Los autores desean agradecer a Ikiel Ptito por su apoyo técnico. Estamos muy agradecidos a Frank Ervin, Palmour Roberta y el personal de Laboratorios de Ciencias del Comportamiento fundación con sede en San Cristóbal, de las Indias Occidentales, por su continuo apoyo a nuestro trabajo de primates.

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. Se recomienda que, poco después del sacrificio de los ojos se inyectan con un fijador justo debajo de la lente y se almacena en el fijador. </li><li> 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 desangrado. Esto es seguido por una solución de paraformaldehído al 4% en PBS durante 5 minutos (aproximadamente 1 litro). </li></ol> Parte 2: Extracción del globo ocular de la cavidad orbital <ol><li> Para facilitar el acceso al globo ocular, se recomienda retirar primero el cerebro. Una vez que el cerebro se ha quitado el hueso órbita de paredes delgadas es evidente. Utilice la gubia ósea para chips lentamente la pared de la órbita. Cortar los músculos oculares con un bisturí y extirpar el tejido conectivo del globo ocular. Corte cuidadosamente el nervio óptico, esto puede ser utilizado en los estudios de microscopía electrónica. El globo del ojo ya debería ser liberado de la cavidad orbital. </li></ol> Parte 3: Disección de la retina de la ojera <ol><li> Coloque el ojo en una placa de Petri con PBS para mantener la retina de secado. Un microscopio de disección o una mesa montada aumento de pie de luz es muy útil en la disección, pero no una necesidad. Retire la córnea mediante la reducción de la esclerótica de cerca en el perímetro de la córnea a nivel de la ora serrata, con un par de tijeras de primavera y quitar la lente con la pinza. </li><li> Use un pincel, pinzas y tijeras de primavera para eliminar la retina de la esclerótica. Esto se hace mediante la separación de la retina de la esclerótica y luego cortar la esclerótica de distancia con las tijeras. Uno debe llevar a cabo este proceso en pequeños incrementos para no dañar o romper el tejido de la retina. La esclerótica no se separa fácilmente del nervio óptico remanente para cortar cuidadosamente la esclerótica alrededor del nervio óptico. </li><li> En este punto de la retina ha conservado su forma curva y tiene que ser aplastada para el muestreo. Antes del aplanamiento de la retina, el humor vítreo, que tiene la consistencia de la gelatina, se puede quitar en un bulto. Para aplanar la retina en una diapositiva, hacer varios cortes radiales con una hoja de bisturí. El humor vítreo residual ahora se puede quitar con papel de filtro ordinario y un pincel. La capa de células ganglionares de la retina se expone en este momento lo que es imprescindible que tenga cuidado al retirar el humor vítreo. Si el nervio óptico está todavía unido en el disco óptico, quite el nervio óptico sin necesidad de digitalizar la retina, utilizando un bisturí y un par de tijeras de primavera. Esta es ahora una retina plana de montaje (Figura 1) y la fóvea debe ser evidente que una mancha oscura en la dirección temporal / ventral de la papila óptica. </li></ol> Parte 4: Muestreo <ol><li> Hay una serie de opciones para el muestreo de la retina. Aquí vamos a describir la preparación y toma de muestras flatmount isodentric. En ambos procedimientos flatmount la retina con la capa de fibra óptica fuera de la diapositiva. Si la intención es examinar la capa de células ganglionares de la retina en la preparación flatmount, es necesario quitar uno o blanquear el epitelio pigmentado. Para quitar el epitelio pigmentado utilizar un pincel ligeramente para eliminar parte de esta capa, lo que tiende a dañar la capa de fotorreceptores. Blanqueo implica sumergir la retina en una solución de permanganato de potasio (0,25%) durante 1 hora, lavar en agua destilada, y la limpieza en ácido oxálico (5%) durante 5 minutos 2. </li><li> Si la intención es analizar la distribución de células y la morfología de cada capa en toda la retina, entonces le sugerimos muestreo isométrica de la retina. La retina se debe flatmounted en un portaobjetos y se mantiene húmeda, con PBS. Para el muestreo isométrica cortar pequeños trozos de tejido equidistante de la papila óptica en las direcciones nasal, temporal, superior e inferior (Figura 1). No es necesario para blanquear el epitelio pigmentado en esta preparación. Estas piezas ahora se puede seccionar en el plano coronal con un criostato, vibratome o ultramicrotomo en función de la pregunta de investigación. Para la sección criostato, las piezas deben ser crioprotegieron en sacarosa al 30% durante la noche y se congeló en el contexto de un medio de montaje en hielo seco. Por otra parte, las muestras se pueden incrustar en agar y en rodajas en una vibratome. El criostato y preparaciones vibratome fiable dará secciones tan delgadas como 4 m. Si las secciones más finas son necesarias (por ejemplo, para la preparación del microscopio electrónico), es necesario preparar las muestras para el ultramicrotomo. Para ello, las secciones deben ser después de la fijada en tetróxido de osmio durante 1 hora bajo el fumehood. Esto es seguido por deshidratación en una serie gradual de etanol (50, 70, 95, 95, 100, 100%) y el 100% de óxido de propileno. El tejidose incrusta en Epon (incrustar-812 Kit de incorporación). </li></ol> Parte 5: Resultados del representante: En nuestro laboratorio, que habitualmente realizan técnicas de inmunohistoquímica sobre retinas cryosectioned (Figura 2). En este caso estamos interesados ​​en la isodensidades de los receptores cannabinoides (CB1) en la retina del primate. También examinamos los efectos de la exposición prenatal al alcohol en el sistema visual de los primates. Con este fin, estamos interesados ​​en la densidad celular y el espesor de la capa en la fóvea y en la retina periférica. Para lograr esto, integrar las piezas de la retina en Epon, el tramo a 700 nm en un ultramicrotomo, y se tiñen con azul de toluidina al 1% (Figura 3). <img src="/files/ftp_upload/1261/fig1.jpg" alt="la figura 1" /> Figura 1 Retina Flatmount. Cortes radiales se utilizan para aplanar la retina. <img src="/files/ftp_upload/1261/fig2.jpg" alt="la figura 2" /> Figura 2 inmunotinción. Criosección de la retina periférica immunostained de CB1 en las células ganglionares de la retina están fuertemente marcadas con un etiquetado en las capas nucleares interna y externa. El espesor de esta sección es de 14 metros y se tiñeron en la diapositiva. RG - capa ganglionares de la retina; IP - capa plexiforme interna, IN - capa nuclear interna, OP - capa externa plexiforme; ON - la capa nuclear externa. <img src="/files/ftp_upload/1261/fig3.jpg" alt="la figura 3" /> Figura 3 Fovea. Corte a través de la fóvea que se incrustó en Epon y en rodajas en una ultramicrotrome a 700 nm. La capa de fotorreceptores (PR) para alinear las secciones. Tenga en cuenta que toda la extensión de los fotorreceptores se pueden identificar lo que indica un ángulo adecuado en el plano coronal. Mediciones de densidad se tomaron a 300 m, 500 m, y 800 m del centro de la fosa foveal utilizando el sistema de imagen Bioquant. RG - capa ganglionares de la retina; IP - capa plexiforme interna, IN - capa nuclear interna, OP - capa externa plexiforme; ON - la capa nuclear externa, barra de escala = 50 m

