Soutenu par IEN octroi EY014850.

1. Préparation de Sylgard couverts vaisselle, chambres expérimentales, et les lames de rasoir <ol><li> 35 mm Falcon boîtes de Pétri recouvert d&#39;élastomère Sylgard sont nécessaires pour le bon à découper une rétine isolée d&#39;obtenir des cellules photoréceptrices unique. L&#39;élastomère est préparé selon les instructions du fournisseur et une petite quantité est versé dans chaque plat pour couvrir le fond avec une couche. Remplacer le plat couvre après le revêtement et les stocker. Dans quelques jours, «l&#39;élastomère durcit et les plats sont prêts. </li><li> Photorécepteurs isolés besoin de coller au fond de la chambre expérimentale de sorte qu&#39;ils sont immobilisés au cours d&#39;une expérience d&#39;imagerie. Ce résultat est obtenu par revêtement au fond des chambres de poly-L-lysine ou la poly-L-ornithine. Ajouter 200 ul de solution à 0,01% soit un par chambre, et couvrent les chambres avec une serviette en papier pour les protéger de la poussière. Après que la solution a séché, laver les chambres avec de l&#39;eau distillée et conserver dans une boîte fermée. Utiliser dans les 2 semaines. </li><li> Pour nettoyer les chambres à la fin d&#39;une expérience, les laver avec de l&#39;éthanol à 100% pour enlever l&#39;huile de l&#39;objectif à immersion et les débris cellulaires. Pour enlever les débris cellulaires, l&#39;utilisation du coton-tige et frottez soigneusement le fond de la chambre. Ensuite, lavez à l&#39;eau distillée et laisser sécher avant les chambres à un nouveau revêtement. </li><li> Couper les lames de rasoir à double tranchant en petits morceaux (8 d&#39;une lame) avec un cutter en métal. </li></ol> 2. Préparation des solutions <ol><li> La composition des solutions dépend de l&#39;espèce. Pour les amphibiens, les années Ringer a (en mmol / L): 110 NaCl, KCl 2,5, 1,6 MgCl 2, 1 CaCl 2, 5 HEPES, pH = 7,55. Le pH doit être ajusté à la valeur finale avec NaOH. Pour les mammifères, la sonnerie des a (en mmol / L): 130 NaCl, 5 KCl, MgCl 2 0,5, CaCl 2 2, 25 hemisodium-HEPES, pH = 7,40. La solution de Ringer peut être gardé bien fermé à température ambiante pendant quelques mois. </li><li> Solution de glucose Stock, 1 mol / L, conservés à -20 ° C pour éviter la prolifération bactérienne. </li><li> Le jour de l&#39;expérience, ajouter à la solution de glucose à l&#39;Ringer à une concentration finale de 5 mmol / L. A la fin de la journée, jetez la sonnerie de qui contient du glucose, car il pourrait se développer des bactéries. </li></ol> 3. Isolement des rétines <ol><li> Pour l&#39;excision correcte d&#39;une rétine, il est important que l&#39;animal soit adaptés à l&#39;obscurité pendant au moins 2-3 heures avant le sacrifice. Les animaux doivent être adaptés à l&#39;obscurité dans un récipient approprié ventilé dans la chambre noire. </li><li> Remplir deux boîtes de Pétri de 35 mm à mi-chemin avec une solution de Ringer. </li><li> Sacrifier l&#39;animal sous une lumière rouge sombre et de supprimer les yeux. Par la suite, toutes les procédures sont menées sous la lumière infrarouge en utilisant un microscope à dissection ou une caméra avec un moniteur vidéo. </li><li> Retirez tout tissu restant en dehors de la surface de l&#39;œil. Couper et enlever la partie antérieure, puis transférer l&#39;œilleton dans l&#39;une des boîtes de Pétri remplie de Ringer. </li><li> Enlever le vitré et la rétine séparer soigneusement du reste de l&#39;œilleton par pincement ou couper les pièces jointes. Soulevez doucement la rétine et de la séparer totalement de l&#39;œilleton. </li><li> Transférer la rétine pour le deuxième plat de Pétri avec une pipette en plastique. Gardez le plat contenant de la rétine dans une boîte étanche à la lumière. </li></ol> 4. Isolement des cellules photoréceptrices seule <ol><li> Toutes les procédures sont menées sous la lumière infrarouge. Coupez un petit morceau de la rétine et le transférer avec une pipette en plastique pour un plat Sylgard-couvert. Le volume de la solution contenant le morceau rétine devrait être d&#39;environ 250 uL. </li><li> Prenez un petit morceau de lame de rasoir, avec le support de lame - le bord de la lame doit être à environ 45 ° au titulaire. Aplatir le morceau de la rétine sur la couche Sylgard et en utilisant la lame hachez le morceau de la rétine finement tout en la gardant collé à la couche Sylgard. </li><li> Transférer 200 ul de la solution contenant les cellules à une chambre expérimentale - laisser aucun morceau restant de la rétine dans le plat Sylgard-couvert. Gardez la chambre avec les cellules isolées dans une boîte étanche à la lumière. </li><li> Attendez 10 min pour les cellules