Ce travail a été financé par le NIH subventions DA15169 et DA035224 à HEM

Toutes les manipulations des animaux et la récolte de tissus a été réalisée en conformité avec les lignes directrices de l&#39;Université de Comité institutionnel de protection des animaux Utilisez Massachusetts Medical School (IACUC), suite à l&#39;approbation protocole # A1506 (Melikian, PI). Solutions requises Liquide céphalo-rachidien artificiel (ACSF) - Assurez frais tous les jours NaCl 125 mM, KCl 2,5, 1,2 mM NaH 2 PO 4, 1,2 mM de MgCl2, 2,4 mM de CaCl2, 26 mM NaHCO 3, et 11 mM de glucose Note: Préparer ACSF comme une solution stock de 10x, à l&#39;exclusion de NaHCO 3 et de glucose. Faites des solutions de travail 1x tous les jours de la 10x stock, complétant avec frais glucose NaHCO 3 et. ACSF saccharose supplémenté (SACSF) - Assurez frais tous les jours MM succès 250s&#39;est levé, 2,5 mM de KCl, 1,2 mM NaH 2 PO 4, 1,2 mM de MgCl2, 2,4 mM de CaCl2, 26 mM NaHCO 3, et 11 mM de glucose Note: Préparer SACSF comme une solution stock de 10x, à l&#39;exclusion de NaHCO 3 et de glucose. Faites des solutions de travail 1x tous les jours de la 10x stock, complétant avec frais glucose NaHCO 3 et. Sulfo-N-hydroxysuccinyle-SS-biotine (sulfo-NHS-SS-biotine, Pierce Chemical Company) Les solutions mères devraient être de 200 mg / ml dans le DMSO et sont résistantes à de multiples cycles de gel / dégel. Des portions aliquotes sont conservées à -20 ° C. L&#39;ester succinyl est rapidement hydrolysé en solution aqueuse, donc les solutions de travail doit être préparée immédiatement avant l&#39;application de tranches. Tranche Quench Solution ACSF supplémenté avec 100 mM de glycine RIPA Lysis Buffer Tris 10 mM, pH 7,4, NaCl 150 mM, EDTA 1,0 mM, 1% de Triton-X-100, 0,1% de SDS, 1% de désoxycholate de Na RIPA avec inhibiteurs de la protéase (RIPA / PI) - Assurez frais tous les jours RIPA additionné de 1 pM de leupeptine, 1 pM de pepstatine, 1 uM d&#39;aprotinine et 1 mM de fluorure de phénylméthyl sulfonyle. Une. Préparer tranches de cerveau <ol><li> Faire SACSF 1x frais et 1x ACSF </li><li> Réfrigérer SACSF sur la glace dans un petit bol. Ceci sera utilisé pour maintenir le cerveau de souris fraîchement récoltées. </li><li> Saturer l&#39;ACSF et SACSF avec de l&#39;oxygène par barbotage avec 95% / 5% de O 2 / CO 2, 20 min sur de la glace. </li><li> P30-38 souris doivent être utilisées pour la viabilité des tissus optimale. Sacrifier des animaux par dislocation cervicale et décapitation, et éliminer rapidement les cerveaux en prérefroidie, SACSF oxygéné. </li><li> L&#39;utilisation d&#39;un microtome vibrant, faire 300 um sections du cerveau dans la région d&#39;intérêt. </li><li> Si vous le souhaitez, les tranches peuvent encore être disséqués avant la récupération pour enrichir certaines régions du cerveau ou droit distinct et hémisphère gauche pour les utiliser comme contrôle et tranches expérimentaux, respectivement. </li><li> En utilisant une pipette Pasteur polie au feu, transférer tranches de filet à fond chambres prévues dans des plaques à 24 puits. </li><li> Permettre aux tranches de récupérer pendant 40 min, 31 ° C dans l&#39;ACSF oxygénée, avec continuelle, léger bullage. </li></ol> 2. Traitement de la toxicomanie (le cas échéant) et Slice Biotinylation <ol><li> Après récupération, laver tranches 3x préchauffé (37 ° C), l&#39;ACSF oxygéné bouillonnement en permanence avec 95% / 5% O 2 / CO 2. </li><li> Ajouter composés d&#39;essai et incuber avec oxygénation continue. <ol><li> Pour plus de commodité, ajouter un 1/10 ème volume de 10x médicament concentré et mélanger en inversant doucement. </li><li> Secouer doucement les plaques dans un bain d&#39;eau à la température souhaitée. </li></ol></li><li> Suite à un traitement médicamenteux, rapidementrefroidissement des tranches de lavage 3x dans la glace ACSF froid. </li></ol> 3. Les protéines de surface biotinyler <ol><li> Préparer 1,0 mg / ml de sulfo-NHS-SS-biotine dans la glace ACSF froide