Ce travail a été soutenu par les Instituts canadiens de recherche en santé du Canada (IRSC), la famille Townsend, et Jack Brown et la famille d&#39;Alzheimer Research Foundation (WAS). WS est le titulaire de la Chaire de recherche du Canada sur la maladie d&#39;Alzheimer. PTTL est soutenu par les sciences naturelles et en conseil de recherche en génie et la Fondation Michael Smith pour des bourses de recherche en santé.

1. Fixation des cerveaux de souris et de cryosectioning <ol><li> Les souris transgéniques sont sacrifiés à l&#39;aide de gaz de dioxyde de carbone, suivie par la décapitation et le cerveau extrait. </li><li> Le cerveau est disséqué longitudinalement au centre. La moitié du cerveau seront surgelés sur glace sèche pour l&#39;analyse biochimique. L&#39;autre sera submergé dans du paraformaldéhyde 4% (PFA) pour au moins 48 heures à 4 ° C. </li><li> Après fixation au PFA 4% pendant au moins 48 heures, le cerveau hémi est transféré directement dans la solution de saccharose à 30% et placés à 4 ° C. Au point, le cerveau hémi doit être flottant dans la solution de saccharose. Quand le cerveau hémi coulé au fond, nécessitant habituellement environ 48 heures, il est prêt à être incorporé dans PTOM pour cryosectioning. </li><li> Avant cryosectioning, monter une fine couche de PTOM sur le bouton et laisser congeler à -20 ° C. Retirez le cervelet du reste du cerveau hémi et placer sur la couche de gelée octobre Recouvrir immédiatement le cerveau avec des hémi PTOM et permettent une lente congeler à -20 ° C. Une fois l&#39;enrobage PTOM Le cerveau a tourné opaque, il est prêt à être sectionné. </li><li> Définissez l&#39;épaisseur de chaque section à 30 um. Recueillir chaque tranche dans un récipient avec D&#39;Olomos solution. </li></ol> 2. Procédure d&#39;immunohistochimie pour plaques séniles (gratuit sections flottantes) <ol><li> Traiter chaque section dans l&#39;acide formique à 88% pendant 15 minutes. Après traitement à l&#39;acide formique, la section sera temporairement tournera opaque et peut apparaître à se ratatiner. </li><li> Retirez chaque section dans une plaque et laver avec Tx-PBS (0,3% de Triton X-100 dans du PBS) pendant 5 min x 3 avec agitation douce. </li><li> Peroxyde bloc endogène bloquant 0,5% H 2 O 2 en Tx-PBS, RT 30 min en remuant doucement. </li><li> Lavez section avec Tx-PBS pendant 10 min x 3 avec agitation douce. </li><li> La section de bloc avec 5% non gras du lait écrémé dissous dans Tx-PBS à température ambiante pendant 1 heure en agitant doucement. </li><li> Incuber sections dans l&#39;anticorps primaire (biotinylé 4G8, 1:500-1:2000, Sceau) dilué dans Tx-PBS contenant 5% de lait à 4 ° C pendant une nuit sous agitation douce. </li><li> Lavez section avec Tx-PBS pendant 10 min x 3 avec agitation douce. </li><li> Préparer le protocole du fabricant ABC solution suivante (Elite ABC, Vector Laboratories,). Incuber sections dans le mélange de ABC pendant 30 min en remuant doucement. </li><li> Lavez section avec Tx-PBS pendant 10 min x 3 avec agitation douce. </li><li> Préparer 3,3 &#39;-diaminobenzidine tétrachlorhydrate (DAB) (Vector Laboratories) le protocole du fabricant. Incuber le mélange de sections DAB pendant 5-10 min. À ce stade, vous pourrez observer une couleur brunâtre se développer sur chaque section. </li><li> Lavez section avec Tx-PBS pendant 10 min x 3 avec agitation douce. </li><li> Coupes de cerveau flottant sont montées sur des lames recouvertes de gélatine, pour aider à l&#39;adhérence des tissus. Les articles sont ensuite séchés à l&#39;air suivie par compensation avec une série de xylène et monté dans Entellen. </li><li> Les plaques sont visualisés en microscopie optique à un grossissement de 40X et sont quantifiés en évaluant le nombre de plaques signifie par tranche pour chaque souris. </li></ol> 3. Thioflavine S coloration procédure de détection de plaques séniles <ol><li> Le sacrifice de la souris et la procédure d&#39;extraction du cerveau sont, comme indiqué dans les sections 1.1 à 1.5. </li><li> Mont 4 à 5 coupes de cerveau hémi sur une lame de verre et laisser sécher à l&#39;air complètement avant la coloration. </li><li> Laver les lames à l&#39;éthanol 70% pendant 1 minute. </li><li> Laver les lames avec de l&#39;éthanol 80% pendant 1 minute. </li><li> Incuber les lames dans la solution filtrée (filtre de 0,2 um) thioflavine S (1% dans 80% d&#39;éthanol) pendant 15 minutes. (Solution thioflavine S doit être filtrée avant chaque utilisation). Remarque: protéger la thioflavine S de la lumière et à protéger les lames colorées de la lumière. </li><li> Laver les lames avec de l&#39;éthanol 80% pendant 1 minute. </li><li> Laver les lames avec de l&#39;éthanol 1 minute à 70%. </li><li> Laver à l&#39;eau distillée deux fois. </li><li> Mont diapositives en milieu aqueux de montage et permettent lames sécher dans l&#39;obscurité pendant au moins 2 heures, suivie par la lamelle d&#39;étanchéité avec du vernis à ongles transparent. Les sections colorées doivent être stockés dans l&#39;obscurité à 4 ° C. </li><li> La fluorescence verte plaques colorées pourrait être visualisées avec microscropy fluorescentes. Analyser des diapositives dans une semaine à cause de la coloration va s&#39;estomper avec le temps. </li></ol> 4. Les résultats représentatifs Dans notre étude récente sur l&#39;efficacité du médicament anti-épileptique et l&#39;acide stabilisateur de l&#39;humeur valproïque (VPA) à inhiber la formation de plaques séniles, nous avons utilisé l&#39;immunomarquage ci-dessus décrites et thioflavine S coloration des procédures pour identifier les plaques séniles chez la souris modèle AD (3). La figure 1 représente une caractéristique de 9 mois APP23 souris transgénique, teinté d&#39;anti-Aß utilisant 4G8 biotinylé et détectée par la méthode ABC. Plaques séniles sont clairement marquées avec l&#39;anticorps et sont indiqués par des flèches blanches. Utilisant une coloration thioflavine S, nous avons détecté dépôt de plaque neuritique 2 mois APP23xPS TRAnsgenic souris (figure 2). Thioflavine S liée plaques séniles contenant Aßi apparaît sous microscopie à fluorescence verte (indiquées par des flèches blanches). Nous avons montré que le traitement APV, la formation de plaques séniles est significativement réduite (3). Dans une autre étude, nous avons étudié l&#39;hypoxie lien moléculaire sous-jacente et la pathogenèse AD. Encore une fois, en utilisant à la fois coloration immunohistochimique 4G8 anti-Aß et coloration thioflavine S, nous avons constaté que l&#39;hypoxie a facilité la formation de plaques séniles contenant Aß (4). <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="Figure 1"/> Figure 1. L&#39;analyse immunohistochimique de formation de plaques séniles chez des souris transgéniques après JC. APP23 souris à l&#39;âge de 9 mois ont été sacrifiés et ont été disséqués, fixe, et sectionné. Plaques séniles dans le hippocampe ont été détectés en utilisant un anticorps anti-4G8 Aßi et ont été développés en utilisant les méthodes ABC et DAB. Les plaques ont été visualisées par microscopie optique en 40X. Les flèches blanches au point de plaques séniles Aß. <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="Figure 2"/> Figure 2. Coloration thioflavine S de plaques séniles chez les souris AD modélisés. APP23xPS45 souris doublement transgéniques à 8 semaines ont été sacrifiés et ont été disséqués, fixe, et sectionné. Fléaux neuritique dans l&#39;hippocampe sont ont été confirmés en utilisant coloration fluorescente thioflavine S et visualisées par microscopie à un grossissement de 40X. Les flèches blanches indiquent les plaques séniles thioflavine S tachés.

