Questo lavoro è stato sostenuto dal Canadian Institutes of Health Research (CIHR), la Famiglia Townsend, e Jack Brown e Famiglia Alzheimer Research Foundation (a WAS). WS è titolare del Canada Research Chair nella malattia di Alzheimer. PTTL è supportato dal Scienze Naturali e Direttore di Ricerca di Ingegneria e del Michael Smith Fondazione per la borsa di studio Health Research.

1. Fissazione dei cervelli del mouse e criosezionamento <ol><li> Topi transgenici sono sacrificati con anidride carbonica, seguita dalla decapitazione e il cervello estratto. </li><li> Il cervello è sezionato longitudinalmente al centro. La metà del cervello saranno istantaneamente congelati in ghiaccio secco per l&#39;analisi biochimica. L&#39;altro sarà sommersa nel 4% paraformaldeide (PFA) per almeno 48 ore a 4 ° C. </li><li> Dopo la fissazione in PFA 4% per almeno 48 ore, il cervello emi sono trasferiti direttamente nella soluzione di saccarosio al 30% e posto a 4 ° C. Al punto, il cervello dovrebbe essere emi galleggiante nella soluzione di saccarosio. Quando il cervello Hemi affondò sul fondo, di solito richiede circa 48 ore, è pronto per essere incorporato in ottobre per criosezionamento. </li><li> Prima criosezionamento, montare un sottile strato di ottobre sulla manopola e far congelare a -20 ° C. Rimuovere il cervelletto dal resto del cervello e emi posto sullo strato di gelato ottobre Coprire immediatamente il cervello emi con OCT e permettono di congelare lentamente a -20 ° C. Una volta che l&#39;embedding ottobre il cervello si è trasformato opaco, è pronto per essere sezionato. </li><li> Impostare lo spessore di ogni sezione a 30 micron. Raccogliere ogni fetta in un contenitore con la soluzione D&#39;Olomos. </li></ol> 2. Procedura di immunoistochimica per placche neuritiche (gratuito sezioni flottante) <ol><li> Trattare ogni sezione in 88% di acido formico per 15 minuti. Dopo il trattamento con acido formico, la sezione sarà temporaneamente volta opaco e può apparire avvizzire. </li><li> Rimuovere ogni sezione in un piatto e lavare con Tx-PBS (0,3% Triton X-100 in PBS) per 5 min x 3 agitando delicatamente. </li><li> Perossido di blocco endogeno blocco dello 0,5% H 2 O 2 in Tx-PBS, RT 30 minuti agitando delicatamente. </li><li> Lavare con Tx-PBS per 10 min x 3 agitando delicatamente. </li><li> Sezione di blocco con 5% senza grassi del latte scremato sciolto in Tx-PBS a temperatura ambiente per 1 ora agitando delicatamente. </li><li> Incubare le sezioni in anticorpo primario (biotinilato 4g8, 1:500-1:2000, Sigillo) diluito in Tx-PBS contenente 5% del latte a 4 ° C per una notte agitando delicatamente. </li><li> Lavare con Tx-PBS per 10 min x 3 agitando delicatamente. </li><li> Preparare protocollo del produttore soluzione ABC successivo (Elite ABC, Vector Laboratories). Incubare le sezioni in miscela ABC per 30 minuti agitando delicatamente. </li><li> Lavare con Tx-PBS per 10 min x 3 agitando delicatamente. </li><li> Preparare 3,3 &#39;-diaminobenzidina tetraidrocloruro (DAB) (Vector Laboratories) protocollo del produttore successivo. Incubare le sezioni in miscela DAB per 5-10 min. A questo punto, si potrà osservare un colore brunastro svilupparsi su ogni sezione. </li><li> Lavare con Tx-PBS per 10 min x 3 agitando delicatamente. </li><li> Free-floating sezioni cervello sono montate su vetrini rivestiti di gelatina, per aiutare con l&#39;adesione dei tessuti. Le sezioni sono poi essiccate all&#39;aria seguita da compensazione con una serie di xilene e montati in Entellen. </li><li> Placche sono visualizzati al microscopio ottico con ingrandimento 40X e sono quantificati valutando la media della conta targa per