Ce travail a été soutenu par l&#39;Institut national sur la surdité et autres troubles de communication (NIDCD) programme intra-muros.

1. Transfection Neuron <ol><li> Culture embryonnaire des neurones de rat jour 18 (E18) hippocampique sur le poly-D-lysine MatTek de 35 mm à fond de verre plats 1. Les 16-18 jours in vitro (DIV), les neurones transfecter utilisant le kit Clontech Calphos transfection mammifères. Tout d&#39;abord, remplacer le milieu de culture avec 1,5 ml <a href="http://www.invitrogen.com/site/us/en/home/Products-and-Services/Applications/Cell-Culture/Mammalian-Cell-Culture/Classical_Media/dmem.html">Medium Eagle modifié par Dulbecco</a> (DMEM) par 35 mm, plat 0,5 heures avant la transfection. Enregistrer le milieu de culture d&#39;origine dans un tube de 15 ml stériles pour plus tard (étape 1.6) utilisation. </li><li> Mélanger 10 g pEGFP-N1 ADN plasmidique avec H 2 O stérile (Clontech) et 12,4 ul 2 M solution de calcium (Clontech) pour un volume total de 100 pl. </li><li> Ajouter le mélange de l&#39;étape 1.2) à 100 ul 2 x HBS goutte à goutte tout en vortex 2 x HBS à vitesse moyenne. </li><li> Laissez reposer le mélange à la température ambiante pendant 20 minutes puis ajouter le mélange final de l&#39;étape 1.3) dans du DMEM-incubées neurones. </li><li> Mettez les neurones de nouveau dans l&#39;incubateur 37 ° C pendant 1-1,5 heures. </li><li> Retirez le phosphate contenant du calcium moyen, puis laver les cellules avec du DMEM à trois reprises. Avant de retourner la boîte de culture à l&#39;incubateur, l&#39;échange du milieu DMEM avec le milieu de culture d&#39;origine. </li></ol> 2. FRAP expérimentation sur une colonne vertébrale <ol><li> Les neurones sont utilisés pour l&#39;expérience FRAP deux à quatre jours après la transfection. </li><li> Remplacez le milieu de culture de la 35-mm à fond de verre plat, en mettant immédiatement en ajoutant la solution de Tyrode préchauffé, qui contient (en mM) NaCl 145, KCl 5, HEPES 10, 10 Glucose, Glycine 0,005, CaCl 2 2,6, et MgCl 2 1.3 (pH ajusté à 7,4 avec NaOH). </li><li> Un Zeiss LSM 710 microscope confocal est utilisé pour l&#39;expérience du FRAP. Le système TempModule Zeiss est utilisé pour contrôler la température (37 ° C), l&#39;humidité et le CO 2 (5%) du système de travail. Assurez-vous que le réservoir de CO 2 est connecté et la bouteille d&#39;eau, qui est utilisé pour l&#39;équilibrage de l&#39;humidité, est rempli d&#39;eau. </li><li> Trouver une dendrite transfectées avec la maturité de 100 × objectif (αplan-Apochromat 100 × / 1,46 huile). Si les cellules transfectées dans le plat sont rares, la recherche d&#39;une cellule souhaitée avec l&#39;objectif de 40 × (Plan-Neofluar 40 × / 1,3 huile), puis passer à l&#39;objectif de 100 × pour capturer des images. </li><li> Utilisez 5 x zoom optique et une résolution de 256 × 256 pixels pour l&#39;image d&#39;une courte pièce de dendrites avec des épines de plusieurs. Pour capturer des images, utilisez la vitesse nominale 9 (pixel temporisation 3,15 msec) qui prend 0,5 secondes pour terminer un scan. Le sténopé est fixé à 2 pm pour obtenir une forte fluorescence. Lors de la prise d&#39;images, essayez d&#39;utiliser la transmission par laser basse, par exemple 1-5%, pour éviter de photoblanchiment toute l&#39;image. </li><li> Sélectionnez la colonne vertébrale de l&#39;intérêt. Dans notre expérience, nous choisissons épines champignons avec leurs diamètres de la tête du rachis de ~ 1 um. </li><li> Pour faire une expérience FRAP, prendre 5 images de contrôle avant le blanchiment, puis l&#39;eau de Javel de la colonne vertébrale d&#39;intérêt 10 fois à la transmission par laser nominale de 100% [La puissance laser à l&#39;argon est de 30 mW, avec 50% (15 MW) dans la ligne 488 au laser, et mW environ 3-4 atteint le microscope par la ligne 488 au laser], puis capturer une série d&#39;images, immédiatement après le blanchiment. Pour cette expérience, les images sont capturées chaque seconde 1 pendant 15 secondes après le blanchiment. L&#39;intervalle de temps doit être ajustée en fonction de