<ol>
	<li>Glenner, G. G., Wong, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alzheimer's+disease:+initial+report+of+the+purification+and+characterization+of+a+novel+cerebrovascular+amyloid+protein.">Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein.</a> Biochem Biophys Res Commun. 120, 885-890 (1984).</li><li>Hardy, J., Selkoe, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amyloid+hypothesis+of+Alzheimer's+disease:+progress+and+problems+on+the+road+to+therapeutics.">The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics.</a> Science. 297, 353-356 (2002).</li><li>Qing, H., He, G., Ly, P. T., Fox, C. J., Staufenbiel, M., Cai, F., Zhang, Z., Wei, S., Sun, X., Chen, C. H., Zhou, W., Wang, K., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Valproic+acid+inhibits+Abeta+production,+neuritic+plaque+formation,+and+behavioral+deficits+in+Alzheimer's+disease+mouse+models.">Valproic acid inhibits Abeta production, neuritic plaque formation, and behavioral deficits in Alzheimer's disease mouse models.</a> J Exp Med. 205, 2781-2789 (2008).</li><li>Sun, X., He, G., Qing, H., Zhou, W., Dobie, F., Cai, F., Staufenbiel, M., Huang, L. E., Song, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+facilitates+Alzheimer's+disease+pathogenesis+by+up-regulating+BACE1+gene+expression.">Hypoxia facilitates Alzheimer's disease pathogenesis by up-regulating BACE1 gene expression.</a> Proc Natl Acad Sci U S A. 103, 18727-18732 (2006).</li></ol>

