Les travaux pris en charge par NIH AG027297, un prix de la moelle épinière et le Kentucky Research Trust blessure à la tête, et un don de la Fondation Kleberg.

<ol>
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La production de cette vidéo article a été parrainé par Leica Microsystems.

Les étapes décrites dans ce protocole aidera à assurer que les dissections du cerveau sont effectués au moins aussi rapidement et efficacement en âge, comme chez les rats adultes jeunes. Nous avons aussi fournir suffisamment de détails pour le débutant de créer leur propre tranche d&#39;études sur la LTP et LTD. Si une exploration plus poussée du vieillissement et des changements AD dans la fonction synaptique et la plasticité est l&#39;un de vos objectifs, il ya au moins deux autres questions méthodologiques, évoqué ci-dessus, qui méritent un examen plus approfondi. D&#39;abord, plusieurs laboratoires ont montré que le Ca 2 +: Mg2 + ratio de l&#39;enregistrement de l&#39;ACSF peut avoir un effet marqué sur la plasticité synaptique dans l&#39;induction des coupes d&#39;hippocampe 2,10,24,25. Dans le LCR chez les mammifères, le Ca 2 +: Mg2 + taux est d&#39;environ un (voir par exemple 26). Toutefois, l&#39;ACSF Ca 2 +: Mg2 + ratios plus proche de 2 sont couramment utilisés dans les études de tranche de la fonction synaptique et la plasticité. Dans les premières études, cette pratique a probablement été adapté pour optimiser l&#39;induction de la LTP, puis est ensuite devenu la routine pour toutes les études de plasticité. Toutefois, cette pratique peut être problématique dans les études de vieillissement et AD en raison des différences bien caractérisées dans les neurones de Ca 2 + règlement. Plus précisément, l&#39;afflux de Ca 2 + et / ou Ca 2 + induite par Ca 2 +-release est élevée chez les rats âgés et / ou des souris modèles AD lors de l&#39;activation neuronale 3,27-31. L&#39;induction de LTD est particulièrement sensible aux changements subtils dans les niveaux de Ca + 2 ACSF. Notre protocole, qui utilise 2 mM de Ca 2 + et 2 mM Mg 2 +, communément traduit par LTD pour personnes âgées, mais pas de jeunes animaux adultes 2, tandis que les études utilisant une Ca 2 +: Mg2 ratio de + près de deux, ont observé LTD robustes chez les adultes dans le absence d&#39;une différence de 2,10 âge ou en conjonction avec réduction LTD chez les rats âgés 32. Ces observations soulignent la nécessité d&#39;examiner attentivement ACSF Ca 2 + et Mg 2 + lors de la comparaison des niveaux de Ca 2 +-dépendante la plasticité chez les animaux adultes jeunes et d&#39;âge. Le deuxième problème méthodologique concerne la forte dépendance de la LTP sur dépolarisation postsynaptique 33 et possibles du vieillissement / génotype des différences dans la force synaptique. Dans une expérience typique de LTP, de base et de l&#39;intensité de stimulation LTP est généralement ajustée pour produire un demi maximal (ou de trois-quarts maximale) EPSP amplitude. Le problème potentiel est que les rats âgés et APP/PS1 souris montrent habituellement réduite force synaptique par rapport à leurs homologues de type plus jeunes et / ou sauvages, ce qui signifie que les valeurs de base EPSP sera également moindre chez les rats âgés et APP/PS1 souris. Les petits RPEB peut se traduire à moins dépolarisation lors de la stimulation LTP, résultant en une probabilité réduite pour induire la LTP 33. En raison de cette confondent potentiel, il est difficile de déterminer si ces animaux présentent un déficit de débit, un déficit de plasticité, ou les deux. C&#39;est, dans les mécanismes d&#39;induction LTP âgés et / ou APP/PS1 souris peuvent être fonctionnellement intactes (pas de déficit plasticité), mais insuffisamment stimulés (déficit débit) dans ces conditions. Cette distinction est cruciale, car les mécanismes de débit et les mécanismes de la plasticité peut réagir très différemment à un traitement spécifique pharmacologique. Nous nous efforçons de minimiser l&#39;impact de débit réduit sur l&#39;induction de la LTP en normalisant l&#39;amplitude EPSP au même niveau (par exemple 1 mV) à travers toutes les tranches avant la stimulation LTP. D&#39;autres stratégies peuvent être efficaces aussi bien (par exemple l&#39;utilisation de la pince de tension ou de courant pour égaliser le potentiel de membrane entre les groupes lors de la stimulation LTP), et devrait être considéré lors des enquêtes LTP dans ces modèles animaux.