immédiatement avant l&#39;étiquetage. </li><li> Ajouter 0,75 ml sulfo-NHS-SS-biotine à des tranches et des tranches incuber sur de la glace, 45 min. </li><li> Laver les tranches trois fois rapidement avec ACSF froid de glace, puis incuber pendant 10 min dans la glace ACSF froid sur la glace. </li><li> Laver les tranches trois fois avec un tampon tranche de refroidissement froid de glace et incuber avec 0,75 ml de tampon de refroidissement de la tranche deux fois, 25 min, sur la glace pour étancher libre sulfo-NHS-SS-biotine. </li></ol> 4. Préparer Lysats de tissus <ol><li> Laver les tranches trois fois dans la glace ACSF froid et transférer chaque tranche dans un tube à centrifuger à l&#39;aide d&#39;une pipette Pasteur polie au feu. </li><li> Culot doucement tranche par centrifugation 200 xg, 1 min et soigneusement restant aspirer ACSF. </li><li> Ajouter 400 ul glace froid RIPA / PI et briser le tissu par aspiration et refoulement fois par un pépin de P200ette. </li><li> Transfert dissocié tranche / RIPA dans un nouveau tube et incuber 30 min, 4 ° C, rotation, pour compléter la lyse. </li><li> Sédimenter les débris cellulaires par centrifugation, 18000 xg, 15 min, 4 ° C. </li><li> Déterminer les concentrations en protéines du lysat en utilisant le dosage des protéines BCA, avec de l&#39;albumine de sérum bovin (BSA) comme étalon. </li></ol> 5. Isoler biotinylés protéines <ol><li> Optimiser perle / ratio de protéine. Remarque: Les niveaux d&#39;expression de protéines individuelles peuvent varier considérablement selon les différentes régions du cerveau. A moins que la totalité de la protéine biotinylée dans une quantité donnée d&#39;un lysat de tissu est capturé, il n&#39;est pas possible de détecter avec précision les changements potentiels dans l&#39;expression de surface. Par conséquent, il est impératif de déterminer empiriquement la perle / taux de protéines totales optimale pour une région protéine / cerveau particulier avant de se lancer sur une nouvelle étude tranche de biotinylation. <ol><li> Incuber 25 billes d&#39;agarose de streptavidine ul avec de plus en pluss de lysat de tissu (gamme approximative 25-200 pg), puis procéder comme décrit ci-dessous pour la liaison, de lavage et d&#39;élution étapes. </li><li> Quantifier les bandes immunoréactives résultant et choisir un rapport billes / lysat dans le domaine linéaire de la liaison qui permettra de quantifier précisément soit augmenté ou diminué expression de la protéine de surface. </li></ol></li><li> Préparer Perles streptavidine-agarose <ol><li> Déterminer le volume de billes total nécessaire pour tous les échantillons. Préparer un volume de perles suffisante pour ce montant, plus un supplémentaires (soit 4 échantillons à 25 pl / échantillon = 100 perles de pl + un supplémentaires = 125 perles ul. </li><li> Vortex streptavidine perle actions et pipette sur désiraient volume. Si vous utilisez une pipette P200, couper l&#39;extrémité de la pointe pour empêcher le blocage ou perle dommages. </li><li> Lavez perles 3 fois dans 0,5-1,0 ml RIPA / PI pour éliminer les conservateurs, les vortex après chaque addition RIPA et la collecte des perles entre les lavages par centrifugation 18 000 xg, 1 min, à la salle humeurture. </li><li> Aspirer tampon entre les lavages à l&#39;aide d&#39;une pipette Pasteur en verre fixé à une fiole à vide. Une pointe de P200 en plastique fixé à l&#39;extrémité de la pipette Pasteur en verre offre un contrôle plus précis lors de l&#39;aspiration. Il n&#39;est pas nécessaire d&#39;enlever absolument tout le tampon pour les deux premiers lavages, car elle risque de perles d&#39;aspiration dans la pipette. Après le lavage final, éliminer autant que possible l&#39;excès de tampon sans aspirer les billes. </li><li> Ajouter RIPA / PI à apporter perles à leur volume initial, aspiration et refoulement de remettre en suspension. Évitez vortex à ce stade, comme des perles vont coller à la paroi du tube. </li><li> perles aliquotes dans des