de s&#39;installer, puis ajouter 2-3 ml de Ringer. A ce stade, les cellules isolées peuvent être chargées avec un colorant fluorescent particulière (par exemple Fura-2) selon le protocole de chargement colorant. </li><li> Les cellules peuvent maintenant être prises pour la platine du microscope pour des expériences. </li></ol> 5. L&#39;imagerie par fluorescence <ol><li> Transfert de la chambre à l&#39;étape du microscope à épifluorescence. Perfusion solution Ajuster, sondes de température, éclairage infrarouge, etc </li><li> Fermez les rideaux et commencer à expérimenter. Allumez la lumière infrarouge dans la cage de microscope, se concentrer sur le fond de la chambre expérimentale, et déplacer la scène à la recherche de cellules. </li></ol> 6. Résultat Représentants: Fig. 1 montre la morphologie de la tige isolés saine et des cônes photorécepteurs obtenus avec ce protocole d&#39;une salamandre (Ambystoma tigrinum) rétine. Salamandre cellules ont été largement utilisés pour des études d&#39;imagerie par fluorescence seule cellule en raison de leur grande taille et leur capacité à survivre pendant plusieurs heures après l&#39;isolement de la rétine. En outre, à partir d&#39;une rétine de salamandre, on peut obtenir régulièrement la tige et des cônes photorécepteurs. Un critère important pour la santé des cellules est la présence d&#39;un ellipsoïde intacte (Fig. 1), la partie de la cellule où les mitochondries sont concentrés. Lorsque les cellules sont considérées dans l&#39;optique DAPI, cette concentration des mitochondries donne un signal fort de fluorescence (Fig. 2) en raison de la présence de NADH. Absence d&#39;un ellipsoïde intacte est un signe d&#39;une cellule endommagée, généralement impropres à l&#39;expérimentation. Fig. 3 montre une salamandre endommagé tige des photorécepteurs, avec un corps cellulaire gonflé et un noyau de condensation. Ces cellules d&#39;affichage beaucoup plus faible fluorescence sous l&#39;optique DAPI, mais vu sous l&#39;optique FITC montrent un signal DCP forte dans la région de l&#39;ellipsoïde (provenant de nucléotides flavine oxydée et flavoprotéines). Un autre critère pour la santé des photorécepteurs isolés, c&#39;est leur capacité à générer des tout-trans rétinol (vitamine A) dans leurs segments externes lors de la stimulation par la lumière. La génération de la vitamine A exige des quantités importantes de NADPH, qui dépend d&#39;une machinerie métabolique intacte. Figures 4 et 5 montrent la formation de la vitamine A dans les segments externes des photorécepteurs grenouille intact et la tige de la souris, respectivement. <img src="/files/ftp_upload/2789/2789fig1.jpg" alt="Figure 1"/> Figure 1. Healthy seule photorécepteurs tige et le cône. Les cellules ont été isolées à partir d&#39;une rétine salamandre tigrée. Phototransduction prend place dans le segment externe et de l&#39;ellipsoïde est très dense avec des mitochondries. Les bâtonnets sont responsables de la vision faible lumière, les cônes pour la vision de la lumière. <img src="/files/ftp_upload/2789/2789fig2.jpg" alt="Figure 2"/> Figure 2. Fluorescence de la vie de salamandre tige et le cône. Ce sont adaptés à l&#39;obscurité des cellules, montrant la fluorescence du NADH forts dans leur ellipsoïdes respectifs et pas de vitamine A par fluorescence significative dans leurs segments externes. La capture de l&#39;image de fluorescence représente leur première exposition à la lumière visible après la période d&#39;adaptation à l&#39;obscurité. <img src="/files/ftp_upload/2789/2789fig3.jpg" alt="Figure 3"/> Figure 3. Damaged salamandre tige photorécepteur. Le corps de la cellule gonflée et le noyau condensé sont révélateurs de dommages. La cellule est oxydé et il est minime signal de NADH (optique DAPI), mais le signal beaucoup plus fort DCP (FITC optique). <img src="/files/ftp_upload/2789/2789fig4.jpg" alt="Figure 4"/> Figure 4. Grenouille tige avec du NADH et le rétinol. Ceci est un photorécepteur grenouille saine tige montrant la fluorescence du NADH forte dans la région de l&#39;ellipsoïde. Avant exposition à la lumière de fluorescence est minime dans le segment externe. Après exposition à la lumière, il ya une augmentation significative de la fluorescence segment externe due à la formation de la vitamine A. <img src="/files/ftp_upload/2789/2789fig5.jpg" alt="Figure 5"/> Figure 5. Souris tige avec le rétinol. Ceci est un photorécepteur de souris saines tige montrant la fluorescence significative segment externe, après exposition à la lumière due à la formation de la vitamine A. Les régions ellipsoïde de photorécepteurs tige de souris ne montrent pas un signal fort de fluorescence.

