Les auteurs tiennent à remercier pour son Ikiel Ptito support technique. Nous sommes reconnaissants à Frank Ervin, Roberta Palmour et le personnel des laboratoires de sciences du comportement de la Fondation situé à St-Kitts, dans les Antilles, pour leur soutien continu de notre travail primates.

Partie 1: Pré-traitement des tissus <ol><li> Les tissus doivent être bien perfusés avec du paraformaldéhyde, glutaraldéhyde, ou du formol. Ceci peut être réalisé grâce à la perfusion transcardial norme généralement utilisée pour la récolte d&#39;autres organes. Il est recommandé que, peu après le sacrifice des yeux être injecté avec fixateur juste sous la lentille et stockés dans un fixateur. </li><li> Dans l&#39;étude actuelle, l&#39;objet a été profondément sédatés avec le chlorhydrate de kétamine (10 mg / kg, im), euthanasiés avec une overdose de pentobarbital sodique (25 mg / kg, iv) et perfusé transcardiaque avec PBS 0,1 M jusqu&#39;à ce que complètement exsangue. Il est suivi par une solution de paraformaldéhyde 4% dans du PBS pendant 5 min (~ 1 litre). </li></ol> Partie 2: Suppression de l&#39;œil de la cavité orbitaire <ol><li> Pour faciliter l&#39;accès à l&#39;œil, il est recommandé d&#39;enlever d&#39;abord le cerveau. Une fois que le cerveau a été enlevée de l&#39;os en orbite à paroi mince est évident. Utilisez la pince gouge os à ronger lentement la paroi de l&#39;orbite. Coupez les muscles oculaires avec un scalpel et retirez le tissu conjonctif du globe oculaire. Découpez soigneusement le nerf optique, ce qui peut être utilisée dans les études de microscopie électronique. Le globe oculaire devrait maintenant être libéré de la cavité orbitaire. </li></ol> Partie 3: Disséquer la rétine de l&#39;œilleton <ol><li> Placez l&#39;oeil dans une boîte de Pétri avec du PBS pour maintenir la rétine de sécher. Un microscope à dissection ou sur une table se loupe lumière est utile dans la dissection, mais pas une nécessité. Retirez la cornée en coupant la sclérotique près du périmètre de la cornée au niveau de l&#39;ora serrata avec une paire de ciseaux à ressort et enlever la lentille avec la pince. </li><li> Utilisez un pinceau, des pinces, des ciseaux et de printemps pour enlever la rétine de la sclère. Cela se fait en séparant la rétine de la sclère, puis couper la sclère écart avec les ciseaux. Il faut mener à bien ce processus dans de petits incréments afin de ne pas endommager ou déchirer le tissu rétinien. La sclérotique n&#39;est pas facilement séparées du reste du nerf optique si soigneusement coupé la sclère autour du nerf optique. </li><li> A ce point de la rétine a conservé sa forme incurvée et doit être aplatie pour l&#39;échantillonnage. Avant aplatissement de la rétine, l&#39;humeur vitrée, qui a la consistance de la gelée, peuvent être retirés dans un morceau. Pour aplatir la rétine sur une diapositive, faire plusieurs coupures radiales avec une lame de bistouri. L&#39;humeur vitrée résiduelle peut maintenant être enlevée avec du papier filtre ordinaire et d&#39;un pinceau. La couche de cellules ganglionnaires de la rétine est exposée à ce point il est donc impératif d&#39;être doux pour retirer le corps vitré. Si le nerf optique est encore attaché à la papille optique, retirez le nerf optique, sans déchirure de la rétine à l&#39;aide d&#39;un scalpel et d&#39;une paire de ciseaux à ressort. C&#39;est désormais une rétine support plat (figure 1) et la fovéa devrait être évident que une tache sombre dans la direction temporelle / ventrale du disque optique. </li></ol> Partie 4: Échantillonnage <ol><li> Il ya un certain nombre d&#39;options pour le prélèvement de la rétine. Ici, nous allons décrire la préparation et l&#39;échantillonnage flatmount isodentric. Pour les deux procédures flatmount la rétine avec la couche de fibres optiques loin de la glissière. Si l&#39;intention est d&#39;examiner la couche de cellules ganglionnaires de la rétine dans la préparation flatmount, il est nécessaire de supprimer ou eau de Javel l&#39;épithélium pigmenté. Pour enlever l&#39;épithélium pigmenté utiliser un pinceau légèrement pour enlever une partie de cette couche, ce qui tend à endommager la couche des photorécepteurs. Blanchiment consiste à tremper dans une solution de la rétine de permanganate de potassium (0,25%) pendant 1 heure, laver à l&#39;eau distillée, et la compensation de l&#39;acide oxalique (5%) pendant 5 minutes. 