différentes protéines de ciblage et de différents modèles expérimentaux (Fig. 1). </li><li> Enregistrer des images. </li></ol> 3. L&#39;analyse des données <ol><li> Ouvrez les images avec logiciel ImageJ. </li><li> Alignez la pile d&#39;images en utilisant l&#39;outil aligner («stacks - brassage &#39;plugins&#39; → → &#39;aligner les tranches dans la pile&#39; → &#39;corps rigide&#39;« traduction »et / ou) dans ImageJ, de sorte que la colonne vertébrale d&#39;intérêt ne flottent pas , en d&#39;autres termes - il reste dans la même position sur l&#39;image. </li><li> Mesure relative intensité de la fluorescence de la colonne vertébrale d&#39;intérêt (F s), une région transfectées mais non blanchie (contrôle), et une région de fond (F b) dans les images laps de temps, en utilisant l&#39;outil &#39;intensité V Temps Moniteur &quot;de ImageJ (« plugins &#39;→&#39; stacks - brassage &#39;→&#39; intensité v Heure Monitor &#39;). La région de contrôle pourrait être un morceau de bien ciblée dendrites distales. La région de fond est une région non-fluorescent. </li><li> Calculer le taux de photoblanchiment (r) en comparant la fluorescence de la région de contrôle avant (F C0) et après (F c) photoblanchiment. r = F C / F C0. </li><li> Normaliser l&#39;intensité de fluorescence de la colonne vertébrale cible (F) comme suit: F = (F s - F b) / R. </li><li> Courbe d&#39;ajustement de l&#39;intensité de la fluorescence de la colonne vertébrale cible avec une équation d&#39;une phase exponentielle de logiciel GraphPad Prism (fig. 2) ou Other un logiciel similaire. </li><li> Calculer la fraction mobile (f m) et la fraction immobile (f i) par les équations suivantes: f m = F ∞ / F 0, où F ∞ est l&#39;intensité de fluorescence après guérison complète, et F 0 est l&#39;intensité de fluorescence avant photoblanchiment. f i = 1 - F M. </li></ol> 4. Les résultats représentatifs: Dans cette étude, nous procédons à une expérience sur le FRAP matures neurones de l&#39;hippocampe. À 18-22 DIV, les épines de champignons sont déjà formées. Grâce à notre méthode, les changements dynamiques de l&#39;intensité de fluorescence dans une petite région, comme une colonne vertébrale, peut être enregistrée. Pour analyser le processus de récupération de fluorescence de l&#39;EGFP, nous prenons 5 images que les contrôles avant le blanchiment et ensuite une image toutes les 1 secondes immédiatement après blanchiment pendant 15 secondes. La résolution de l&#39;image est suffisante pour une analyse quantitative. Les profils de recouvrement de fluorescence des protéines étiquetées par fluorescence sont hautement reproductibles. Nous avons également montrer brièvement comment définir les fractions mobiles et immobiles d&#39;une protéine de fluorescence, en utilisant le logiciel ImageJ et Prism Graphpad. La méthode FRAP et de l&#39;analyse que nous présentons ici peut être largement utilisé dans les neurosciences, la biologie cellulaire et d&#39;autres études. <img src="/files/ftp_upload/2568/2568fig1.jpg" alt="Figure 1" /> Figure 1. FRAP mesures de fluorescence dans EGFP une colonne vertébrale à partir d&#39;un neurone en culture d&#39;hippocampe. Les flèches rouges indiquent les temps de photoblanchiment. Photographies représentent la même zone avant (Pre) et à 0, 1, 5, 10, 15 secondes après photoblanchiment. La région de la colonne vertébrale, de contrôle et de fond sont marqués avec les lettres S, C et B, respectivement. Les neurones ont été maintenues à 37 ° C pendant l&#39;expérience. La barre d&#39;échelle, une um. <img src="/files/ftp_upload/2568/2568fig2.jpg" alt="Figure 2" /> Figure 2. FRAP courbes de EGFP par fluorescence sur une période de 15 secondes. La ligne verte représente la courbe d&#39;origine, la ligne rouge montre la courbe normalisée. Les points sur les courbes montrent la FRAP chaque seconde 1. Les courbes ont été montés par une phase équations exponentielles. La fluorescence moyenne avant photoblanchiment a été compté comme 100%. Dans cette expérience, la fraction mobile (MF) est de 94% et la fraction immobile (SI) est de 6%.