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Si la santé des cellules isolées ne sont pas obtenus, le problème réside soit avec l&#39;isolement ou la santé de la rétine ou avec son hachage. Généralement, après avoir enlevé le devant de l&#39;œil et le corps vitré, la rétine ascenseurs facilement hors de l&#39;épithélium pigmentaire. Si ce n&#39;est pas, essayez de le retirer à partir de la périphérie de l&#39;œilleton. S&#39;il est encore difficile de séparer, une possibilité probable est que l&#39;animal n&#39;a pas été adaptés à l&#39;ob...

<table border="1"><tbody><tr><th> Nom </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires </th></tr><tr><td> Chambre noire (100-150 pi 2) </td><td></td><td></td><td></td></tr><tr><td> Les feux rouges 19 </td><td></td><td></td><td> Les magasins en ligne </td></tr><tr><td> Sources de lumière infrarouge et les téléspectateurs d&#39;images infrarouges </td><td> FJW Optical Systems, Inc </td><td></td><td></td></tr><tr><td> Loupe binoculaire 19 </td><td></td><td></td><td> Muni de téléspectateurs à infrarouge </td></tr><tr><td> Microscope à épifluorescence enfermé dans une cage étanche à la lumière 19 </td><td></td><td></td><td></td></tr><tr><td> Des outils de dissection (ciseaux, pinces, porte-lame) </td><td> Roboz ou Outils Fine Science </td><td></td><td></td></tr><tr><td> Élastomère Sylgard </td><td> Essex (Charlotte, Caroline du Nord) </td><td> Kit Sylgard élastomère 184 </td><td></td></tr><tr><td> Poly-L-ornithine (0,01%) </td><td> Sigma-Aldrich </td><td> P4957 </td><td></td></tr><tr><td> Poly-L-lysine (0,1%) </td><td> Sigma-Aldrich </td><td> P8920 </td><td> Diluer à 0,01% </td></tr><tr><td> Chambres expérimentales </td><td> Warner Instruments (Hamden, CT) </td><td> D3512P </td><td></td></tr><tr><td> Boîtes de Pétri, pipettes en plastique </td><td> Fisher Scientific </td><td></td><td></td></tr></tbody></table>

preparation of living isolated vertebrate photoreceptor cells for fluorescence imaging