2 </li><li> Si l&#39;intention est d&#39;examiner la distribution de cellules et la morphologie de chaque couche à travers la rétine, alors nous vous suggérons d&#39;échantillonnage isométrique de la rétine. La rétine doit être flatmounted sur une lame et maintenu humide avec du PBS. Pour l&#39;échantillonnage isométrique coupé de petits morceaux de tissus à égale distance de la papille optique dans le nez, les directions temporelles, supérieure et inférieure (Figure 1). Il n&#39;est pas nécessaire de blanchir l&#39;épithélium pigmenté dans cette préparation. Ces pièces peuvent désormais être sectionnée dans le plan frontal avec un cryostat, vibratome ou ultramicrotome en fonction de la question de recherche. Pour sectionner cryostat, les pièces doivent être cryoprotégés de saccharose à 30% du jour au lendemain et congelé dans le contexte d&#39;un milieu de montage sur glace sèche. Alternativement, les échantillons peuvent être intégrés dans l&#39;agar et tranché sur une vibratome. Le cryostat et préparations vibratome de manière fiable le rendement des sections aussi fine que 4 m. Si des sections plus minces sont nécessaires (par exemple pour la préparation au microscope électronique), il est nécessaire de préparer les échantillons pour les ultramicrotome. Pour ce faire, les sections doivent être post-fixe dans le tétroxyde d&#39;osmium pendant 1 heure sous la hotte. Elle est suivie par une déshydratation dans une série d&#39;éthanol à (50, 70, 95, 95, 100, 100%) et l&#39;oxyde de propylène de 100%. Le tissuest incorporé dans l&#39;Epon (Embed-812 Kit incorporation). </li></ol> Partie 5: Résultats du représentant: Dans notre laboratoire, nous avons l&#39;habitude de réaliser immunohistochimie sur la rétine cryosectioned (figure 2). Dans ce cas, nous sommes intéressés à l&#39;isodensité des récepteurs cannabinoïdes (CB1) dans la rétine des primates. Nous examinons également les effets de l&#39;exposition prénatale éthanol sur le système visuel des primates. À cette fin, nous sommes intéressés à la densité des cellules et l&#39;épaisseur de la couche dans la fovéa et de la rétine périphérique. Pour ce faire, nous intégrons des morceaux de la rétine dans l&#39;Epon, tranche à 700nm sur un ultramicrotome, et tacher avec 1% de bleu de toluidine (figure 3). <img src="/files/ftp_upload/1261/fig1.jpg" alt="Figure 1" /> Retina Figure Flatmount 1. Coupes radiales sont utilisés pour aplatir la rétine. <img src="/files/ftp_upload/1261/fig2.jpg" alt="Figure 2" /> Figure 2 Immunocoloration. Cryosection de rétine périphérique immunocolorées pour CB1 où les cellules ganglionnaires de la rétine sont fortement étiqueté avec la mention de certains dans les couches intérieure et extérieure nucléaire. L&#39;épaisseur de cette section est de 14 m et colorées sur la diapositive. RG - couche ganglionnaires de la rétine; IP - couche plexiforme interne; EN - couche intérieure nucléaire; OP - couche externe plexiforme; ON - couche externe nucléaire. <img src="/files/ftp_upload/1261/fig3.jpg" alt="Figure 3" /> Figure 3 Fovea. Section à travers la fovéa qui a été incorporé dans l&#39;Epon et tranché sur une ultramicrotrome à 700nm. La couche des photorécepteurs (PR) a été utilisé pour aligner les sections. Notez que toute l&#39;étendue des photorécepteurs peuvent être identifiées en indiquant un angle approprié dans le plan coronal. Les mesures de densité ont été prises à 300 m, 500 m et 800 m du centre de la fosse fovéale utilisant le système d&#39;imagerie Bioquant. RG - couche ganglionnaires de la rétine; IP - couche plexiforme interne; EN - couche intérieure nucléaire; OP - couche externe plexiforme; ON - couche externe nucléaire; barre d&#39;échelle = 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 préparation de la rétine comme un wholemount permet l&#39;analyse de la topographie et la répartition spatiale soit de la couche des cellules ganglionnaires ou les cellules endothéliales des vaisseaux sanguins de la rétine 3. La quantification de la densité cellulaire dans la périphérie de la rétine des primates est facilement réalisée. Cependant, dans les régions et les périfovéolaires fovéale, l&#39;empilement de couches multiples dans la couche des cellules ganglionnaires obstrue la quant...