Toutes les expériences ont été menées en conformité avec l&#39;Université de la Colombie Soins des animaux-Britannique et le comité de l&#39;utilisation et les lignes directrices des IRSC.

L&#39;immunohistochimie utilisant l&#39;anticorps biotinylé étiquetés taches 4G8 plaques séniles dans la MA modélisé souris avec une spécificité. Le résultat coloration permet la quantification et la comparaison de la charge de la plaque entre les différents groupes de traitement. Il ya plusieurs étapes cruciales qui pourraient affecter le résultat. Parce que les cerveaux ne sont pas hémi perfusé avant l&#39;extraction du crâne, les soins devraient être utilisés pendant le processus d&#39;extraction afin de prévenir les dommages au cerveau hémi. Par ailleurs, puisque le cerveau est passivement hémi perfusé, nous vous recommandons d&#39;incubation de 4% PFA pour au moins 48 heures à 4 ° C avant de plonger dans la solution de saccharose à 30%. Alternativement, la perfusion transcardial pourrait être fait avant l&#39;extraction du cerveau. Si le cerveau est fixé correctement, il doit avoir une texture caoutchouteuse. Dans le cas où le cerveau n&#39;est pas fixée correctement, les sections se déchirer dans la solution D&#39;Olomos ou pendant les procédures de coloration. L&#39;anticorps monoclonal 4G8 est réactif aux résidus d&#39;acides aminés 17-24 de Aß et reconnu l&#39;épitope dans la séquence de base (VFFAE). Depuis 4G8 proviennent d&#39;espèces de souris, elle tend à donner une coloration de fond plus élevé. Ainsi, nous avons choisi d&#39;incuber les sections à la dilution indiquée par des considérations de parvenir à un vrai signal, tout en minimisant la coloration de fond. En plus d&#39;immunomarquage pour la détection des plaques séniles en utilisant l&#39;anticorps 4G8, une méthode de coloration thioflavine S est aussi utilisé pour identifier les plaques. Thioflavine S est un mélange de colorant homogène qui résulte de la méthylation de déhydrothiotoluidine avec de l&#39;acide sulfonique. Thioflavine S non sélective lie le contenu des feuillets bêta de protéines, comme celles en oligomères amyloïdes. Lors de la liaison, thioflavine subit un décalage bleue caractéristique de son spectre d&#39;émission. Inversement contraignante thioflavine S pour les formes monomériques ne sera pas susciter un décalage vers le bleu et ne pouvaient pas être détectés avec microscope à fluorescence. Coloration thioflavine S offre une alternative rapide à l&#39;écran de l&#39;amyloïde comme l&#39;intensité de fluorescence permet une bonne visualisation de petites quantités de dépôts amyloïdes. Cependant, un inconvénient de coloration thioflavine S est le manque de spécificité. Beaucoup de composants d&#39;autres tissus, y compris contenant des feuillets bêta étendue, comme fibrinoids, hyalines, la kératine, etc, ont une affinité plutôt pour ce colorant.