<ol>
	<li>Zheng, C. Y., Petralia, R. S., Wang, Y. X., Kachar, B., Wenthold, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SAP102+is+a+highly+mobile+MAGUK+in+spines.">SAP102 is a highly mobile MAGUK in spines.</a> J Neurosci. 30, 4757-4766 (2010).</li><li>Grati, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+turnover+of+stereocilia+membrane+proteins:+evidence+from+the+trafficking+and+mobility+of+plasma+membrane+Ca(2+)-ATPase+2.">Rapid turnover of stereocilia membrane proteins: evidence from the trafficking and mobility of plasma membrane Ca(2+)-ATPase 2.</a> J Neurosci. 26, 6386-6395 (2006).</li><li>Liu, L. N., Aartsma, T. J., Thomas, J. C., Zhou, B. C., Zhang, Y. Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+Analysis+on+Red+Alga+Reveals+the+Fluorescence+Recovery+Is+Ascribed+to+Intrinsic+Photoprocesses+of+Phycobilisomes+than+Large-Scale+Diffusion.">FRAP Analysis on Red Alga Reveals the Fluorescence Recovery Is Ascribed to Intrinsic Photoprocesses of Phycobilisomes than Large-Scale Diffusion.</a> PLoS ONE. 4, e5295-e5295 (2009).</li><li>Wiedenmann, J., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live-cell+imaging+with+EosFP+and+other+photoactivatable+marker+proteins+of+the+GFP+family.">Live-cell imaging with EosFP and other photoactivatable marker proteins of the GFP family.</a> Expert Rev Proteomics. 3, 361-374 (2006).</li><li>Shaner, N. C., Patterson, G. H., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+fluorescent+protein+technology.">Advances in fluorescent protein technology.</a> J Cell Sci. 120, 4247-4260 (2007).</li><li>Sprague, B. L., McNally, J. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FRAP+analysis+of+binding:+proper+and+fitting.">FRAP analysis of binding: proper and fitting.</a> Trends Cell Biol. 15, 84-91 (2005).</li></ol>

Analyse du PAF a été largement utilisée in vivo et in vitro 1-2 études. Cette technique utilise couramment la GFP <a href="http://en.wikipedia.org/wiki/Fusion_protein" title="Protéine de fusion">des protéines de fusion</a> , mais elle pourrait également utiliser les protéines de fusion algue rouge 3. Cette analyse est sensible et peut être utilisée pour caractériser la mobilité des protéines étiquetées GFP. Pour produire une analyse FRAP sens, il est important d&#39;éviter de photoblanchiment inutiles avant et pendant l&#39;expérience de FRAP. Il ya deux façons d&#39;y parvenir. Premièrement, le processus de rechercher et d&#39;observer les neurones expérimentale devrait être rapide. Surtout, l&#39;observation des neurones avec un objectif 100X pour une longue période blanchit de manière significative la fluorescence. Deuxièmement, la puissance du laser à balayage élevée et fréquente souvent à accroître la possibilité de photoblanchiment. Ainsi, il sera nécessaire de calculer le taux de photoblanchiment dans une région de contrôle puis de normaliser la courbe du PAF dans la région expérimentale. La méthode de normalisation a été décrite dans le protocole (voir l&#39;étape 3.3 à 3.5 pour plus de détails). L&#39;étape photoblanchiment est également essentielle pour assurer des résultats FRAP bon. Dans cette expérience, nous avons l&#39;eau de Javel de la colonne vertébrale d&#39;intérêt 10 fois à 100% de transmission laser. Cette condition est suffisante pour blanchir la fluorescence d&#39;une colonne vertébrale au niveau de base dans une préparation fixée. Ainsi, nous avons mis de l&#39;intensité de fluorescence pour le même nombre que de fond à 0 secondes après photoblanchiment. Selon la vitesse de la première analyse, une quantité importante de récupération de fluorescence peut-être déjà détecté lors de la protéine d&#39;intérêt est très mobile. Beaucoup de sondes protéine fluorescente ont été développées pour étudier la dynamique des protéines avec des FRAP. Une approche complémentaire est, par exemple, d&#39;utiliser la GFP photoactivables (PA-GFP), ou des variantes photoconvertible. Ensemble, avec la technique du FRAP, ces outils deviennent indispensables pour vivre des études d&#39;imagerie cellulaire 4-6.