Dans la rétine des vertébrés, phototransduction, la conversion de la lumière en un signal électrique, est effectué par la tige et le cône cellules photoréceptrices 1-4. Rod photorécepteurs sont responsables de la vision dans la pénombre, des cônes de lumière. Phototransduction prend place dans le segment externe de la cellule de photorécepteur, un compartiment spécialisé qui contient une forte concentration de pigment visuel, le détecteur de lumière primaire. Le pigment visuel est composé d&#39;un chromophore, 11 - cis rétinal, attaché à une protéine, l&#39;opsine. Un photon absorbé par le pigment visuel isomérise le chromophore de 11 - cis-trans. Cette photoisomérisation provoque un changement conformationnel dans le pigment visuel qui déclenche une cascade de réactions aboutissant à un changement dans le potentiel de membrane, et entraînant la transduction du stimulus lumineux en un signal électrique. La reprise de la cellule de la stimulation lumineuse implique la désactivation des intermédiaires activés par la lumière, et le rétablissement du potentiel de membrane. Ca 2 + module l&#39;activité de plusieurs enzymes impliquées dans la phototransduction, et sa concentration est réduite lors de la stimulation lumineuse. De cette façon, le Ca 2 + joue un rôle important dans le rétablissement de la cellule de la stimulation lumineuse et son adaptation à la lumière de fond. Une autre partie essentielle du processus de récupération est la régénération du pigment visuel qui a été détruit pendant la lumière de détection par le photoisomérisation de ses 11 - chromophore cis-trans 5-7. Cette régénération commence par la libération de tous les trans-rétinienne par le pigment photoactivé, laissant derrière lui l&#39;opsine apo-protéines. Les libérés tout-trans rétinal est rapidement réduit dans une réaction en utilisant le NADPH au rétinol tout trans, et se combine avec l&#39;opsine 11 frais - rétinien cis introduits dans le segment externe pour réformer le pigment visuel. Tous les trans-rétinol est alors transférée hors du segment externe et dans les cellules voisines par la protéine porteuse rétinoïdes Interphotoreceptor spécialisés de reliure (IRBP). L&#39;imagerie de fluorescence des cellules photoréceptrices unique peut être utilisée pour étudier leur physiologie et biologie cellulaire. Ca 2 + sensibles colorants fluorescents peuvent être utilisés pour examiner en détail l&#39;interaction entre les segments externes Ca 2 + et les changements réponse à la lumière de 8 à 12 ainsi que le rôle du segment intérieur de Ca 2 + magasins dans l&#39;homéostasie du Ca 2 + 13,14 . Les colorants fluorescents peuvent également être utilisés pour mesurer la concentration en Mg 2 + 15, le pH et comme traceurs de compartiments aqueux et la membrane 16. Enfin, la fluorescence intrinsèque des tout-trans rétinol (vitamine A) peuvent être utilisés pour suivre la cinétique de sa formation et l&#39;élimination des cellules photoréceptrices seule 17-19.

Watch this Scientific Journal Video about Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging at JoVE.com

Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

Une méthode est décrite pour la préparation des cellules photoréceptrices de vie simple de différentes espèces de vertébrés pour l&#39;imagerie de fluorescence. La méthode peut être utilisée pour l&#39;image de la fluorescence des fluorophores endogènes, tels que le NADH ou la vitamine A, ou celle de manière exogène ajouté des colorants fluorescents sensibles à Ca<sup&gt; 2 +</sup&gt; Ou d&#39;autres facteurs.

Préparation de vie isolées cellules photoréceptrices vertébrés pour l&#39;imagerie de fluorescence

In the vertebrate retina, phototransduction, the conversion of light to an electrical signal, is carried out by the rod and cone photoreceptor cells1-4.  ...

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Neuroscience

12.2K vues.  Medical University of South Carolina. Une méthode est décrite pour la préparation des cellules photoréceptrices de vie simple de différentes espèces de vertébrés pour l'imagerie de fluorescence. La méthode peut être utilisée pour l'image de la fluorescence des fluorophores endogènes, tels que le NADH ou la vitamine A, ou celle de manière exogène ajouté des colorants fluorescents sensibles à Ca 2 + Ou d'autres facteurs.