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td></tr><tr><td> NaCl </td><td> Fisher </td><td> BP358-1 </td></tr><tr><td> KCl </td><td> Fisher </td><td> BP366-500 </td></tr><tr><td> KH 2 PO 4 (monobasique) </td><td> Sigma </td><td> P5379-100G </td></tr><tr><td> MgSO 4 </td><td> Sigma </td><td> M2643-500G </td></tr><tr><td> CaCl 2 (dihydrate) </td><td> Sigma </td><td> C3306-250G </td></tr><tr><td> NaHCO 3 </td><td> Fisher </td><td> S233-500 </td></tr><tr><td> C 6 H 12 O 6 (dextrose) </td><td> Fisher </td><td> BP350-1 </td></tr></tbody></table> Réactifs Tableau 1. Requise <table border="1"><tbody><tr><td> Nom de l&#39;équipement </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Erlenmeyer </td><td> Fisher </td><td> FB-500-2000 FB-500-1000 </td><td></td></tr><tr><td> Aquarium Bulleur </td><td></td><td></td><td> Utilisé pour les médias oxygénant. Disponible dans la plupart des magasins d&#39;animaux </td></tr><tr><td> 50 ml en verre bécher </td><td> Fisher </td><td> 02-540G </td><td> Pour storarge cerveau dans l&#39;ACSF </td></tr><tr><td> Parafilm </td><td> Fisher </td><td> 13-374-10 </td><td></td></tr><tr><td> Guillotine pour petits animaux </td><td> World Precision Instruments (WPI) </td><td> PACD-M </td><td></td></tr><tr><td> Serviette en papier plat </td><td></td><td></td><td></td></tr><tr><td> # 11 lames Feather chirurgicale </td><td> Fisher </td><td> 08 à 916-5B </td><td></td></tr><tr><td> Beebee Ciseaux d&#39;os </td><td> Outils Fine Science (FST) </td><td> 16044-10 </td><td></td></tr><tr><td> Lempert Rongeurs </td><td> Roboz </td><td> RS-8321 </td><td> Utilisez pour les rats </td></tr><tr><td> Friedman-Pearson Rongeurs </td><td> TSF </td><td> 16020-14 </td><td> Utilisez pour les souris ou les rats </td></tr><tr><td> Outil de Hippocampus </td><td> TSF </td><td> 10099-15 </td><td></td></tr><tr><td> Cuillère </td><td></td><td></td><td> Une cuillère à café en plastique fera </td></tr><tr><td> Spatule </td><td> Fisher </td><td> 21 à 401-25A </td><td> Spatule </td></tr><tr><td> Chirurgical ciseaux à iris </td><td> TSF </td><td> 14058-09 </td><td></td></tr><tr><td> pipettes de transfert en plastique </td><td> Fisher </td><td> 13-711-43 </td><td></td></tr><tr><td> 110mm papier filtre Whatman </td><td> Fisher </td><td> 09-805E </td><td> Cat Whatman. 1001-110 </td></tr><tr><td> Verre de Petri </td><td> Fisher </td><td></td><td></td></tr><tr><td> Leica VT1000P Manuel vibrant microtome </td><td> Vibratome </td><td></td><td></td></tr><tr><td> 0.1mm FA-10 S Feather lame </td><td> Ted Pella </td><td> 121-9 </td><td> 0.1mm FA-10 S Feather lame </td></tr><tr><td> Verre borosilicate Pasteur Pipet (avec poire en caoutchouc) </td><td> Fisher </td><td> 13-678-20A </td><td> Pour transférer des tranches: La pointe est cassée et polies au feu pour la plus grande