<table border="1"><tbody><tr><th> Nom du réactif et de l&#39;équipement </th><th> Société </th><th> Numéro de catalogue </th></tr><tr><td> Paraformaldéhyde </td><td> Sigma-Aldrich </td><td> P6148-1KG </td></tr><tr><td> Polyvinylpyrrolidene </td><td> Sigma-Aldrich </td><td> PVP40-100G </td></tr><tr><td> Saccharose </td><td> Fisher Scientific </td><td> S5-3 </td></tr><tr><td> L&#39;éthylène glycol </td><td> Sigma-Aldrich </td><td> 324 558-2L </td></tr><tr><td> Tissue-Tek octobre Compound </td><td> Sakura </td><td> 4583 </td></tr><tr><td> 88% d&#39;acide formique </td><td> Fisher Scientific </td><td> A118P-500 </td></tr><tr><td> Triton X-100 </td><td> ICN Biomedicals </td><td> 807426 </td></tr><tr><td> H2O2 </td><td> Sigma-Aldrich </td><td> H-1009 </td></tr><tr><td> Biotine 4G8 Anticorps </td><td> Cedarlane </td><td> SIG-39240-500 </td></tr><tr><td> Elite ABC kit </td><td> Vecteur </td><td> PK-6100 </td></tr><tr><td> Imm PACT DAB </td><td> Vecteur </td><td> SK-4105 </td></tr><tr><td> Xylène </td><td> Fisher Scientific </td><td> X4-4 </td></tr><tr><td> Entellan </td><td> EM Science </td><td> 65037-71 </td></tr><tr><td> * Superfrost Plus Lames de microscope </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Cryostat </td><td> Leica </td><td> CM-3050-S </td></tr><tr><td> MX35 Premier microtome lame </td><td> Thermo Scientific </td><td> 3052835 </td></tr><tr><td> Thioflavine S </td><td> Sigma-Aldrich </td><td> T1892-25G </td></tr></tbody></table>

detection of neuritic plaques in alzheimer's disease mouse model

La maladie d&#39;Alzheimer (MA) est le trouble neurodégénérative la plus fréquente conduisant à la démence. Formation de plaques séniles est l&#39;une des caractéristiques pathologiques de la maladie d&#39;Alzheimer. Le composant central de plaques séniles est une petite protéine appelée protéine filamenteuse β-amyloïde (Aß) 1, qui est dérivé du clivage protéolytique séquentiel de la protéine précurseur bêta-amyloïde (APP) par β-sécrétase et γ-sécrétase. L&#39;hypothèse amyloïde implique que Aγ contenant des plaques que le mécanisme sous-jacent dans la pathologie de la MA toxiques 2. L&#39;analyse post-mortem de la présence de plaques séniles confirme le diagnostic de la MA. Pour approfondir notre compréhension de la neurobiologie Aγ dans la pathogenèse de AD, différentes souches de souris exprimant AD liés à des mutations dans les gènes APP humaine ont été générés. Selon la gravité de la maladie, ces souris se développera plaques séniles à des âges différents. Ces souris sont des outils précieux pour étudier le développement pathogenèse et de médicaments qui pourraient affecter la voie de traitement APP et la formation des plaques séniles. Dans ce protocole, nous employons une méthode immunohistochimique pour la détection spécifique de plaques séniles chez la souris modèle AD. Nous allons discuter spécifiquement de la préparation d&#39;extraire le cerveau et demi, la fixation de paraformaldéhyde, cryosectioning, et deux méthodes pour détecter les plaques névrotique chez des souris transgéniques AD: détection immunohistochimique utilisant la méthode ABC et DAB et détection par fluorescence en utilisant la méthode de coloration thiofalvin S.

Watch this Scientific Journal Video about Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model at JoVE.com

Detection of Neuritic Plaques in Alzheimer&#039;s Disease Mouse Model

Une des caractéristiques pathologiques de la MA est la formation de plaques amyloïdes β protéines neuritique positif. Dans ce protocole, nous décrivons deux méthodes pour détecter les plaques séniles dans le modèle de souris transgéniques AD: détection immunohistochimique utilisant la méthode ABC et DAB et détection par fluorescence en utilisant la méthode de coloration thioflavine S.

La détection de plaques séniles dans le modèle d&#39;Alzheimer La maladie de la souris

Alzheimer's disease (AD) is the most common neurodegenerative disorder leading to dementia. Neuritic plaque formation is one of the pathological hallmarks ...

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JoVE Journal

Neuroscience

Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

36.4K vues.  The University of British Columbia. Une des caractéristiques pathologiques de la MA est la formation de plaques amyloïdes β protéines neuritique positif. Dans ce protocole, nous décrivons deux méthodes pour détecter les plaques séniles dans le modèle de souris transgéniques AD: détection immunohistochimique utilisant la méthode ABC et DAB et détection par fluorescence en utilisant la méthode de coloration thioflavine S.