Vidéo: Preparation of Living Isolated Vertebrate Photoreceptor Cells for Fluorescence Imaging

An Isolated Retinal Preparation to Record Light Response from Genetically Labeled Retinal Ganglion Cells

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1. This information is then segregated into what are known as the ON and OFF pathways. The photoreceptors signal light information to the bipolar cells (BCs), which depolarize in response to increases (On BCs) or decreases (Off BCs) in light intensity. This segregation of light information is maintained at the level of the retinal ganglion cells (RGCs), which have dendrites stratifying in either the Off sublamina of the inner plexiform layer (IPL), where they receive direct excitatory input from Off BCs, or stratifying in the On sublamina of the IPL, where they receive direct excitatory input from On BCs. This segregation of information regarding increases or decreases in illumination (the On and Off pathways) is conserved and signaled to the brain in parallel.
The RGCs are the output cells of the retina, and are thus an important cell to study in order to understand how light information is signaled to visual nuclei in the brain. Advances in mouse genetics over recent decades have resulted in a variety of fluorescent reporter mouse lines where specific RGC populations are labeled with a fluorescent protein to allow for identification of RGC subtypes 2 3 4 and specific targeting for electrophysiological recording. Here, we present a method for recording light responses from fluorescently labeled ganglion cells in an intact, isolated retinal preparation. This isolated retinal preparation allows for recordings from RGCs where the dendritic arbor is intact and the inputs across the entire RGC dendritic arbor are preserved. This method is applicable across a variety of ganglion cell subtypes and is amenable to a wide variety of single-cell physiological techniques.

This article provides a description of how to dissect and record from the isolated retinal preparation in mouse. In particular, we describe how to record light responses from a fluorescently labeled ganglion cell population and subsequently identify and analyze its morphology.

Une préparation isolée rétinienne pour enregistrer la réponse de Lumière génétiquement marqués cellules ganglionnaires rétiniennes

The first steps in vertebrate vision take place when light stimulates the rod and cone photoreceptors of the retina 1.  This information is then ...

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Neurosciences

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Imagerie des cellules vivantes des cellules sensorielles de précurseurs d&#39;organes en Intact Drosophile Les chrysalides

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Detection of Microregional Hypoxia in Mouse Cerebral Cortex by Two-photon Imaging of Endogenous NADH Fluorescence

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. Here we present a visualized experimental and methodological protocol to directly visualize microregional tissue hypoxia and to infer perivascular oxygen gradients in the mouse cortex. It is based on the non-linear relationship between nicotinamide adenine dinucleotide (NADH) endogenous fluorescence intensity and oxygen partial pressure in the tissue, where observed tissue NADH fluorescence abruptly increases at tissue oxygen levels below 10 mmHg1. We use two-photon excitation at 740 nm which allows for concurrent excitation of intrinsic NADH tissue fluorescence and blood plasma contrasted with Texas-Red dextran. The advantages of this method over existing approaches include the following: it takes advantage of an intrinsic tissue signal and can be performed using standard two-photon in vivo imaging equipment; it permits continuous monitoring in the whole field of view with a depth resolution of ~50 &mu;m. We demonstrate that brain tissue areas furthest from cerebral blood vessels correspond to vulnerable watershed areas which are the first to become functionally hypoxic following a decline in vascular oxygen supply. This method allows one to image microregional cortical oxygenation and is therefore useful for examining the role of inadequate or restricted tissue oxygen supply in neurovascular diseases and stroke.

Here we describe a method to directly visualize microregional tissue hypoxia in the mouse cortex in vivo. It is based on concurrent two-photon imaging of nicotinamide adenine dinucleotide (NADH) and the cortical microcirculation. This method is useful for high resolution analysis of tissue oxygen supply.