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the gateway to the brain: dissecting the primate eye

Le système visuel chez l&#39;homme est considéré comme la passerelle vers le monde et joue un rôle principal dans la pléthore de sensorielle, les processus perceptifs et cognitifs. Il n&#39;est donc pas surprenant que la qualité de la vision est liée à la qualité de vie. Malgré des recherches cliniques et fondamentales généralisé entourant les causes de troubles visuels, de nombreuses formes de déficiences visuelles, telles que la rétinite pigmentaire et la dégénérescence maculaire, le manque de traitements efficaces. Les primates non humains ont le plus proche caractéristiques générales du développement de l&#39;œil à celui des humains. Non seulement ils ont une anatomie similaire vasculaire, mais parmi les autres mammifères, les primates ont la caractéristique unique d&#39;avoir une région de la rétine temporale spécialisés pour l&#39;acuité visuelle élevée, la fovéa<sup&gt; 1</sup&gt;. Nous décrivons ici une technique générale pour disséquer la rétine des primates pour fournir des tissus pour l&#39;histologie de la rétine, l&#39;immunohistochimie, microdissection laser, ainsi que la lumière et la microscopie électronique. Avec l&#39;utilisation prolongée du primate non-humain comme un modèle de translation, notre espoir est qu&#39;une meilleure compréhension de la rétine se donnent un aperçu des approches efficaces en direction d&#39;atténuer ou d&#39;inverser l&#39;impact négatif des troubles visuels sur la qualité de vie des personnes touchées.

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

Le primate non-humain est une espèce importante translationnelle pour notre compréhension du développement et du vieillissement. L&#39;organisation anatomique de la rétine des primates peuvent fournir des indications importantes sur les conditions normales et pathologiques chez l&#39;homme.

La Passerelle au cerveau: Disséquer les yeux Primat

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 vues.  University of Montreal. Le primate non-humain est une espèce importante translationnelle pour notre compréhension du développement et du vieillissement. L'organisation anatomique de la rétine des primates peuvent fournir des indications importantes sur les conditions normales et pathologiques chez l'homme.

Vidéo: 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.

Dissection et d&#39;enregistrement de la jonction neuromusculaire 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 ...

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Biologie

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.

Objectif transplantation chez le poisson zèbre et son application dans l&#39;analyse de mutants Eye

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.

Disséquer le cerveau des primates non humains dans l&#39;espace stéréotaxique

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.

Sachant ce qui compte: Stéréologie impartial dans le cerveau des primates non humains

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.

Un journaliste Roman DP pour faciliter la visualisation du disque Eye Imaginal au Drosophile</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.

L&#39;échantillonnage de sang de la veine caudale latérale de la Rat

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-Clear : Un tissu méthode de compensation pour l’imagerie en trois dimensions du tissu adipeux

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.

Inverser la Dissection et données DiceCT Reveal autrement cachés dans l’évolution du visage de primates

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.

Une méthode de sortie élevée pour isoler les péricartétes cérébraux de la souris

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.

Un guide étape par étape de l’électroanténnographie des moustiques

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 ...