Vidéo: Detection of Neuritic Plaques in Alzheimer's Disease Mouse Model

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). &beta;-amyloid (A&beta;), a peptide produced in high amounts in AD, is known to reduce Long-Term Potentiation (LTP), a cellular correlate of learning and memory. Indeed, LTP impairment caused by A&beta; is a useful experimental paradigm for studying synaptic dysfunctions in AD models and for screening drugs capable of mitigating or reverting such synaptic impairments. Studies have shown that A&beta; produces the LTP disruption preferentially via its oligomeric form. Here we provide a detailed protocol for impairing LTP by perfusion of oligomerized synthetic A&beta;1-42 peptide onto acute hippocampal slices. In this video, we outline a step-by-step procedure for the preparation of oligomeric A&beta;1-42. Then, we follow an individual experiment in which LTP is reduced in hippocampal slices exposed to oligomerized A&beta;1-42 compared to slices in a control experiment where no A&beta;1-42 exposure had occurred.

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

One feature of Alzheimer's Disease is the elevation of A&beta;1-42 peptide. Here we provide a protocol for preparing synthetic A&beta;1-42 oligomers, which impairs hippocampal Long-Term Potentiation, a cellular correlate of memory. This procedure is useful for investigating mechanisms of A&beta;-induced pathology and drug screening.

Préparation des oligomères β-amyloïde 1-42 Et induction de la plasticité synaptique Dépréciation sur les coupes d&#39;hippocampe

Impairment of synaptic connections is likely to underlie the subtle amnesic changes occurring at the early stages of Alzheimer s Disease (AD). ...

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Neurosciences

Selection of Aptamers for Amyloid &beta;-Protein, the Causative Agent of Alzheimer&#39;s Disease

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis difficult1. Definite AD diagnosis is achieved only postmortem, thus establishing presymptomatic, early diagnosis of AD is crucial for developing and administering effective therapies2,3.
Amyloid &beta;-protein (A&beta;) is central to AD pathogenesis. Soluble, oligomeric A&beta; assemblies are believed to affect neurotoxicity underlying synaptic dysfunction and neuron loss in AD4,5. Various forms of soluble A&beta; assemblies have been described, however, their interrelationships and relevance to AD etiology and pathogenesis are complex and not well understood6. Specific molecular recognition tools may unravel the relationships amongst A&beta; assemblies and facilitate detection and characterization of these assemblies early in the disease course before symptoms emerge. Molecular recognition commonly relies on antibodies. However, an alternative class of molecular recognition tools, aptamers, offers important advantages relative to antibodies7,8. Aptamers are oligonucleotides generated by in-vitro selection: systematic evolution of ligands by exponential enrichment (SELEX)9,10. SELEX is an iterative process that, similar to Darwinian evolution, allows selection, amplification, enrichment, and perpetuation of a property, e.g., avid, specific, ligand binding (aptamers) or catalytic activity (ribozymes and DNAzymes).
Despite emergence of aptamers as tools in modern biotechnology and medicine11, they have been underutilized in the amyloid field. Few RNA or ssDNA aptamers have been selected against various forms of prion proteins (PrP)12-16. An RNA aptamer generated against recombinant bovine PrP was shown to recognize bovine PrP-&beta;17, a soluble, oligomeric, &beta;-sheet-rich conformational variant of full-length PrP that forms amyloid fibrils18. Aptamers generated using monomeric and several forms of fibrillar &beta;2-microglobulin (&beta;2m) were found to bind fibrils of certain other amyloidogenic proteins besides &beta;2m fibrils19. Ylera et al. described RNA aptamers selected against immobilized monomeric A&beta;4020. Unexpectedly, these aptamers bound fibrillar A&beta;40. Altogether, these data raise several important questions. Why did aptamers selected against monomeric proteins recognize their polymeric forms? Could aptamers against monomeric and/or oligomeric forms of amyloidogenic proteins be obtained? To address these questions, we attempted to select aptamers for covalently-stabilized oligomeric A&beta;4021 generated using photo-induced cross-linking of unmodified proteins (PICUP)22,23. Similar to previous findings17,19,20, these aptamers reacted with fibrils of A&beta; and several other amyloidogenic proteins likely recognizing a potentially common amyloid structural aptatope21. Here, we present the SELEX methodology used in production of these aptamers21.

Selection of Aptamers for Amyloid &amp;#946;-Protein, the Causative Agent of Alzheimer&#039;s Disease

Aptamers are short ribo-/deoxyribo-oligonucleotides selected by in-vitro evolution methods based on affinity for a specific target. Aptamers are molecular recognition tools with versatile therapeutic, diagnostic, and research applications. We demonstrate methods for selection of aptamers for amyloid &beta;-protein, the causative agent of Alzheimer's disease.