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<td>35 mm glass-bottom dishes </td>
<td>&nbsp;</td>
<td>MatTek</td>
<td>P35GC-1.0-14-C</td>
<td>&nbsp;</td>
<tr>
<td>CalPhos Mammalian Transfection Kit</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>631312</td>
<td>&nbsp;</td>
<tr>
<td>pEGFP-N1 plasmid</td>
<td>&nbsp;</td>
<td>Clontech</td>
<td>6085-1</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss confocal microscope</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>LSM 710</td>
<td>&nbsp;</td>
<tr>
<td>Zeiss TempModule system</td>
<td>&nbsp;</td>
<td>Zeiss</td>
<td>N.A.</td>
<td>&nbsp;</td>
<tr>
<td>ImageJ software</td>
<td>&nbsp;</td>
<td>NIH</td>
<td>N.A.</td>
<td>&nbsp;</td>
<tr>
<td>Graphpad Prism software</td>
<td>&nbsp;</td>
<td>Graphpad software</td>
<td>N.A.</td>
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fluorescence recovery after photobleaching (frap) of fluorescence tagged proteins in dendritic spines of cultured hippocampal neurons

FRAP a été utilisée pour quantifier la mobilité de la GFP-protéines étiquetées. L&#39;utilisation d&#39;un laser d&#39;excitation forte, la fluorescence d&#39;une protéine GFP-étiqueté est blanchi dans la région d&#39;intérêt. La fluorescence de la région récupère lorsque l&#39;écru GFP-protéine marquée de l&#39;extérieur de la région se diffuse dans la région d&#39;intérêt. La mobilité de la protéine est ensuite analysé en mesurant le taux de recouvrement de fluorescence. Cette technique pourrait être utilisée pour caractériser la mobilité des protéines et le taux de roulement. Dans cette étude, nous exprimons le vecteur (enhanced green fluorescent protein) EGFP dans des cultures de neurones de l&#39;hippocampe. Utilisation du microscope confocal Zeiss 710, nous photobleach du signal de fluorescence de la protéine GFP dans une colonne unique, puis de prendre des images laps de temps pour enregistrer le recouvrement de fluorescence après photoblanchiment. Enfin, nous estimons que le pourcentage des fractions mobiles et immobiles de la GFP dans les épines, en analysant les données d&#39;imagerie utilisant ImageJ et logiciels Graphpad. Ce protocole FRAP montre comment effectuer une expérience FRAP base ainsi que la façon d&#39;analyser les données.

Watch this Scientific Journal Video about Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons at JoVE.com

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP a été utilisée pour quantifier la mobilité des protéine de fluorescence verte (GFP)-protéines étiquetées dans les cellules cultivées. Nous avons examiné les fractions mobiles / immobiles de la GFP en analysant le pourcentage de recouvrement de fluorescence après photoblanchiment. Dans cette étude, le FRAP a été réalisée à épines des neurones de l&#39;hippocampe.

Récupération de fluorescence après photoblanchiment (FRAP) de fluorescence protéines marquées dans les épines dendritiques des neurones de l&#39;hippocampe de culture

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

fluorescence-recovery-after-photobleaching-frap-fluorescence-tagged

Research

JoVE Journal

Neuroscience

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

54.1K vues.  National Institutes of Health, Bethesda. FRAP a été utilisée pour quantifier la mobilité des protéine de fluorescence verte (GFP)-protéines étiquetées dans les cellules cultivées. Nous avons examiné les fractions mobiles / immobiles de la GFP en analysant le pourcentage de recouvrement de fluorescence après photoblanchiment. Dans cette étude, le FRAP a été réalisée à épines des neurones de l'hippocampe.