<ol>
	<li>Hendrickson, A., Kupfer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+histogenesis+of+the+fovea+in+the+macaque+monkey.">The histogenesis of the fovea in the macaque monkey.</a> Invest Ophthalmol Vis Sci. 15, 746-756 (1976).</li><li>Herbin, M., Boire, D., 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=Size+and+distribution+of+retinal+ganglion+cells+in+the+St.+Kitts+green+monkey+(Cercopithecus+aethiops+sabeus).">Size and distribution of retinal ganglion cells in the St. Kitts green monkey (Cercopithecus aethiops sabeus).</a> J Comp Neurol. 383, 459-472 (1997).</li><li>Stone, 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> The Wholemount Handbook. , (1981).</li><li>Herbin, M., Boire, D., Theoret, H., 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=Transneuronal+degeneration+of+retinal+ganglion+cells+in+early+hemispherectomized+monkeys.">Transneuronal degeneration of retinal ganglion cells in early hemispherectomized monkeys.</a> Neuroreport. 10, 1447-1452 (1999).</li><li>Krebs, W., Krebs, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Primate Retina and Choroid Atlas of Fine Structure in Man and Monkey. , (1991).</li><li>Pow, D., Sullivan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+kinesis,+neurite+sprouting+and+abnormal+axonal+projections+of+cone+photoreceptors+in+the+aged+and+AMD-afflicted+human+retina.">Nuclear kinesis, neurite sprouting and abnormal axonal projections of cone photoreceptors in the aged and AMD-afflicted human retina.</a> Exp Eye Res. 84, 850-857 (2007).</li></ol>

La preparación de la retina como un wholemount permite el análisis de la topografía y la distribución espacial de cualquiera de la capa de células ganglionares y las células endoteliales de los vasos sanguíneos de la retina 3. La cuantificación de la densidad de las células en la periferia de la retina de los primates se logra fácilmente. Sin embargo, en las regiones perifoveal y foveal, el apilamiento de múltiples capas en la capa de células ganglionares obstruye la cuantificación. Para evitar e...