Sélection d&#39;aptamères pour amyloïde β-protéine, l&#39;agent causal de la maladie d&#39;Alzheimer

Alzheimer's disease (AD) is a progressive, age-dependent, neurodegenerative disorder with an insidious course that renders its presymptomatic diagnosis ...

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#39;s Disease Model Mice

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including acquisition of spatial memoryand long-term spatial memory 1. The MWM is a relatively simple procedure typically consisting of six day trials, the main advantage being the differentiation between the spatial (hidden-platform) and non-spatial (visible platform) conditions 2-4. In addition, the MWM testing environment reduces odor trail interference 5. This has led the task to be used extensively in the study of the neurobiology and neuropharmacology of spatial learning and memory. The MWM plays an important role in the validation of rodent models for neurocognitive disorders such as Alzheimer&#x2019;s Disease 6, 7. In this protocol we discussed the typical procedure of MWM for testing learning and memory and data analysis commonly used in Alzheimer&#x2019;s disease transgenic model mice.

Morris Water Maze Test for Learning and Memory Deficits in Alzheimer&#039;s Disease Model Mice

The Morris Water Maze is a behavioral task to test hippocampal-dependent learning and memory. It has been widely used in the study of neurobiology, neuropharmacology and neurocognitive disorders in rodent models.

Test Morris Water Maze pour les déficits d&#39;apprentissage et de mémoire chez la souris modèle de la maladie d&#39;Alzheimer

The Morris Water Maze (MWM) was first established by neuroscientist Richard G. Morris in 1981 in order to test hippocampal-dependent learning, including ...

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

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

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

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

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

Stereotaxic Infusion of Oligomeric Amyloid-beta into the Mouse Hippocampus

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the over-production of amyloid-beta and the deposition of amyloid-beta plaques in brain regions of the afflicted individuals. Throughout the years scientists have generated numerous Alzheimer&#8217;s disease mouse models that attempt to replicate the amyloid-beta pathology. Unfortunately, the mouse models only selectively mimic the disease features. Neuronal death, a prominent effect in the brains of Alzheimer&#8217;s disease patients, is noticeably lacking in these mice. Hence, we and others have employed a method of directly infusing soluble oligomeric species of amyloid-beta - forms of amyloid-beta that have been proven to be most toxic to neurons - stereotaxically into the brain. In this report we utilize male C57BL/6J mice to document this surgical technique of increasing amyloid-beta levels in a select brain region. The infusion target is the dentate gyrus of the hippocampus because this brain structure, along with the basal forebrain that is connected by the cholinergic circuit, represents one of the areas of degeneration in the disease. The results of elevating amyloid-beta in the dentate gyrus via stereotaxic infusion reveal increases in neuron loss in the dentate gyrus within 1 week, while there is a concomitant increase in cell death and cholinergic neuron loss in the vertical limb of the diagonal band of Broca of the basal forebrain. These effects are observed up to 2 weeks. Our data suggests that the current amyloid-beta infusion model provides an alternative mouse model to address region specific neuron death in a short-term basis. The advantage of this model is that amyloid-beta can be elevated in a spatial and temporal manner.

Here, we present a protocol for direct stereotaxic brain infusion of amyloid-beta. This methodology provides an alternative in vivo mouse model to address the short-term effects of amyloid-beta on brain neurons.

Infusion stéréotaxique des oligomères bêta-amyloïde dans les Hippocampus souris

Alzheimer&#8217;s disease is a neurodegenerative disease affecting the aging population. A key neuropathological feature of the disease is the ...

Generalized Psychophysiological Interaction (PPI) Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer's Disease