Vidéo: Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

L&#39;analyse de la morphologie des épines dendritiques dans les neurones du SNC de culture

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

Recherche

Neurosciences

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Induire la croissance dendritique dans des cultures de neurones sympathiques

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Diffusion latérale et l&#39;exocytose des protéines membranaires dans des neurones en culture évaluée à l&#39;aide de récupération de fluorescence et de fluorescence de perte de photoblanchiment

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Voltage-sensitive Dye Recording from Axons, Dendrites and Dendritic Spines of Individual Neurons in Brain Slices

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding how the brain works. The primary function of any nerve cell is to process electrical signals, usually from multiple sources. Electrical properties of neuronal processes are extraordinarily complex, dynamic, and, in the general case, impossible to predict in the absence of detailed measurements. To obtain such a measurement one would, ideally, like to be able to monitor, at multiple sites, subthreshold events as they travel from the sites of origin on neuronal processes and summate at particular locations to influence action potential initiation. This goal has not been achieved in any neuron due to technical limitations of measurements that employ electrodes. To overcome this drawback, it is highly desirable to complement the patch-electrode approach with imaging techniques that permit extensive parallel recordings from all parts of a neuron. Here, we describe such a technique - optical recording of membrane potential transients with organic voltage-sensitive dyes (Vm-imaging) - characterized by sub-millisecond and sub-micrometer resolution. Our method is based on pioneering work on voltage-sensitive molecular probes 2. Many aspects of the initial technology have been continuously improved over several decades 3, 5, 11. Additionally, previous work documented two essential characteristics of Vm-imaging. Firstly, fluorescence signals are linearly proportional to membrane potential over the entire physiological range (-100 mV to +100 mV; 10, 14, 16). Secondly, loading neurons with the voltage-sensitive dye used here (JPW 3028) does not have detectable pharmacological effects. The recorded broadening of the spike during dye loading is completely reversible 4, 7. Additionally, experimental evidence shows that it is possible to obtain a significant number (up to hundreds) of recordings prior to any detectable phototoxic effects 4, 6, 12, 13. At present, we take advantage of the superb brightness and stability of a laser light source at near-optimal wavelength to maximize the sensitivity of the Vm-imaging technique. The current sensitivity permits multiple site optical recordings of Vm transients from all parts of a neuron, including axons and axon collaterals, terminal dendritic branches, and individual dendritic spines. The acquired information on signal interactions can be analyzed quantitatively as well as directly visualized in the form of a movie.

An imaging technique for monitoring of membrane potential changes with sub-micrometer spatial and sub-millisecond temporal resolution is described. The technique, based on laser excitation of voltage-sensitive dyes, allows measurements of signals in axons and axon collaterals, terminal dendritic branches, and individual dendritic spines.

Enregistrement colorant sensible à la tension d&#39;axones, des dendrites et des épines dendritiques des neurones individuels dans des tranches de cerveau

Understanding the biophysical properties and functional organization of single neurons and how they process information is fundamental for understanding ...

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imagerie pHluorin-étiqueté insertion du récepteur de la membrane plasmique dans les neurones primaires de souris en culture

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange (ABE)

Palmitoylation is a post-translational lipid modification involving the attachment of a 16-carbon saturated fatty acid, palmitate, to cysteine residues of substrate proteins through a labile thioester bond [reviewed in1]. Palmitoylation of a substrate protein increases its hydrophobicity, and typically facilitates its trafficking toward cellular membranes. Recent studies have shown palmitoylation to be one of the most common lipid modifications in neurons1, 2, suggesting that palmitate turnover is an important mechanism by which these cells regulate the targeting and trafficking of proteins. The identification and detection of palmitoylated substrates can therefore better our understanding of protein trafficking in neurons.
Detection of protein palmitoylation in the past has been technically hindered due to the lack of a consensus sequence among substrate proteins, and the reliance on metabolic labeling of palmitoyl-proteins with 3H-palmitate, a time-consuming biochemical assay with low sensitivity. Development of the Acyl-Biotin Exchange (ABE) assay enables more rapid and high sensitivity detection of palmitoylated proteins2-4, and is optimal for measuring the dynamic turnover of palmitate on neuronal proteins. The ABE assay is comprised of three biochemical steps (Figure 1): 1) irreversible blockade of unmodified cysteine thiol groups using N-ethylmaliemide (NEM), 2) specific cleavage and unmasking of the palmitoylated cysteine's thiol group by hydroxylamine (HAM), and 3) selective labeling of the palmitoylated cysteine using a thiol-reactive biotinylation reagent, biotin-BMCC. Purification of the thiol-biotinylated proteins following the ABE steps has differed, depending on the overall goal of the experiment.
Here, we describe a method to purify a palmitoylated protein of interest in primary hippocampal neurons by an initial immunoprecipitation (IP) step using an antibody directed against the protein, followed by the ABE assay and western blotting to directly measure palmitoylation levels of that protein, which is termed the IP-ABE assay. Low-density cultures of embryonic rat hippocampal neurons have been widely used to study the localization, function, and trafficking of neuronal proteins, making them ideally suited for studying neuronal protein palmitoylation using the IP-ABE assay. The IP-ABE assay mainly requires standard IP and western blotting reagents, and is only limited by the availability of antibodies against the target substrate. This assay can easily be adapted for the purification and detection of transfected palmitoylated proteins in heterologous cell cultures, primary neuronal cultures derived from various brain tissues of both mouse and rat, and even primary brain tissue itself.