La détection de l&#39;hypoxie dans le cortex de souris microrégionale cérébrale par imagerie à deux photons de fluorescence endogène NADH

The brain's ability to function at high levels of metabolic demand depends on continuous oxygen supply through blood flow and tissue oxygen diffusion. ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural progenitor cells with astrocytic features (called SVZ astrocytes), neuroblasts, and intermediate progenitor cells. Neuroblasts born in the SVZ tangentially migrate a great distance to the olfactory bulb, where they differentiate into interneurons. Intercellular signaling through adhesion molecules and diffusible signals play important roles in controlling neurogenesis. Many of these signals trigger intercellular calcium activity that transmits information inside and between cells. Calcium activity is thus reflective of the activity of extracellular signals and is an optimal way to understand functional intercellular signaling among SVZ cells.
Calcium activity has been studied in many other regions and cell types, including mature astrocytes and neurons. However, the traditional method to load cells with calcium indicator dye (i.e. bath loading) was not efficient at loading all SVZ cell types. Indeed, the cellular density in the SVZ precludes dye diffusion inside the tissue. In addition, preparing sagittal slices will better preserve the three-dimensional arrangement of SVZ cells, particularly the stream of neuroblast migration on the rostral-caudal axis.
Here, we describe methods to prepare sagittal sections containing the SVZ, the loading of SVZ cells with calcium indicator dye, and the acquisition of calcium activity with time-lapse movies. We used Fluo-4 AM dye for loading SVZ astrocytes using pressure application inside the tissue. Calcium activity was recorded using a scanning confocal microscope allowing a precise resolution for distinguishing individual cells. Our approach is applicable to other neurogenic zones including the adult hippocampal subgranular zone and embryonic neurogenic zones. In addition, other types of dyes can be applied using the described method.

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described. The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye diffusion.

Préparation des tranches aiguës zone sous-ventriculaire pour l&#39;imagerie calcique

The subventricular zone (SVZ) is one of the two neurogenic zones in the postnatal brain. The SVZ contains densely packed cells, including neural ...

Visualization of Endosome Dynamics in Living Nerve Terminals with Four-dimensional Fluorescence Imaging

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis muscle of the garter snake. Raw data comprises time-lapse sequences of 3D z-stacks. Each stack contains 4-20 images acquired with epifluorescence optics at focal planes separated by 400-1,500 nm. Steps in the acquisition of image stacks, such as adjustment of focus, switching of excitation wavelengths, and operation of the digital camera, are automated as much as possible to maximize image rate and minimize tissue damage from light exposure. After acquisition, a set of image stacks is deconvolved to improve spatial resolution, converted to the desired 3D format, and used to create a 4D "movie" that is suitable for variety of computer-based analyses, depending upon the experimental data sought. One application is study of the dynamic behavior of two classes of endosomes found in nerve terminals-macroendosomes (MEs) and acidic endosomes (AEs)-whose sizes (200-800 nm for both types) are at or near the diffraction limit. Access to 3D information at each time point provides several advantages over conventional time-lapse imaging. In particular, size and velocity of movement of structures can be quantified over time without loss of sharp focus. Examples of data from 4D imaging reveal that MEs approach the plasma membrane and disappear, suggesting that they are exocytosed rather than simply moving vertically away from a single plane of focus. Also revealed is putative fusion of MEs and AEs, by visualization of overlap between the two dye-containing structures as viewed in each three orthogonal projections.

Four-dimensional (4D) imaging is utilized to study the behavior and interactions among two types of endosomes in living vertebrate nerve terminals. Movement of these small structures is characterized in three dimensions, permitting confirmation of events such as endosome fusion and exocytosis.

Visualisation des Endosome Dynamics dans Vivre terminaisons nerveuses avec quatre dimensions d&#39;imagerie par fluorescence

Four-dimensional (4D) light imaging has been used to study behavior of small structures within motor nerve terminals of the thin transversus abdominis ...