ouverture </td></tr><tr><td> Boîte de 35 mm Culture Polysterine </td><td> Corning </td><td> 430588 </td><td> Utilisé pour la collecte des tranches après dissection </td></tr></tbody></table> Outils de tableau 2. Et des matériaux pour la dissection <table border="1"><tbody><tr><td> Équipement / matériel </td><td> Société </td><td> Commentaires (optionnel) </td></tr><tr><td> Tenir la chambre </td><td> Custom Built </td><td></td></tr><tr><td> P-97 Extracteur horizontal pipette </td><td> Sutter Instrument Co. </td><td></td></tr><tr><td> Table d&#39;isolation vibratoire </td><td> Technique Manufacturing Corporation (TMC) </td><td></td></tr><tr><td> Cage de Faraday </td><td> Fabriquée </td><td></td></tr><tr><td> Pyrex Aspirateur Bouteille avec Sidearm Bas (Produit # 1220-1L) </td><td> Corning </td><td></td></tr><tr><td> Gravity-contolled mis IV avec régulateur (Produit # 2C8891) </td><td> Baxter </td><td></td></tr><tr><td> Ligne d&#39;aspiration centrale </td><td></td><td> Disponible dans la plupart des laboratoires modernes </td></tr><tr><td> 95% 2 / 5% CO 2 mélange de gaz O </td><td> Écossaist-Gross Co. </td><td></td></tr><tr><td> TygonTM Lab tube Pour O 2 / CO 2 de livraison </td><td> Fisher Scientific </td><td> Non toxique, non-oxydant, vient dans une variété de tailles. </td></tr><tr><td> Eclipse E600FN Microscope </td><td> Nikon </td><td> avec des objectifs 10x et 40x, près infrarouge filtre, et la GFP, DS-RED2 filtres </td></tr><tr><td> Refroidir Aligner ES Appareil Photo Numérique </td><td> Photometrics </td><td> Refroidir Aligner ES Appareil Photo Numérique </td></tr><tr><td> X-Cite illuminateur fluorescent </td><td> EXFO </td><td> X-Cite illuminateur fluorescent </td></tr><tr><td> Platform Microscope </td><td> Siskiyou </td><td> Personnalisé assemblés </td></tr><tr><td> RC-22 submersibles d&#39;enregistrement de chambre (Produit # 64-0228) </td><td> Warner Instruments (WI) </td><td> Nécessite P-1 adaptateur plateforme et le stade (Produit # 64-0277 De Warner) </td></tr><tr><td> Contrôleur de température TC2BIP 2/3Ch </td><td> Microcontrols cellulaire </td><td> Contrôleur de température TC2BIP 2/3Ch </td></tr><tr><td> 4 manipulateur miniature axe manuel </td><td> Siskiyou </td><td></td></tr><tr><td> Fil Iridium Platine (0,002 po) (Item # PTT0203) </td><td> WPI </td><td></td></tr><tr><td> A365 Isolateur Stimulus </td><td> WPI </td><td> A365 Isolateur Stimulus </td></tr><tr><td> Multiclamp amplificateur 700b </td><td> Axon Instruments </td><td></td></tr><tr><td> Digidata 1322A convertisseur A / D </td><td> Axon Instruments </td><td></td></tr><tr><td> Logiciels pClamp </td><td> Axon Instruments </td><td></td></tr><tr><td> Personal Computer (Pentium 4) </td><td> Dell </td><td></td></tr></tbody></table> Tableau 3. Équipements d&#39;électrophysiologie et de matériaux