tubes à centrifuger en utilisant un P200 avec la pointe coupée. Introduire à la pipette de haut en bas à plusieurs reprises entre le prélèvement d&#39;assurer perles restent uniformément suspension et dispersées entre les tubes. </li></ol></li><li> Protéines biotinylées Bind pour streptavidine perles <ol><li> Distribuer des lysats de cellules dans les tubes contenant les perles d&#39;agarose. Pour les expériences EPM<html> re de multiples échantillons sont comparés, assurez-vous d&#39;utiliser la même quantité de protéines pour chaque échantillon des comparaisons précises. </li><li> Ajouter supplémentaire RIPA / PI à apporter des échantillons à un volume minimal de 200 pi. Cela assure que les échantillons se mélangent de manière adéquate lors de l&#39;incubation et aussi unifie les concentrations de protéines dans les échantillons. </li><li> Placer les tubes dans un dispositif de rotation de tube et mélanger pendant une nuit à 4 ° C. </li><li> Dans des tubes séparés, délivrer un équivalent du volume de lysat total utilisé pour chaque échantillon. En variante, si les volumes d&#39;échantillon sont élevées, une fraction du volume de lysat total peut être réservé à la place, à accommoder les volumes de charge maximales sur des gels de SDS-PAGE. Ceux-ci seront utilisés pour normaliser l&#39;expression de surface de la quantité de protéine totale. </li><li> Ajouter soit un volume égal de 2x ou de 1/5 volume de 6x tampon d&#39;échantillon SDS-PAGE et soit incuber à 4 ° C, en parallèle avec les échantillons de perles, ou de conserver à -20 ° C jusqu&#39;à ce que les échantillons sont analysés. </li></ol></li></ol>ove_title &quot;&gt; 6 Elute. et analyser des échantillons <ol><li> Un culot de perles par centrifugation 18000 x g, 2 min, à la température ambiante. </li><li> Aspirer le surnageant et laver perles trois fois avec 0,75 ml RIPA. Pour minimiser les pertes de perle, laissez un petit volume de la tête de tampon ci-dessus perles entre les lavages. Après le dernier lavage, éliminer autant que possible RIPA, sans perturber le culot de billes. </li><li> Éluer les protéines biotinylées à partir de billes de streptavidine, en réduisant la liaison disulfure. Ajouter 25 pi de 2x SDS-PAGE réduisant le tampon d&#39;échantillon, vortex et ainsi faire tourner les échantillons de 30 min, température ambiante. Remarque: Beaucoup de protéines membranaires ont une forte tendance à s&#39;agréger à l&#39;ébullition dans un tampon d&#39;échantillon SDS-PAGE, d&#39;altérer gravement leur mobilité électrophorétique. Si cela est le cas pour la protéine objet de l&#39;enquête, les échantillons éviter de chauffage et, à la place, éluer lentement à la température ambiante. Si l&#39;ébullition est absolument nécessaire (par exemple, si une autre protéine évalué en parallèle nécessite ébullition), sample tampon peut être complété avec 2 M d&#39;urée (concentration finale) afin de minimiser l&#39;agrégation. Bien qu&#39;ils soient efficaces dans la réduction de l&#39;agrégation, dans certains cas, de l&#39;urée compromet souvent les apparences de la bande. </li><li> Analyser des échantillons par immunoblot. </li><li> Décongeler les échantillons de lysats totaux et tourner en parallèle avec des échantillons de perles, 30 min, la température ambiante. </li><li> Protéines séparées sur des gels SDS-PAGE. </li><li> Identifiez-protéine (s) d&#39;intérêt par immuno. <ol><li> Assurez-vous que les bandes sont détectées dans la gamme linéaire de détection, pour la quantification adéquate. </li><li> Pour assurer que le réactif de biotinylation n&#39;a pas eu accès à des protéines intracellulaires par les cellules endommagées / compromis. Le mieux est accompli par immunoblot en parallèle pour une protéine intracellulaire spécifique du type de cellule à l&#39;étude. </li><li> Quantifier la densité de la bande et de calculer la densité relative de la surface de la protéine comme un pour cent du niveau d&#39;expression de protéine totale. </li></ol></li></ol>