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of correlation of the BOLD signal in spatially independent regions of the brain defines the functional connectivity of those regions. During a cognitive fMRI task, a psychophysiological interaction (PPI) analysis can be used to examine changes in the functional connectivity during specific contexts defined by the cognitive task. An example of such a task is one that engages the memory system, asking participants to learn pairs of unrelated words (encoding) and recall the second word in a pair when presented with the first word (retrieval). In the present study, we used this type of associative memory task and a generalized PPI (gPPI) analysis to compare changes in hippocampal connectivity in older adults who are carriers of the Alzheimer's disease (AD) genetic risk factor apolipoprotein-E epsilon-4 (APOE&#949;4). Specifically, we show that the functional connectivity of subregions of the hippocampus changes during encoding and retrieval, the two active phases of the associative memory task. Context-dependent changes in functional connectivity of the hippocampus were significantly different in carriers of APOE&#949;4 compared to non-carriers. PPI analyses make it possible to examine changes in functional connectivity, distinct from univariate main effects, and to compare these changes across groups. Thus, a PPI analysis may reveal complex task effects in specific cohorts that traditional univariate methods do not capture. PPI analyses cannot, however, determine directionality or causality between functionally connected regions. Nevertheless, PPI analyses provide powerful means for generating specific hypotheses regarding functional relationships, which can be tested using causal models. As the brain is increasingly described in terms of connectivity and networks, PPI is an important method for analyzing fMRI task data that is in line with the current conception of the human brain.

Generalized Psychophysiological Interaction PPI Analysis of Memory Related Connectivity in Individuals at Genetic Risk for Alzheimer&#039;s Disease

This manuscript describes how to implement a psychophysiological interaction analysis to reveal task-dependent changes in functional connectivity between a selected seed region and voxels in other regions of the brain. Psychophysiological interaction analysis is a popular method to examine task effects on brain connectivity, distinct from traditional univariate activation effects.

Analyse d’Interaction psychophysiologique généralisée (PPI) de mémoire qui connectivité chez les personnes à risque génétique pour la maladie d’Alzheimer

In neuroimaging, functional magnetic resonance imaging (fMRI) measures the blood-oxygenation-level dependent (BOLD) signal in the brain. The degree of ...

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer's Mouse Models

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying Alzheimer's disease, Parkinson's disease, depression, and anxiety. Traditionally, microdialysis has been the most common (lauded) technique to examine neurotransmitter changes that occur in these disorders. But because microdialysis has the ability to measure slow 1-20 minute changes across large areas of tissue, it has the disadvantage of invasiveness, potentially destroying intrinsic connections within the brain and a slow sampling capability. A relatively newer technique, the microelectrode array (MEA), has numerous advantages for measuring specific neurotransmitter changes within discrete brain regions as they occur, making for a spatially and temporally precise approach. In addition, using MEAs is minimally invasive, allowing for measurement of neurotransmitter alterations in vivo. In our laboratory, we have been specifically interested in changes in the neurotransmitter, glutamate, related to Alzheimer's disease pathology. As such, the method described here has been used to assess potential hippocampal disruptions in glutamate in a transgenic mouse model of Alzheimer's disease. Briefly, the method used involves coating a multi-site microelectrode with an enzyme very selective for the neurotransmitter of interest and using self-referencing sites to subtract out background noise and interferents. After plating and calibration, the MEA can be constructed with a micropipette and lowered into the brain region of interest using a stereotaxic device. Here, the method described involves anesthetizing rTg(TauP301L)4510 mice and using a stereotaxic device to precisely target sub-regions (DG, CA1, and CA3) of the hippocampus.

Using Enzyme-based Biosensors to Measure Tonic and Phasic Glutamate in Alzheimer&#039;s Mouse Models

Here, we describe the setup, software navigation, and data analysis for a spatially and temporally precise method of measuring tonic and phasic extracellular glutamate changes in vivo using enzyme-linked microelectrode arrays (MEA).

En utilisant biocapteurs à base d&#39;enzymes pour mesurer Tonic et Phasic Glutamate dans les modèles murins d&#39;Alzheimer

Neurotransmitter disruption is often a key component of diseases of the central nervous system (CNS), playing a role in the pathology underlying ...

Neurobehavioral Assessments in a Mouse Model of Neonatal Hypoxic-ischemic Brain Injury

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI brain injury by studying neurobehavioral functions in these mice compared to non-operated (i.e., normal) mice. During the study, Rice-Vannucci&#39;s method was used to induce neonatal HI brain damage in postnatal day 7-10 (P7-10) mice. The HI operation was performed on the pups by unilateral carotid artery ligation and exposure to hypoxia (8% O2 and 92% N2 for 90 min). One week after the operation, the damaged brains were evaluated with the naked eye through the semi-transparent skull and were categorized into subgroups based on the absence (&#34;no cortical injury&#34; group) or presence (&#34;cortical injury&#34; group) of cortical injury, such as a lesion in the right hemisphere. On week 6, the following neurobehavioral tests were performed to evaluate the cognitive and motor functions: passive avoidance task (PAT), ladder walking test, and grip strength test. These behavioral tests are helpful in determining the effects of neonatal HI brain injury and are used in other mouse models of neurodegenerative diseases. In this study, neonatal HI brain injury mice showed motor deficits that corresponded to right hemisphere damage. The behavioral test results are relevant to the deficits observed in human neonatal HI patients, such as cerebral palsy or neonatal stroke patients. In this study, a mouse model of neonatal HI brain injury was established and showed different degrees of motor deficits and cognitive impairment compared to non-operated mice. This work provides basic information on the HI mouse model. MRI images demonstrate the different phenotypes, separated according to the severity of brain damage by motor and cognitive tests.