Vibratome Sectioning Mouse Retina to Prepare Photoreceptor Cultures

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in contact with the retinal pigmented epithelium in the most distant part on the retina considering the direction of light, and the ganglion cells in the most proximal distance. This architecture allows the isolation of the photoreceptor layer by vibratome sectioning. The dissected neural retina of a mouse aged 8 days is flat-embedded in 4% gelatin on top of a slice of 20% gelatin photoreceptor layer facing down. Using a vibratome and a double edged razor blade, the 100 &#181;m thick inner retina is sectioned. This section contains the ganglion cells and the inner layer with notably the bipolar cells. An intermediary section of 15 &#181;m is discarded before 200 &#181;m of the outer retina containing the photoreceptors is recovered. The gelatin is removed by heating at 37 &#176;C. Pieces of outer layer are incubated in 500 &#181;l of Ringer&#39;s solution with 2 units of activated papain for 20 min at 37 &#176;C. The reaction is stopped by adding 500 &#181;l 10% fetal calf serum (FCS) in Dulbecco&#39;s Modified Eagle Medium (DMEM), then 25 units of DNAse I is added before centrifugation at RT, washed several times to remove serum and the cells are resuspended in 500 &#181;l of DMEM and seeded at 1 x 105 cells/cm2. The cells are grown to 5 days in vitro and their viability scored using live/dead assay. The purity of the culture is first determined by microscopic observation during the experiment. The purity is then validated by seeding and fixing cells on a histological slide and analyzing using a rabbit polyclonal anti-SAG, a photoreceptor marker and mouse monoclonal anti-RHO, a rod photoreceptor specific marker. Alternatively, the photoreceptor layer (97% rods) can be used for gene or protein expression analysis and for transplantation.

Neural retina of a mouse aged 8 days is on top of a 4% gelatin block. After isolation of the photoreceptor layer (200 &#181;m) by vibratome, the photoreceptors are seeded after mechanical and enzymatic dissociation for culture. The photoreceptor layer can be used for molecular, biochemical analyses or transplantation.

Vibratome sectionnement rétine de souris pour préparer des cultures photoréceptrices

The retina is a part of the central nervous system that has organized architecture, with neurons in layers from the photoreceptors, both rods and cones in ...

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to blindness in many retinal diseases. Calcium (Ca2+), a key second messenger in photoreceptor signaling and metabolism, has been proposed to be indirectly linked with photoreceptor degeneration in various animal models. Systematically studying these aspects of cone physiology and pathophysiology has been hampered by the difficulties of electrically recording from these small cells, in particular in the mouse where the retina is dominated by rod photoreceptors. To circumvent this issue, we established a two-photon Ca2+ imaging protocol using a transgenic mouse line that expresses the genetically encoded Ca2+ biosensor TN-XL exclusively in cones and can be crossbred with mouse models for photoreceptor degeneration. The protocol described here involves preparing vertical sections (&#8220;slices&#8221;) of retinas from mice and optical imaging of light stimulus-evoked changes in cone Ca2+ level. The protocol also allows &#8220;in-slice measurement&#8221; of absolute Ca2+ concentrations; as the recordings can be followed by calibration. This protocol enables studies into functional cone properties and is expected to contribute to the understanding of cone Ca2+ signaling as well as the potential involvement of Ca2+ in photoreceptor death and retinal degeneration.

Imaging Ca2+ Dynamics in Cone Photoreceptor Axon Terminals of the Mouse Retina

We describe a protocol to monitor Ca2+ dynamics in the axon terminals of cone photoreceptors using an ex-vivo&#160;slice preparation of the mouse retina. This protocol allows comprehensive studies of cone Ca2+ signaling in an important mammalian model system, the mouse.

Imagerie Ca<sup&gt; 2+</sup&gt; Dynamics dans les terminaux cône photorécepteur Axon de la rétine de souris

Retinal cone photoreceptors (cones) serve daylight vision and are the basis of color discrimination. They are subject to degeneration, often leading to ...

Assay Development for High Content Quantification of Sod1 Mutant Protein Aggregate Formation in Living Cells

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc superoxide dismutase 1 (SOD1). The structural instability of SOD1 and the detection of SOD1-positive inclusions in familial-ALS patients supports a potential causal role for misfolded and/or aggregated SOD1 in ALS pathology. In this study, we describe the development of a cell-based assay designed to quantify the dynamics of SOD1 aggregation in living cells by high content screening approaches. Using lentiviral vectors, we generated stable cell lines expressing wild-type and mutant A4V SOD1 tagged with yellow fluorescent protein and found that both proteins were expressed in the cytosol without any sign of aggregation. Interestingly, only SOD1 A4V stably expressed in HEK-293, but not in U2OS or SH-SY5Y cell lines, formed aggregates upon proteasome inhibitor treatment. We show that it is possible to quantify aggregation based on dose-response analysis of various proteasome inhibitors, and to track aggregate-formation kinetics by time-lapse microscopy. Our approach introduces the possibility of quantifying the effect of ALS mutations on the role of SOD1 in aggregate formation as well as screening for small molecules that prevent SOD1 A4V aggregation.