<ol>
	<li>Conner, S. D., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+portals+of+entry+into+the+cell.">Regulated portals of entry into the cell.</a> Nature. 422, 37-44 (2003).</li><li>Zastrow, M., Williams, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulating+neuromodulation+by+receptor+membrane+traffic+in+the+endocytic+pathway.">Modulating neuromodulation by receptor membrane traffic in the endocytic pathway.</a> Neuron. 76, 22-32 (2012).</li><li>Leto, D., Saltiel, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+glucose+transport+by+insulin:+traffic+control+of+GLUT4.">Regulation of glucose transport by insulin: traffic control of GLUT4.</a> Nat. Rev. Mol. Cell Biol. 13, 383-396 (2012).</li><li>Liu, Y. W., Lukiyanchuk, V., Schmid, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Common+membrane+trafficking+defects+of+disease-associated+dynamin+2+mutations.">Common membrane trafficking defects of disease-associated dynamin 2 mutations.</a> Traffic. 12, 1620-1633 (2011).</li><li>Li, X., DiFiglia, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+recycling+endosome+and+its+role+in+neurological+disorders.">The recycling endosome and its role in neurological disorders.</a> Prog. Neurobiol. , 127-141 (2012).</li><li>Sandvig, K., Pust, S., Skotland, T., van Deurs, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-independent+endocytosis:+mechanisms+and+function.">Clathrin-independent endocytosis: mechanisms and function.</a> Curr. Opin. Cell Biol. 23, 413-420 (2011).</li><li>Kumari, S., Mg, S., Mayor, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endocytosis+unplugged:+multiple+ways+to+enter+the+cell.">Endocytosis unplugged: multiple ways to enter the cell.</a> Cell Res. 20, 256-275 (2010).</li><li>Barry, M. F., Ziff, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Receptor+trafficking+and+the+plasticity+of+excitatory+synapses.">Receptor trafficking and the plasticity of excitatory synapses.</a> Curr. Opin. Neurobiol. 12, 279-286 (2002).</li><li>Bredt, D. S., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+at+excitatory+synapses.">AMPA receptor trafficking at excitatory synapses.</a> Neuron. 40, 361-379 (2003).</li><li>Kerchner, G. A., Nicoll, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silent+synapses+and+the+emergence+of+a+postsynaptic+mechanism+for+LTP.">Silent synapses and the emergence of a postsynaptic mechanism for LTP.</a> Nat. Rev. Neurosci. 9, 813-825 (2008).</li><li>Malinow, R., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AMPA+receptor+trafficking+and+synaptic+plasticity.">AMPA receptor trafficking and synaptic plasticity.</a> Annu. Rev. Neurosci. 25, 103-126 (2002).</li><li>Borgland, S. L., Malenka, R. C., Bonci, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+and+chronic+cocaine-induced+potentiation+of+synaptic+strength+in+the+ventral+tegmental+area:+electrophysiological+and+behavioral+correlates+in+individual+rats.">Acute and chronic cocaine-induced potentiation of synaptic strength in the ventral tegmental area: electrophysiological and behavioral correlates in individual rats.</a> J. Neurosci. 24, 7482-7490 (2004).</li><li>Dong, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cocaine-induced+potentiation+of+synaptic+strength+in+dopamine+neurons:+Behavioral+correlates+in+GluRA(-/-)+mice.">Cocaine-induced potentiation of synaptic strength in dopamine neurons: Behavioral correlates in GluRA(-/-) mice.</a> PNAS. 101, 14282-14287 (2004).</li><li>Hyman, S. E., Malenka, R. C., Nestler, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+mechanisms+of+addiction:+the+role+of+reward-related+learning+and+memory.">Neural mechanisms of addiction: the role of reward-related learning and memory.