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the gateway to the brain: dissecting the primate eye

El sistema visual en el ser humano se considera la puerta de entrada a todo el mundo y desempeña un papel principal en la gran cantidad de procesos sensoriales, perceptivos y cognitivos. Por lo tanto, no es sorprendente que la calidad de la visión está ligada a la calidad de vida. A pesar de la investigación clínica y básica general sobre las causas de los trastornos visuales, muchas formas de discapacidad visual, como la retinitis pigmentosa y la degeneración macular, la falta de tratamientos efectivos. Los primates no humanos tienen más características generales del desarrollo del ojo a la de los seres humanos. No sólo tienen una anatomía vascular similar, pero entre los otros mamíferos, los primates tienen la característica única de tener una región en la retina temporal especializados de alta agudeza visual, la fóvea<sup&gt; 1</sup&gt;. A continuación se describe una técnica general para la disección de la retina de primates para proporcionar tejidos para histología, inmunohistoquímica, microdisección láser captura, así como la luz y microscopía electrónica. Con el uso extendido de los primates no humanos, como un modelo de traducción, nuestra esperanza es que una mejor comprensión de la retina se proporciona una visión de enfoques eficaces para atenuar o revertir los efectos negativos de los trastornos visuales en la calidad de vida de las personas afectadas.

Watch this Scientific Journal Video about The Gateway to the Brain: Dissecting the Primate Eye at JoVE.com

The Gateway to the Brain: Dissecting the Primate Eye

Los primates no humanos es una importante especie de traslación para nuestra comprensión del desarrollo y el envejecimiento. La organización anatómica de la retina de primates pueden proporcionar información importante sobre las condiciones normales y patológicas en los seres humanos.

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 ...

the-gateway-to-the-brain-dissecting-the-primate-eye

Research

JoVE Journal

Biology

14.1K vistas.  University of Montreal. Los primates no humanos es una importante especie de traslación para nuestra comprensión del desarrollo y el envejecimiento. La organización anatómica de la retina de primates pueden proporcionar información importante sobre las condiciones normales y patológicas en los seres humanos.

Video: The Gateway to the Brain: Dissecting the Primate Eye

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 ...

Investigación

Biología

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Trasplante de lentes en el pez cebra y su aplicación en el análisis de mutantes de ojos

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Dissecting the Non-human Primate Brain in Stereotaxic Space

The use of non-human primates provides an excellent translational model for our understanding of developmental and aging processes in humans1-6. In addition, the use of non-human primates has recently afforded the opportunity to naturally model complex psychiatric disorders such as alcohol abuse7. Here we describe a technique for blocking the brain in the coronal plane of the vervet monkey (Chlorocebus aethiops sabeus) in the intact skull in stereotaxic space. The method described here provides a standard plane of section between blocks and subjects and minimizes partial sections between blocks. Sectioning a block of tissue in the coronal plane also facilitates the delineation of an area of interest. This method provides manageable sized blocks since a single hemisphere of the vervet monkey yields more than 1200 sections when slicing at 50&mu;m. Furthermore by blocking the brain into 1cm blocks, it facilitates penetration of sucrose for cyroprotection and allows the block to be sliced on a standard cryostat.

The non-human primate is an important translational species for our understanding of the normal processing of the brain. The anatomical organization of the primate brain can provide important insights into normal and pathological conditions in humans.

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 ...

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 ...

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries made using this system have taken advantage of the spatiotemporal nature of photoreceptor differentiation in the developing eye imaginal disc. To use this system it is first necessary for the researcher to learn to identify and dissect the eye disc. We describe a novel RFP reporter to aid in the identification of the eye disc and the visualization of specific cell types in the developing eye. We detail a methodology for dissection of the eye imaginal disc from third instar larvae and describe how the eye-RFP reporter can aid in this dissection. This eye-RFP reporter is only expressed in the eye and can be visualized using fluorescence microscopy either in live tissue or after fixation without the need for signal amplification. We also show how this reporter can be used to identify specific cells types within the eye disc. This protocol and the use of the eye-RFP reporter will aid researchers using the Drosophila eye to address fundamentally important biological questions.

A Novel RFP Reporter to Aid in the Visualization of the Eye Imaginal Disc in Drosophila

We describe a novel red fluorescent protein (RFP) reporter that is expressed specifically in the Drosophila eye. We detail a methodology for dissection of the eye imaginal disc and how this reporter can be used to aid in the dissection and identification of specific cell types in the developing eye.

Una novela RFP reportero para ayudar en la visualización del disco imaginal del ojo en Drosophila</em

The Drosophila eye is a powerful model system for studying areas such as neurogenesis, signal transduction and neurodegeneration. Many of the discoveries ...

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, ...