We describe a method to quantify the aggregation of misfolded proteins. Our protocol details lentiviral induced stable cell line generation, automated confocal imaging, and image analysis of protein aggregates. As an illustrative application, we studied the effect of small molecules in promoting SOD1 aggregation in a time- and dose-dependent manner.

Développement de dosage pour une Quantification contenue élevée de Formation globale de protéine mutante Sod1 dans les cellules vivantes

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that can be caused by inherited mutations in the gene encoding copper-zinc ...

Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are laterally packed in the outermost layer of the retina, the photoreceptor layer, resulting in alignment of analogous subcellular compartments. Traditionally, tangential sectioning of the frozen flat-mounted retina has been used to study the movement and localization of proteins within different rod compartments. However, the high curvature of the rod-dominant mouse retina makes tangential sectioning challenging. Motivated by the study of protein transport between compartments, we developed two peeling methods that reliably isolate the rod outer segment (ROS) and other subcellular compartments for western blots. Our relatively quick and simple techniques deliver enriched and subcellular-specific fractions to quantitatively measure the distribution and redistribution of important photoreceptor proteins in normal rods. Moreover, these isolation techniques can also be easily adapted to isolate and quantitatively investigate the protein composition of other cellular layers within both healthy and degenerating retinae.

This protocol presents two techniques to isolate the subcellular compartments of murine rod photoreceptors for protein analysis. The first method utilizes live retinae and cellulose filter paper to separate rod outer segments, while the second employs lyophilized retinae and adhesive tape to peel away rod inner and outer segment layers.

Deux méthodes de peeling pour l’isolement des compartiments des cellules photoréceptrices dans la rétine de la souris pour l’analyse des protéines

Rod photoreceptors are highly polarized sensory neurons with distinct compartments. Mouse rods are long (~80 &#181;m) and thin (~2 &#181;m) and are ...

Millisecond Hydrogen/Deuterium-Exchange Mass Spectrometry for the Study of Alpha-Synuclein Structural Dynamics Under Physiological Conditions

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks of Parkinson's disease. Yet, much of its biological activity, as well as its aggregation, centrally involves the soluble monomer form of the protein. Elucidation of the molecular mechanisms of aSyn biology and pathophysiology requires structurally highly resolved methods and is sensitive to biological conditions. Its natively unfolded, meta-stable structures make monomeric aSyn intractable to many structural biology techniques. Here, the application of one such approach is described: hydrogen/deuterium-exchange mass spectrometry (HDX-MS) on the millisecond timescale for the study of proteins with low thermodynamic stability and weak protection factors, such as aSyn. At the millisecond timescale, HDX-MS data contain information on the solvent accessibility and hydrogen-bonded structure of aSyn, which are lost at longer labeling times, ultimately yielding structural resolution up to the amino acid level. Therefore, HDX-MS can provide information at high structural and temporal resolutions on conformational dynamics and thermodynamics, intra- and inter-molecular interactions, and the structural impact of mutations or alterations to environmental conditions. While broadly applicable, it is demonstrated how to acquire, analyze, and interpret millisecond HDX-MS measurements in monomeric aSyn.

The structural ensemble of monomeric alpha-synuclein affects its physiological function and physicochemical properties. The present protocol describes how to perform millisecond hydrogen/deuterium-exchange mass spectrometry and subsequent data analyses to determine conformational information on the monomer of this intrinsically disordered protein under physiological conditions.

Spectrométrie de masse à échange hydrogène/deutérium milliseconde pour l’étude de la dynamique structurelle de l’alpha-synucléine dans des conditions physiologiques

Alpha-synuclein (aSyn) is an intrinsically disordered protein whose fibrillar aggregates are abundant in Lewy bodies and neurites, which are the hallmarks ...