</a> Annu. Rev. Neurosci. 29, 565-598 (2006).</li><li>Thomas, M. J., Malenka, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+plasticity+in+the+mesolimbic+dopamine+system.">Synaptic plasticity in the mesolimbic dopamine system.</a> Philos. Trans. R. Soc. Lond. B. Biol. Sci. 358, 815-819 (2003).</li><li>Sorkina, T., Hoover, B. R., Zahniser, N. R., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Constitutive+and+protein+kinase+C-induced+internalization+of+the+dopamine+transporter+is+mediated+by+a+clathrin-dependent+mechanism.">Constitutive and protein kinase C-induced internalization of the dopamine transporter is mediated by a clathrin-dependent mechanism.</a> Traffic. 6, 157-170 (2005).</li><li>Holton, K. L., Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nonclassical,+distinct+endocytic+signals+dictate+constitutive+and+PKC-regulated+neurotransmitter+transporter+internalization.">Nonclassical, distinct endocytic signals dictate constitutive and PKC-regulated neurotransmitter transporter internalization.</a> Nat. Neurosci. 8, 881-888 (2005).</li><li>Loder, M. K., Melikian, H. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dopamine+transporter+constitutively+internalizes+and+recycles+in+a+protein+kinase+C-regulated+manner+in+stably+transfected+PC12+cell+lines.">The dopamine transporter constitutively internalizes and recycles in a protein kinase C-regulated manner in stably transfected PC12 cell lines.</a> J. Biol. Chem. 278, 22168-22174 (2003).</li><li>Melikian, H. E., Buckley, K. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+trafficking+regulates+the+activity+of+the+human+dopamine+transporter.">Membrane trafficking regulates the activity of the human dopamine transporter.</a> J. Neurosci. 19, 7699-7710 (1999).</li><li>Daniels, G. M., Amara, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+trafficking+of+the+human+dopamine+transporter.+Clathrin-mediated+internalization+and+lysosomal+degradation+in+response+to+phorbol+esters.">Regulated trafficking of the human dopamine transporter. Clathrin-mediated internalization and lysosomal degradation in response to phorbol esters.</a> J. Biol. Chem. 274, 35794-35801 (1999).</li><li>Sorkina, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA+interference+screen+reveals+an+essential+role+of+Nedd4-2+in+dopamine+transporter+ubiquitination+and+endocytosis.">RNA interference screen reveals an essential role of Nedd4-2 in dopamine transporter ubiquitination and endocytosis.</a> J. Neurosci. 26, 8195-8205 (2006).</li><li>Eriksen, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+dopamine+transporter+trafficking+in+live+neurons+by+use+of+fluorescent+cocaine+analogs.">Visualization of dopamine transporter trafficking in live neurons by use of fluorescent cocaine analogs.</a> J. Neurosci. 29, 6794-6808 (2009).</li><li>Rao, A., Simmons, D., Sorkin, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+subcellular+distribution+of+endosomal+compartments+and+the+dopamine+transporter+in+dopaminergic+neurons.">Differential subcellular distribution of endosomal compartments and the dopamine transporter in dopaminergic neurons.</a> Mol. Cell Neurosci. 46, 148-158 (2011).</li><li>Zhao, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type-specific+channelrhodopsin-2+transgenic+mice+for+optogenetic+dissection+of+neural+circuitry+function.">Cell type-specific channelrhodopsin-2 transgenic mice for optogenetic dissection of neural circuitry function.</a> Nat. Methods. 8, 745-752 (2011).</li></ol>