Adipo-Clear: A Tissue Clearing Method for Three-Dimensional Imaging of Adipose Tissue

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte precursors, immune cells, fibroblasts, blood vessels, and nerve projections. Although the molecular control of cell type specification and how these cells interact have been increasingly delineated, a more comprehensive understanding of these adipose-resident cells can be achieved by visualizing their distribution and architecture throughout the whole tissue. Existing immunohistochemistry and immunofluorescence approaches to analyze adipose histology rely on thin paraffin-embedded sections. However, thin sections capture only a small portion of tissue; as a result, the conclusions can be biased by what portion of tissue is analyzed. We have therefore developed an adipose tissue clearing technique, Adipo-Clear, to permit comprehensive three-dimensional visualization of molecular and cellular patterns in whole adipose tissues. Adipo-Clear was adapted from iDISCO/iDISCO+, with specific modifications made to completely remove the lipid stored in the tissue while preserving native tissue morphology. In combination with light-sheet fluorescence microscopy, we demonstrate here the use of the Adipo-Clear method to obtain high-resolution volumetric images&#160;of an entire adipose tissue.

Due to the high lipid content, adipose tissue has been challenging to visualize using traditional histological methods. Adipo-Clear is a tissue clearing technique that allows robust labeling and high-resolution volumetric fluorescent imaging of adipose tissue. Here, we describe the methods for sample preparation, pretreatment, staining, clearing, and mounting for imaging.

Adipo-claro: Un tejido claro método de proyección de imagen tridimensional del tejido adiposo

Adipose tissue plays a central role in energy homeostasis and thermoregulation. It is composed of different types of adipocytes, as well as adipocyte ...

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 ...

A High Output Method to Isolate Cerebral Pericytes from Mouse

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood brain barrier formation and physiology is now demonstrated but its molecular basis remains largely unknown. As the pathophysiological role of cerebral pericytes in neurological disorders is intriguing and of great importance, the in vitro models are not only sufficiently appropriate but also able to incorporate different techniques for these studies. Several methods have been proposed as in vitro models for the extraction of cerebral pericytes, although an antibiotic-free protocol with high output is desirable. Most importantly, a method that has increased output per extraction reduces the usage of more animals.
Here, we propose a simple and efficient method for extracting cerebral pericytes with sufficiently high output. The mouse brain tissue homogenate is mixed with a BSA-dextran solution for the separation of the tissue debris and microvascular pellet. We propose a three-step separation followed by filtration to obtain a microvessel rich filtrate. With this method, the quantity of microvascular fragments obtained from 10 mice is sufficient to seed 9 wells (9.6 cm2 each) of a 6-well plate. Most interestingly with this protocol, the user can obtain 27 pericyte rich wells (9.6 cm2 each) in passage 2. The purity of the pericyte cultures are confirmed with the expression of classical pericyte markers: NG2, PDGFR-&#946; and CD146. This method demonstrates an efficient and feasible in vitro tool for physiological and pathophysiological studies on pericytes.

We present a protocol for the extraction of murine cerebral pericytes. Based on an antibiotic-free enrichment oriented pericyte extraction, this protocol is a valuable tool for in vitro studies providing high purity and high yield, thus decreasing the number of experimental animals used.

Un método de alta salida para aislar los pericytes cerebrales del ratón

In recent years, cerebral pericytes have become the focus of extensive research in vascular biology and pathology. The importance of pericytes in blood ...

A Step-by-Step Guide to Mosquito Electroantennography

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when acquiring a blood-meal. To locate a host to feed on, mosquitoes rely on a wide range of sensory cues, including visual, mechanical, thermal, and olfactory. The study details a technique, electroantennography (EAG), that allows researchers to assess whether the mosquitoes can detect individual chemicals and blends of chemicals in a concentration-dependent manner. When coupled with gas-chromatography (GC-EAG), this technique allows to expose the antennae to a full headspace/complex mixture and determines which chemicals present in the sample of interest, the mosquito can detect. This is applicable to host body odors as well as plant floral bouquets or other ecologically relevant odors (e.g., oviposition sites odorants). Here, we described a protocol that permits long durations of preparation responsiveness time and is applicable to both female and male mosquitoes from multiple genera, including Aedes, Culex, Anopheles, and Toxorhynchites mosquitoes. As olfaction plays a major part in mosquito-host interactions and mosquito biology in general, EAGs and GC-EAG can reveal compounds of interest for the development of new disease vector control strategies (e.g., baits). Complemented with behavioral assays, the valence (e.g., attractant, repellent) of each chemical can be determined.

The present article details a step-by-step protocol for successful and low noise electroantennograms in several genera of mosquitoes, including both females and males.

Una guía paso a paso para la electroantenografía de mosquitos

Female mosquitoes are the deadliest animals on earth, claiming the lives of more than 1 million people every year due to pathogens they transmit when ...