fetta per ogni mouse. </li></ol> 3. Thioflavin S colorazione procedura per l&#39;individuazione delle placche neuritiche <ol><li> Sacrificio del mouse e procedure di estrazione del cervello sono quelli indicati in punti da 1.1 a 1.5. </li><li> Montare 4-5 sezioni del cervello emi su un vetrino e lasciare asciugare completamente prima della colorazione. </li><li> Lavare i vetrini con etanolo al 70% per 1 minuto. </li><li> Lavare i vetrini con 80% etanolo per 1 minuto. </li><li> Incubare i vetrini in soluzione filtrata (0,2 micron filtro) thioflavin S (1% a 80% di etanolo) per 15 minuti. (Soluzione Thioflavin S deve essere filtrata prima di ogni utilizzo). Nota: thioflavin S proteggere dalla luce e proteggere i vetrini colorati dalla luce. </li><li> Lavare i vetrini con 80% etanolo per 1 minuto. </li><li> Lavare i vetrini con il 70% etanolo 1 minuto. </li><li> Lavare con acqua distillata due volte. </li><li> Diapositive montare in acquosi di montaggio e permettere che i vetrini ad asciugare al buio per almeno 2 ore, seguita da coprioggetto tenuta con smalto trasparente. Sezioni colorate devono essere conservati al buio a 4 ° C. </li><li> La fluorescenza verde placche colorate possono essere visualizzate con microscropy fluorescenti. Analizzare le diapositive entro una settimana, perché la colorazione svanirà con il tempo. </li></ol> 4. Rappresentante Risultati Nel nostro recente studio sulla efficacia del farmaco anti-epilettico e stabilizzatore dell&#39;umore acido valproico (VPA) per inibire la formazione di placche neuritiche, abbiamo usato il immunocolorazione sopra descritto e thioflavin S colorazione procedure per identificare le placche neuritiche nei topi modello dC (3). La figura 1 rappresenta un tipico 9 mesi APP23 topo transgenico, colorate con 4g8 con anti-Aβ biotinilato e rilevati con il metodo ABC. Placche neuritiche sono chiaramente etichettati con l&#39;anticorpo e sono indicati dalle frecce bianche. Mediante colorazione thioflavin S, abbiamo rilevato deposizione neuritiche targa 2 mesi vecchia APP23xPS traditopi nsgenic (Figura 2). Thioflavin S legato Aβ contenenti placche neuritiche appare verde al microscopio a fluorescenza (indicato da frecce bianche). Abbiamo dimostrato che il trattamento con VPA, la formazione di placche neuritiche è significativamente ridotto (3). In un altro studio, abbiamo studiato l&#39;ipossia molecolare collegamento sottostante e patogenesi dell&#39;Alzheimer. Ancora una volta, utilizzando sia 4g8 anti-Aβ colorazione immunoistochimica e colorazione thioflavin S, abbiamo scoperto che l&#39;ipossia favorito la formazione di placche contenenti Aβ neuritiche (4). <img src="/files/ftp_upload/2831/2831fig1.jpg" alt="Figura 1"/> Figura 1. Analisi immunoistochimica della formazione di placche neuritiche in topi transgenici dC. APP23 topi all&#39;età di 9 mesi sono stati sacrificati e sono stati sezionati, fissa e sezionato. Placche neuritiche nel dell&#39;ippocampo sono state rilevate con un anti-Aβ 4g8 anticorpo e sono stati sviluppati utilizzando i metodi ABC e DAB. Le placche sono state visualizzate mediante microscopia ottica con 40X. Frecce bianche indicano placche neuritiche Aβ. <img src="/files/ftp_upload/2831/2831fig2.jpg" alt="Figura 2"/> Figura 2. Colorazione Thioflavin S di placche neuritiche nei topi dC modellato. APP23xPS45 doppio topi transgenici a 8 settimane di vita sono stati sacrificati e sono stati sezionati, fissa e sezionato. Piaghe neuritiche nel dell&#39;ippocampo sono state confermate mediante colorazione fluorescente thioflavin S e visualizzati al microscopio con ingrandimento 40x. Frecce bianche indicano thioflavin S placche neuritiche macchiato.