Les auteurs n&#39;ont aucun intérêt financier en conflit avec ce travail.

Malgré la connaissance de longue date que le trafic endocytose critique impacts de signalisation synaptique dans le cerveau, il s&#39;est avéré difficile de mesurer quantitativement les changements dans l&#39;expression des protéines de surface des neurones adultes. Dans ce travail, nous rapportons une approche fiable pour étiqueter la protéine de surface ex vivo dans des tranches de cerveau de courte durée. préparations de tranches de cerveau ont une longue histoire d&#39;utilité pour les enregistreme...

Le trafic est un mécanisme d&#39;endocytose cellulaire ubiquitaire qui affine le plasma présentation membranaire d&#39;une variété de protéines membranaires intégrales. L&#39;endocytose fournit des nutriments essentiels pour le milieu intracellulaire et une désensibilise la signalisation du récepteur en réponse à l&#39;activation du récepteur 2. Endocytose recyclage en arrière de la membrane plasmatique peut en outre améliorer la signalisation cellulaire par l&#39;augmentation des niveaux d&#39;expression de protéines à la surface de la cellule 3. En outre, les perturbations de trafic membranaire sont impliqués dans de nombreuses maladies et conditions pathologiques 4,5, soulignant la nécessité d&#39;étudier les mécanismes moléculaires qui gouvernent la protéine endocytose trafic. Alors que de nombreuses protéines utilisent des mécanismes d&#39;internalisation de clathrine dépendante classiques, des preuves de montage au cours des dernières années démontre que les mécanismes d&#39;endocytose clathrine indépendant multiples régissent le potentiel endocytose d&#39;une gamme croissante deprotéines 6,7. Ainsi, la nécessité d&#39;étudier les mécanismes d&#39;endocytose facilitant la traite des systèmes physiologiques pertinentes a considérablement augmenté. Dans le cerveau, le trafic d&#39;endocytose des récepteurs, canaux ioniques et transporteurs de neurotransmetteurs joue un rôle primordial dans l&#39;établissement de la plasticité synaptique 8-11 et la réponse aux drogues d&#39;abus 12-15, en fin de compte un impact de l&#39;excitabilité neuronale et les réponses synaptiques. À ce jour, la majorité des études de trafic neuronales compter sur des systèmes d&#39;expression hétérologues ou neurones primaires en culture, qui ne peut refléter de façon fiable les mécanismes en jeu dans les neurones adultes. Nous rapportons ici une approche qui utilise biotinylation de surface à mesurer quantitativement les niveaux de protéines de surface dans des tranches de cerveau de courte durée provenant de rongeurs adultes. En utilisant cette approche, nous présentons des données qui démontrent que le transporteur de la dopamine du striatum de souris intériorise rapidement reréponse à phorbol ester à médiation par la protéine kinase C (PKC) activation.