preparation of acute hippocampal slices from rats and transgenic mice for the study of synaptic alterations during aging and amyloid pathology

La préparation de tranches de rongeurs hippocampe est peut-être l&#39;outil le plus largement utilisé pour étudier les mammifères fonction synaptique et la plasticité. L&#39;hippocampe peut être extraite facilement et rapidement à partir de rats et de souris et de tranches de rester viables pendant des heures dans oxygénée liquide céphalorachidien artificiel. Par ailleurs, les techniques de base sont electrophysisologic facilement appliquée à l&#39;enquête de la fonction synaptique dans des coupes d&#39;hippocampe et ont fourni certains des meilleurs biomarqueurs pour des troubles cognitifs. La tranche d&#39;hippocampe est particulièrement populaire pour l&#39;étude des mécanismes de plasticité synaptique impliqués dans l&#39;apprentissage et la mémoire. Les changements dans l&#39;induction de la potentialisation à long terme et la dépression (LTP et LTD) de l&#39;efficacité synaptique dans les coupes d&#39;hippocampe (ou leur absence) sont fréquemment utilisés pour décrire le phénotype neurologique des animaux ayant une déficience cognitive et / ou pour évaluer le mécanisme d&#39;action des composés nootropique. Cet article décrit les procédures que nous utilisons pour la préparation de coupes d&#39;hippocampe de rats et de souris transgéniques pour l&#39;étude des altérations synaptiques associés à vieillissement du cerveau et la maladie d&#39;Alzheimer (MA) 1-3. Utilisez des rats âgés et les souris modèle AD peuvent présenter un ensemble unique de défis pour les chercheurs habitués à utiliser des jeunes rats et / ou des souris dans leurs recherches. Les rats âgés ont plus épais et plus dur crânes des tissus conjonctifs que les jeunes rats et des souris, ce qui peut retarder l&#39;extraction du cerveau et / ou de dissection et par conséquent nier ni exagérer réelles différences d&#39;âge dans la fonction synaptique et la plasticité. Le vieillissement et la pathologie amyloïde peut également exacerber les dommages subis pendant l&#39;hippocampe à la procédure de dissection, encore une fois toute complication conclusions tirées de l&#39;évaluation physiologique. Ici, nous discutons des mesures prises pendant la procédure de dissection afin de minimiser ces problèmes. Exemples de réponses synaptiques acquis en «bonne santé» et «malsain» tranches de rats et de souris sont fournis, ainsi que le représentant des expériences plasticité synaptique. L&#39;impact possible d&#39;autres facteurs méthodologiques sur la fonction synaptique dans ces modèles animaux (par exemple composants solution d&#39;enregistrement, les paramètres de stimulation) sont également discutés. Bien que l&#39;accent de cet article est sur l&#39;utilisation des rats âgés et les souris transgéniques, les novices pour trancher la physiologie devrait trouver suffisamment de détails ici pour obtenir commencé sur leurs propres études, en utilisant une variété de modèles de rongeurs.

Watch this Scientific Journal Video about Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology at JoVE.com

Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

Cet article décrit les procédures pour la préparation de coupes d&#39;hippocampe de rats et de souris transgéniques pour l&#39;étude des altérations synaptiques associés au vieillissement du cerveau et liée à l&#39;âge des maladies neurodégénératives comme la maladie d&#39;Alzheimer.

Préparation des coupes d&#39;hippocampe aiguë chez des rats et des souris transgéniques pour l&#39;étude des altérations synaptiques au cours du vieillissement et de l&#39;amylose pathologie

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The ...

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Neuroscience

93.7K vues.  University of Kentucky College of Public Health. Cet article décrit les procédures pour la préparation de coupes d'hippocampe de rats et de souris transgéniques pour l'étude des altérations synaptiques associés au vieillissement du cerveau et liée à l'âge des maladies neurodégénératives comme la maladie d'Alzheimer.