We performed unilateral carotid artery occlusion on postnatal day 7-10 CD-1 mouse pups to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of HI brain injury. We studied neurobehavioral functions in these mice compared to non-operated normal mice.

Évaluations neurocomportementales dans un modèle murin de lésion cérébrale hypoxique-ischémique néonatale

We performed unilateral carotid artery occlusion on CD-1 mice to create a neonatal hypoxic-ischemic (HI) model and investigated the effects of neonatal HI ...

Labeling and Imaging of Amyloid Plaques in Brain Tissue Using the Natural Polyphenol Curcumin

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). Therefore, detection of the presence of A&#946; in AD brain tissue is a valuable tool for developing new treatments to prevent the progression of AD. Several classical amyloid binding dyes, fluorochrome, imaging probes, and A&#946;-specific antibodies have been used to detect A&#946; histochemically in AD brain tissue. Use of these compounds for A&#946; detection is costly and time consuming. However, because of its intense fluorescent activity, high-affinity, and specificity for A&#946;, as well as structural similarities with traditional amyloid binding dyes, curcumin (Cur) is a promising candidate for labeling and imaging of A&#946; plaques in postmortem brain tissue. It is a natural polyphenol from the herb Curcuma longa. In the present study, Cur was used to histochemically label A&#946; plaques from both a genetic mouse model of 5x familial Alzheimer&#39;s disease (5xFAD) and from human AD tissue within a minute. The labeling capability of Cur was compared to conventional amyloid binding dyes, such as thioflavin-S (Thio-S), Congo red (CR), and Fluoro-jade C (FJC), as well as A&#946;-specific antibodies (6E10 and A11). We observed that Cur is the most inexpensive and quickest way to label and image A&#946; plaques when compared to these conventional dyes and is comparable to A&#946;-specific antibodies. In addition, Cur binds with most A&#946; species, such as oligomers and fibrils. Therefore, Cur could be used as the most cost-effective, simple, and quick fluorochrome detection agent for A&#946; plaques.

Curcumin is an ideal fluorophore for labeling and imaging of amyloid beta protein plaques in brain tissue due to its preferential binding to amyloid beta protein as well as its structural similarities with other traditional amyloid binding dyes. It can be used to label and image amyloid beta protein plaques more efficiently and inexpensively than traditional methods.

Étiquetage et imagerie des plaques amyloïdes dans les tissus cérébraux à l'aide de la curcumine polyphénol naturelle

Deposition of amyloid beta protein (A&#946;) in extra- and intracellular spaces is one of the hallmark pathologies of Alzheimer&#39;s disease (AD). ...

Stereological Estimation of Cholinergic Fiber Length in the Nucleus Basalis of Meynert of the Mouse Brain

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a useful method to quantify neuronal profiles of various brain structures. Here we provide a software-based stereology protocol to estimate the total length of cholinergic fibers in the nucleus basalis of Meynert (NBM) of the basal forebrain. The method uses a space ball probe for length estimates. The cholinergic fibers are visualized by choline acetyltransferase (ChAT) immunostaining with the horseradish peroxidase-diaminobenzidine (HRP-DAB) detection system. The staining protocol is also valid for fiber and cell number estimation in various brain regions using stereology software. The stereology protocol can be used for estimation of any linear profiles such as cholinoceptive fibers, dopaminergic/catecholaminergic fibers, serotonergic fibers, astrocyte processes, or even vascular profiles.

Neuronal fiber length within a three-dimensional structure of a brain region is a reliable parameter to quantify specific neuronal structural integrity or degeneration. This article details a stereological quantification method to measure cholinergic fiber length within the nucleus basalis of Meynert in mice as an example.

Estimation stéréologique de la longueur de la fibre cholinergique dans le noyau Basalis de Meynert du cerveau de souris

The length of cholinergic or other neuronal axons in various brain regions are often correlated with the specific function of the region. Stereology is a ...