<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>

Tutti gli esperimenti sono stati condotti in conformità con l&#39;Università di cura degli animali Columbia britannica e il comitato Usa e le linee guida CIHR.

Immunoistochimica utilizzando il biotinilato macchie anticorpo marcato 4g8 placche neuritiche nel AD modellato topi con specificità. Il risultato colorazione consente la quantificazione e il confronto del carico di placca tra i diversi gruppi di trattamento. Ci sono diversi passi cruciali che potrebbero influenzare l&#39;esito. Poiché il cervello emi non sono perfusi prima dell&#39;estrazione dal cranio, la cura deve essere usato durante il processo di estrazione al fine di prevenire danni al cervello emi. Inoltre, poiché il cervello emi è passivamente perfuso, si consiglia di incubazione in 4% PFA per almeno 48 ore a 4 ° C prima immersione nella soluzione di saccarosio al 30%. In alternativa, la perfusione transcardial poteva essere fatto prima di estrarre il cervello. Se il cervello è fissata correttamente, dovrebbe avere una consistenza gommosa. Nel caso in cui il cervello non è fissato bene, le sezioni sarà facile strappare nella soluzione D&#39;Olomos o durante le procedure di colorazione. L&#39;anticorpo monoclonale 4g8 è reattivo ai residui di aminoacidi 17-24 di Aβ e ha riconosciuto l&#39;epitopo nella sequenza core (VFFAE). Dal momento che 4g8 è derivato da specie del mouse, si tende a dare una colorazione di fondo più alto. Così, abbiamo scelto di incubare le sezioni alla diluizione indicata con considerazioni a raggiungere un vero segnale, minimizzando la colorazione di fondo. Oltre a immunocolorazione per neuritiche rilevamento targhe con l&#39;anticorpo 4g8, metodo di colorazione uno thioflavin S è anche utilizzato per identificare le placche. Thioflavin S è una miscela omogenea colorante che risulta dalla metilazione di dehydrothiotoluidine con acido solfonico. Thioflavin S non si lega selettivamente i contenuti foglietto beta di proteine, come quelle in oligomeri amiloide. Su vincolante, thioflavin subisce un cambiamento caratteristico blu del suo spettro di emissione. Al contrario vincolante thioflavin S alle forme monomeriche non suscitare un spostamento verso il blu e non poteva essere rilevata con il microscopio a fluorescenza. Colorazione Thioflavin S offre una valida alternativa per lo screening rapido per amiloide come l&#39;intensità della fluorescenza permette una buona visualizzazione di piccole quantità di depositi di amiloide. Tuttavia, uno svantaggio di colorazione thioflavin S è la mancanza di specificità. Molti altri componenti del tessuto tra cui fogli contenenti beta estese, quali fibrinoids, ialina, cheratina, ecc, hanno un po affinità con questo colorante.

<table border="1"><tbody><tr><th> Nome del reagente e </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Paraformaldeide </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> Saccarosio </td><td> Fisher Scientific </td><td> S5-3 </td></tr><tr><td> Glicole etilenico </td><td> Sigma-Aldrich </td><td> 324558-2L </td></tr><tr><td> Tissue-Tek ottobre Compound </td><td> Sakura </td><td> 4583 </td></tr><tr><td> 88% di acido formico </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> Biotina etichetta 4g8 anticorpi </td><td> Cedarlane </td><td> SIG-39240-500 </td></tr><tr><td> Elite ABC kit </td><td> Vettore </td><td> PK-6100 </td></tr><tr><td> Imm PATTO DAB </td><td> Vettore </td><td> SK-4105 </td></tr><tr><td> Xilene </td><td> Fisher Scientific </td><td> X4-4 </td></tr><tr><td> Entellan </td><td> EM scienza </td><td> 65037-71 </td></tr><tr><td> SuperFrost * Plus preparati microscopici </td><td> Fisher Scientific </td><td> 12-550-15 </td></tr><tr><td> Criostato </td><td> Leica </td><td> CM-3050-S </td></tr><tr><td> MX35 Premier microtomo lama </td><td> Thermo Scientific </td><td> 3052835 </td></tr><tr><td> Thioflavin S </td><td> Sigma-Aldrich </td><td> T1892-25G </td></tr></tbody></table>

detection of neuritic plaques in alzheimer's disease mouse model

Malattia di Alzheimer (AD) è il disturbo più comune neurodegenerativa che porta alla demenza. La formazione di placche neuritiche è una delle caratteristiche patologiche della malattia di Alzheimer. Il componente centrale di placche neuritiche è una piccola proteina chiamata proteina filamentosa β amiloide (Aβ) 1, che è derivata da sequenziale taglio proteolitico della proteina beta-amiloide precursore (APP) da β-secretasi e γ-secretasi. L&#39;ipotesi amiloide comporta che Aγ contenenti placche come il meccanismo sottostante tossici in AD patologia 2. L&#39;analisi post-mortem della presenza di placca neuritiche conferma la diagnosi di Alzheimer. Per ulteriormente la nostra comprensione della neurobiologia Aγ nella patogenesi dell&#39;Alzheimer, vari ceppi di topi che esprimono AD-correlati mutazioni nei geni APP umani sono stati generati. A seconda della gravità della malattia, questi topi si svilupperà placche neuritiche in età diverse. Questi topi servire come preziosi strumenti per lo studio della patogenesi e sviluppo di droghe che potrebbero influenzare il percorso di elaborazione APP e la formazione della placca neuritiche. In questo protocollo, utilizziamo un metodo di immunoistochimica per l&#39;individuazione specifica delle placche neuritiche nei topi modello dC. Noi specificamente discutere della preparazione di estrarre il cervello e mezzo, la fissazione paraformaldeide, criosezionamento, e due metodi per rilevare placche nevrotico nel AD topi transgenici: rilevazione immunoistochimica utilizzando il metodo ABC e DAB e la rilevazione con metodo di colorazione fluorescente 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's Disease Mouse Model

Una delle caratteristiche patologiche di Alzheimer è la formazione di placche di amiloide β neuritiche proteina positivo. In questo protocollo si descrivono due metodi per rilevare placche neuritiche in topi transgenici modello AD: rilevazione immunoistochimica utilizzando il metodo ABC e DAB e la rilevazione con metodo di colorazione fluorescente thioflavin S.