Vidéo: Preparation of Acute Hippocampal Slices from Rats and Transgenic Mice for the Study of Synaptic Alterations during Aging and Amyloid Pathology

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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Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation des neurones à partir de tranches de cerveau de rongeurs pour étudier la transmission synaptique et de l&#39;image terminaisons présynaptiques

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

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

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

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

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

Dual Electrophysiological Recordings of Synaptically-evoked Astroglial and Neuronal Responses in Acute Hippocampal Slices

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs through activation of their ion channels and neurotransmitter receptors, and process information in part through activity-dependent release of gliotransmitters. Furthermore, astrocytes constitute the main uptake system for glutamate, contribute to potassium spatial buffering, as well as to GABA clearance. These cells therefore constantly monitor synaptic activity, and are thereby sensitive indicators for alterations in synaptically-released glutamate, GABA and extracellular potassium levels. Additionally, alterations in astroglial uptake activity or buffering capacity can have severe effects on neuronal functions, and might be overlooked when characterizing physiopathological situations or knockout mice. Dual recording of neuronal and astroglial activities is therefore an important method to study alterations in synaptic strength associated to concomitant changes in astroglial uptake and buffering capacities. Here we describe how to prepare hippocampal slices, how to identify stratum radiatum astrocytes, and how to record simultaneously neuronal and astroglial electrophysiological responses. Furthermore, we describe how to isolate pharmacologically the synaptically-evoked astroglial currents.

The preparation of acute brain slices from isolated hippocampi, as well as the simultaneous electrophysiological recordings of astrocytes and neurons in stratum radiatum during stimulation of schaffer collaterals is described. The pharmacological isolation of astroglial potassium and glutamate transporter currents is demonstrated.

Deux enregistrements électrophysiologiques des synapses-réponses évoquées astrogliales et neuronales aiguës dans des coupes d&#39;hippocampe

Astrocytes form together with neurons tripartite synapses, where they integrate and modulate neuronal activity. Indeed, astrocytes sense neuronal inputs ...

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 ...

Investigation of Synaptic Tagging/Capture and Cross-capture using Acute Hippocampal Slices from Rodents

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity is achieved in neurons within a specific time frame. These long-term plasticity-related processes are the leading candidate models to study the basis of memory formation and persistence at the cellular level. Both STC and cross-tagging involve two serial processes: (1) setting of the synaptic tag as triggered by a specific pattern of stimulation, and (2) synaptic capture, whereby the synaptic tag interacts with newly synthesized plasticity-related proteins (PRPs). Much of the understanding about the concepts of STC and cross-tagging arises from the studies done in CA1 region of the hippocampus and because of the technical complexity many of the laboratories are still unable to study these processes. Experimental conditions for the preparation of hippocampal slices and the recording of stable late-LTP/LTD are extremely important to study synaptic tagging/cross-tagging. This video article describes the experimental procedures to study long-term plasticity processes such as STC and cross-tagging in the CA1 pyramidal neurons using stable, long-term field-potential recordings from acute hippocampal slices of rats.

This video article describes experimental procedures to study long-term plasticity and its associative processes such as synaptic tagging, capture and cross-tagging in the CA1 pyramidal neurons using acute hippocampal slices from rodents.

Enquête de Synaptic étiquetage / capture et de la Croix-capture à l&#39;aide aiguë coupes d&#39;hippocampe des rongeurs

Synaptic tagging and capture (STC) and cross-tagging are two important mechanisms at cellular level that explain how synapse-specificity and associativity ...