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</td>
		</tr>
	</tbody>
</table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

Traite endocytose régulée est le mécanisme central faciliter une variété d&#39;événements de neuromodulation, en contrôlant dynamiquement récepteur, canal ionique, et le transporteur cellule présentation de surface à l&#39;échelle minutes de temps. Il ya une grande diversité de mécanismes qui contrôlent le trafic endocytose de protéines individuelles. Les études portant sur les fondements moléculaires de la traite ont principalement invoqué biotinylation de surface à mesurer quantitativement les changements dans la protéine de membrane expression de surface en réponse à des stimuli exogènes et la manipulation génétique. Cependant, cette approche a été limitée principalement à des cellules en culture, qui peuvent ne pas refléter fidèlement les mécanismes physiologiquement pertinents en jeu dans les neurones adultes. En outre, les approches de cellules en culture peuvent sous-estimer les différences spécifiques à la région dans les mécanismes de la traite. Ici, nous décrivons une approche qui s&#39;étend biotinylation de la surface cellulaire pour la préparation de tranches de cerveau aiguë. Nousdémontrer que cette méthode offre une approche de haute fidélité pour mesurer les changements rapides dans les niveaux de surface de la protéine de la membrane des neurones adultes. Cette approche est susceptible d&#39;avoir une large utilité dans le domaine du trafic d&#39;endocytose neuronale.

Le transporteur de la dopamine des neurones est internalisé en réponse à l&#39;activation de la PKC dans des lignées cellulaires de 16 à 20. Malgré de nombreux rapports démontrant PKC-induites pertes de surface de DAT dans une variété de lignées cellulaires et des systèmes d&#39;expression, il a été difficile de confirmer ce résultat dans les neurones dopaminergiques en culture de 21 à 23. Nous avons utilisé les tranches de striatum de souris pour tester directement si internalise D...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Trafic membranaire neuronal contrôle dynamiquement la membrane plasmique disponibilité des protéines et un impact significatif sur la neurotransmission. À ce jour, il a été difficile de mesurer le trafic endocytose neuronale dans les neurones adultes. Ici, nous décrivons une méthode très efficace, quantitative pour mesurer les changements rapides dans l&#39;expression des protéines de surface ex vivo dans des tranches de cerveau de courte durée.

Tranche de cerveau Biotinylation: Une<em&gt; Ex Vivo</em&gt; Approche de Mesure région spécifique membrane de protéines plasmatiques traite des adultes neurones

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Research

JoVE Journal

Neuroscience

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

12.8K vues.  University of Massachusetts Medical School.  Trafic membranaire neuronal contrôle dynamiquement la membrane plasmique disponibilité des protéines et un impact significatif sur la neurotransmission. À ce jour, il a été difficile de mesurer le trafic endocytose neuronale dans les neurones adultes. Ici, nous décrivons une méthode très efficace, quantitative pour mesurer les changements rapides dans l'expression des protéines de surface ex vivo dans des tranches de cerveau de courte durée. 

Vidéo: Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analyse quantitative des vésicules reconstitution Piscine synaptique dans des cultures de neurones granulaires du cervelet utilisant des colorants FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Recherche

Neurosciences

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 ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

L&#39;utilisation d&#39;un α-bungarotoxine Reliure Tag du site à étudier récepteur GABA A membrane localisation et le trafic

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: aiguë cerveau adulte trancher dans physiologique Température

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Une<em&gt; Ex Vivo</em&gt; Induite par laser Spinal Cord Injury modèle pour évaluer les mécanismes de la dégénérescence axonale dans en temps réel

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo optogénétiques Dissection de circuits Peur en tranches cérébrales

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Images en direct de la protéine GLUT4 traite des neurones hypothalamiques primaire de souris au microscope de déconvolution

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

Une autre approche pour étudier les événements primaires dans la neurodégénérescence utilisant des coupes de cerveau de Rat Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Imagerie calcique pour visualiser le cerveau réponses à signalisation endocrinienne chez la drosophile

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Utilisation de la fluorescence proche infrarouge et de la numérisation haute résolution pour mesurer l’expression des protéines dans le cerveau des rongeurs

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...