Rilevazione di placche neuritiche nel modello di malattia di Alzheimer mouse

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

detection-of-neuritic-plaques-in-alzheimer-s-disease-mouse-model

Research

JoVE Journal

Neuroscience

36.5K Views.  The University of British Columbia. Una delle caratteristiche patologiche di Alzheimer è la formazione di placche di amiloide β neuritiche proteina positivo. In questo protocollo si descrivono due metodi per rilevare placche neuritiche in topi transgenici modello AD: rilevazione immunoistochimica utilizzando il metodo ABC e DAB e la rilevazione con metodo di colorazione fluorescente thioflavin S.

Video: 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 &#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.

Preparazione di oligomeriche β-amiloide 1-42 E induzione di Impairment plasticità sinaptica in fettine di ippocampo

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

Ricerca

Neuroscienze

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 &#946;-Protein, the Causative Agent of Alzheimer'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.

Selezione di Aptamers per β-amiloide proteina, l&#39;agente eziologico della malattia di Alzheimer

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

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has significantly broadened our knowledge of physiological and pathological processes in numerous tissues in vivo 4-7. In studies of the central nervous system (CNS), there has been a broad application of in vivo imaging in the brain, which has produced a plethora of novel and often unexpected findings about the behavior of cells such as neurons, astrocytes, microglia, under physiological or pathological conditions 8-17. However, mostly technical complications have limited the implementation of in vivo imaging in studies of the living mouse spinal cord. In particular, the anatomical proximity of the spinal cord to the lungs and heart generates significant movement artifact that makes imaging the living spinal cord a challenging task.
We developed a novel method that overcomes the inherent limitations of spinal cord imaging by stabilizing the spinal column, reducing respiratory-induced movements and thereby facilitating the use of two-photon microscopy to image the mouse spinal cord in vivo. This is achieved by combining a customized spinal stabilization device with a method of deep anesthesia, resulting in a significant reduction of respiratory-induced movements. This video protocol shows how to expose a small area of the living spinal cord that can be maintained under stable physiological conditions over extended periods of time by keeping tissue injury and bleeding to a minimum. Representative raw images acquired in vivo detail in high resolution the close relationship between microglia and the vasculature. A timelapse sequence shows the dynamic behavior of microglial processes in the living mouse spinal cord. Moreover, a continuous scan of the same z-frame demonstrates the outstanding stability that this method can achieve to generate stacks of images and/or timelapse movies that do not require image alignment post-acquisition. Finally, we show how this method can be used to revisit and reimage the same area of the spinal cord at later timepoints, allowing for longitudinal studies of ongoing physiological or pathological processes in vivo.

In vivo Imaging of the Mouse Spinal Cord Using Two-photon Microscopy

A minimally invasive protocol to stabilize the mouse spinal column and perform repetitive in vivo spinal cord imaging using two-photon microscopy is described. This method combines a spinal stabilization device and an anesthetic regimen to minimize respiratory-induced movements and produce raw imaging data that require no alignment or other post-processing.

Imaging in vivo del midollo spinale di topo Utilizzo di due fotoni Microscopia

In vivo imaging using two-photon microscopy 1 in mice that have been genetically engineered to express fluorescent proteins in specific cell types 2-3 has ...

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'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.

Morris Maze prova d&#39;acqua per deficit di apprendimento e memoria in topi modello della malattia di 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.

Rilevamento di ipossia microregionale nella corteccia cerebrale di topo Imaging a due fotoni di fluorescenza endogena 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. ...

Intracerebroventricular Viral Injection of the Neonatal Mouse Brain for Persistent and Widespread Neuronal Transduction

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

Here we demonstrate a technique for widespread neuronal transduction by intraventricular injection of adeno-associated virus into the neonatal mouse brain. This method provides a rapid and easy way to attain lifelong expression of virally-delivered transgenes.

Intracerebroventricular Iniezione virale del cervello neonatale mouse per persistente e diffusa trasduzione neuronale

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene ...

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.

Infuso stereotassico di Oligomeric amiloide-beta nelle Hippocampus mouse

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'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.

Analisi di interazione generalizzata psicofisiologica (PPI) di memoria relative connettività in individui a rischio genetico per la malattia di 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.

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).

Utilizzando biosensori a base di enzimi misurare Tonic e Phasic glutammato in modelli murini di 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.

Valutazioni di neurobehavioral in un modello murino di ferita di cervello Hypoxic-ischemica neonatale

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 ...