness, without anesthesia or analgesia. Additionally, it can be used to obtain large volume samples (upwards of 1 ml in some rats), and it may be used repeatedly across experimental days. By minimizing the stress response and pain resulting from blood sampling, measures can more accurately reflect the true basal state of the animal, with minimal influence from the sampling procedure itself.

Blood samples are useful for assessing biomarkers of physiological states or disease in vivo. Here we describe the methodology to sample blood from the lateral tail vein in the rat. This method provides rapid samples with minimal pain and invasiveness.

Toma de muestras de sangre de la vena lateral de la cola de la rata

Blood samples are commonly obtained in many experimental contexts to measure targets of interest, including hormones, immune factors, growth factors, ...

Micropuncture of Bowman's Space in Mice Facilitated by 2 Photon Microscopy

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional microscopy to surface nephron features, and 2-photon studies are limited in that interventions can only be assessed at the organ, rather than the nephron level. In particular, micropuncture studies of the glomeruli of mice have been challenged by the paucity of surface glomeruli in mice. To address this limitation in order to pursue studies of aspirate from Bowman&#39;s space in mouse physiologic models, we developed 2-photon glomerular micropuncture. We present a novel surgical preparation that allows lateral access to the kidney while preserving the required vertical imaging column for 2-photon microscopy. Administration of high molecular weight fluorescein isothiocyanate (FITC)-dextran is used to render the renal vasculature and therefore glomeruli visible for 2-photon imaging. A quantum dot-coated pipette is then introduced under stereotactic guidance to a glomerulus selected from the several to many which may be visualized within the imaging window. In this protocol, we provide details of the preparation, materials, and methods necessary to carry out the procedure. This technique facilitates previously-impossible physiologic study of the kidney, including recovery of filtrate from Bowman&#39;s space and all segments of the nephron within the imaging depth limit, about 100 &#181;m below the renal capsule. Pressure, charge and flow may all be measured using the introduced pipette. Here, we provide representative data from liquid chromatography/mass spectrometry performed on aspirate from Bowman&#39;s space. We expect this technique to have wide applicability in renal physiologic investigation.

Micropuncture of Bowman&#039;s Space in Mice Facilitated by 2 Photon Microscopy

We present use of 2-photon microscopy to place a micropipette within Bowman&#39;s urinary space in mice, combining 2 foundational techniques of renal physiology. Use of 2-photon microscopy overcomes critical limitations of conventional microscopy for micropuncture renal physiology studies.

Micropunción del espacio de Bowman en ratones por 2 fotones microscopía

Renal micropuncture and renal 2-photon imaging are seminal techniques in renal physiology. However, micropuncture is limited by dependence on conventional ...

Reverse Dissection and DiceCT Reveal Otherwise Hidden Data in the Evolution of the Primate Face

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with conspecifics at a close range. These displays are achieved by contraction of the mimetic muscles, which are skeletal muscle attached to the dermis of the face. Reverse dissection, removing the full facial mask from the skull and approaching mimetic muscles in reverse, is an effective but destructive way of revealing the morphology of mimetic muscles but it is destructive. DiceCT is a novel mechanism for visualizing skeletal muscles, including mimetic muscles, and isolating individual muscle fascicles for quantitative measurement. Additionally, DiceCT provides a non-destructive mechanism for visualizing muscles. The combined techniques of reverse dissection and DiceCT can be used to assess the evolutionary morphology of mimetic musculature as well as potential contraction strength and velocity in these muscles. This study further demonstrates that DiceCT can be used to accurately and reliably visualize mimetic muscles as well as reverse dissection and provide a non-destructive method for sampling mimetic muscles.

Facial expressions are a mode of visual communication produced by mimetic muscles. Here, we present protocols for the novel techniques of reverse dissection and DiceCT to fully visualize and assess mimetic muscles. These combined techniques can examine both morphological and physiological aspects of mimetic musculature to determine functional aspects.

Inversa, disección y DiceCT revelan lo contrario oculta datos en la evolución de la cara de Primate

Facial expressions, or facial displays, of social or emotional intent are produced by many mammalian taxa as a means of visually communicating with ...

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an application of smFISH to measure the rates of transcription and mRNA degradation in Escherichia coli. As smFISH is based on fixed cells, we perform smFISH at multiple time points during a time-course experiment, i.e., when cells are undergoing synchronized changes upon induction or repression of gene expression. At each time point, sub-regions of an mRNA are spectrally distinguished to probe transcription elongation and premature termination. The outcome of this protocol also allows for analyzing intracellular localization of mRNAs and heterogeneity in mRNA copy numbers among cells. Using this protocol many samples (~50) can be processed within 8 h, like the amount of time needed for just a few samples. We discuss how to apply this protocol to study the transcription and degradation kinetics of different mRNAs in bacterial cells.

Probing mRNA Kinetics in Space and Time in Escherichia coli using Two-Color Single-Molecule Fluorescence In Situ Hybridization

This protocol describes an application of single-molecule fluorescence in situ hybridization (smFISH) to measure the in vivo kinetics of mRNA synthesis and degradation.

Sondeo de mRNA cinética en el espacio y el tiempo en Escherichia coli usando hibridación de fluorescencia de una sola molécula de dos colores

Single-molecule fluorescence in situ hybridization (smFISH) allows for counting the absolute number of mRNAs in individual cells. Here, we describe an ...

Dissecting Mechanoenzymatic Properties of Processive Myosins with Ultrafast Force-Clamp Spectroscopy

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of both conventional and unconventional myosins under load with unprecedented time resolution. In particular, the possibility to probe myosin motors under constant force right after the actin-myosin bond formation, together with the high rate of the force feedback (200 kHz), has shown UFFCS to be a valuable tool to study the load dependence of fast dynamics such as the myosin working stroke. Moreover, UFFCS enables the study of how processive and non-processive myosin-actin interactions are influenced by the intensity and direction of the applied force.
By following this protocol, it will be possible to perform ultrafast force-clamp experiments on processive myosin-5 motors and on a variety of unconventional myosins. By some adjustments, the protocol could also be easily extended to the study of other classes of processive motors such as kinesins and dyneins. The protocol includes all the necessary steps, from the setup of the experimental apparatus to sample preparation, calibration procedures, data acquisition and analysis.

Presented here is a comprehensive protocol to perform ultrafast force-clamp experiments on processive myosin-5 motors, which could be easily extended to the study of other classes of processive motors. The protocol details all the necessary steps, from the setup of the experimental apparatus to sample preparation, data acquisition and analysis.

Disección de las propiedades mecanoenzimáticas de miosinas procesivas con espectroscopia ultrarrápida de pinza de fuerza

Ultrafast force-clamp spectroscopy (UFFCS) is a single molecule technique based on laser tweezers that allows the investigation of the chemomechanics of ...