Detection of Protein Palmitoylation in Cultured Hippocampal Neurons by Immunoprecipitation and Acyl-Biotin Exchange ABE

The reversible addition of palmitate to proteins is an important regulator of intracellular protein trafficking. This is of particular interest in neurons where many synaptic proteins are palmitoylated. We utilize a simple biochemical method to detect palmitoylated proteins in cultured neurons, which can be adapted for multiple cell types and tissues.

Détection de la palmitoylation des protéines dans les neurones de l&#39;hippocampe en culture par immunoprécipitation et Acyl-biotine Exchange (ABE)

Palmitoylation is a post-translational lipid  modification involving the attachment of a 16-carbon saturated fatty acid,  palmitate, to cysteine residues ...

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

Étiquetage différentiel de surface cellulaire et intériorisé protéines après Anticorps alimentation de Live de culture neurones

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light's intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy's theoretical resolution limit of 200 nm.
Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.

This article describes a working protocol to image dendritic spines from hippocampal neurons in vitro using Structured Illumination Microscopy (SIM). Super-resolution microscopy using SIM provides image resolution significantly beyond the light diffraction limit in all three spatial dimensions, allowing the imaging of individual dendritic spines with improved detail.

Imagerie épines dendritiques des neurones de l&#39;hippocampe de rat primaire utilisant structuré Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. ...

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible are badly known. Excitotoxicity, a process believed to contribute to neuron damage induced by both insults, is mediated by activation of glutamate receptors that promotes Ca2+ influx and mitochondrial Ca2+ overload. A substantial change in intracellular Ca2+ homeostasis or remodeling of intracellular Ca2+ homeostasis may favor neuron damage in old neurons. For investigating Ca2+ remodeling in aging we have used live cell imaging in long-term cultures of rat hippocampal neurons that resemble in some aspects aged neurons in vivo. For this end, hippocampal cells are, in first place, freshly dispersed from new born rat hippocampi and plated on poli-D-lysine coated, glass coverslips. Then cultures are kept in controlled media for several days or several weeks for investigating young and old neurons, respectively. Second, cultured neurons are loaded with fura2 and subjected to measurements of cytosolic Ca2+ concentration using digital fluorescence ratio imaging. Third, cultured neurons are transfected with plasmids expressing a tandem of low-affinity aequorin and GFP targeted to mitochondria. After 24 hr, aequorin inside cells is reconstituted with coelenterazine and neurons are subjected to bioluminescence imaging for monitoring of mitochondrial Ca2+ concentration. This three-step procedure allows the monitoring of cytosolic and mitochondrial Ca2+ responses to relevant stimuli as for example the glutamate receptor agonist NMDA and compare whether these and other responses are influenced by aging. This procedure may yield new insights as to how aging influence cytosolic and mitochondrial Ca2+ responses to selected stimuli as well as the testing of selected drugs aimed at preventing neuron cell death in age-related diseases.

Fluorescence and Bioluminescence Imaging of Subcellular Ca2+ in Aged Hippocampal Neurons

Intracellular Ca2+ remodeling in aging may contribute to excitotoxicity and neuron damage, processes mediated by Ca2+ overload. We aimed at investigating Ca2+ remodeling in the aging brain using fluorescence and bioluminescence imaging of cytosolic and mitochondrial Ca2+ in long-term cultures of rat hippocampal neurons, a model of neuronal aging.

Fluorescence et bioluminescence imagerie des Subcellular Ca<sup&gt; 2+</sup&gt; In Aged neurones de l&#39;hippocampe

Susceptibility to neuron cell death associated to neurodegeneration and ischemia are exceedingly increased in the aged brain but mechanisms responsible ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.