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have validated the utility of this method for enhancing neuronal preservation and overall brain slice viability. The implementation of this technique by early adopters has facilitated detailed investigations into brain function using diverse experimental applications and spanning a wide range of animal ages, brain regions, and cell types. Steps are outlined for carrying out the protective recovery brain slice technique using an optimized NMDG artificial cerebrospinal fluid (aCSF) media formulation and enhanced procedure to reliably obtain healthy brain slices for patch clamp electrophysiology. With this updated approach, a substantial improvement is observed in the speed and reliability of gigaohm seal formation during targeted patch clamp recording experiments while maintaining excellent neuronal preservation, thereby facilitating challenging experimental applications. Representative results are provided from multi-neuron patch clamp recording experiments to assay synaptic connectivity in neocortical brain slices prepared from young adult transgenic mice and mature adult human neurosurgical specimens. Furthermore, the optimized NMDG protective recovery method of brain slicing is compatible with both juvenile and adult animals, thus resolving a limitation of the original methodology. In summary, a single media formulation and brain slicing procedure can be implemented across various species and ages to achieve excellent viability and tissue preservation.

Preparation of Acute Brain Slices Using an Optimized N-Methyl-D-glucamine Protective Recovery Method

This protocol demonstrates the implementation of an optimized N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. A single media formulation is used to reliably obtain healthy brain slices from animals of any age and for diverse experimental applications.

Préparation de tranches de cerveau aiguë en utilisant un optimisé N-méthyl-D-glucamine méthode de recouvrement protecteur

This protocol is a practical guide to the N-methyl-D-glucamine (NMDG) protective recovery method of brain slice preparation. Numerous recent studies have ...

Recording Synaptic Plasticity in Acute Hippocampal Slices Maintained in a Small-volume Recycling-, Perfusion-, and Submersion-type Chamber System

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful analysis of synaptic transmission modulation when performing field potential or intracellular recordings. This manuscript describes methodological aspects that might be helpful in improving experimental conditions for the maintenance of acute brain slices and for recording field excitatory postsynaptic potentials in a commercially available submersion chamber with an outflow-carbogenation unit. The outflow-carbogenation helps to stabilize the oxygen level in experiments that rely on the recycling of a small buffer reservoir to enhance the cost-efficiency of drug experiments. In addition, the manuscript presents representative experiments that examine the effects of different carbogenation modes and stimulation paradigms on the activity-dependent synaptic plasticity of synaptic transmission.

This protocol describes the stabilization of the oxygen level in a small volume of recycled buffer and methodological aspects of recording activity-dependent synaptic plasticity in submerged acute hippocampal slices.

Enregistrement de la plasticité synaptique dans des tranches d’hippocampe aiguës maintenues dans un petit volume recyclage - Perfusion- et système de chambre de Submersion-type

Even though experiments on brain slices have been in use since 1951, problems remain that reduce the probability of achieving a stable and successful ...

Single Synapse Indicators of Glutamate Release and Uptake in Acute Brain Slices from Normal and Huntington Mice

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other neurodegenerative disorders, this independence might be compromised due to insufficient glutamate clearance and the resulting spill-in and spill-out effects. Altered astrocytic coverage of the presynaptic terminals and/or dendritic spines as well as a reduced size of glutamate transporter clusters at glutamate release sites have been implicated in the pathogenesis of diseases resulting in symptoms of dys-/hyperkinesia. However, the mechanisms leading to the dysfunction of glutamatergic synapses in HD are not well understood. Improving and applying synapse imaging we have obtained data shedding new light on the mechanisms impeding the initiation of movements. Here, we describe the principle elements of a relatively inexpensive approach to achieve single synapse resolution by using the new genetically encoded ultrafast glutamate sensor iGluu, wide-field optics, a scientific CMOS (sCMOS) camera, a 473 nm laser and a laser positioning system to evaluate the state of corticostriatal synapses in acute slices from age appropriate healthy or diseased mice. Glutamate transients were constructed from single or multiple pixels to obtain estimates of i) glutamate release based on the maximal elevation of the glutamate concentration [Glu] next to the active zone and ii) glutamate uptake as reflected in the time constant of decay (TauD) of the perisynaptic [Glu]. Differences in the resting bouton size and contrasting patterns of short-term plasticity served as criteria for the identification of corticostriatal terminals as belonging to the intratelencephalic (IT) or the pyramidal tract (PT) pathway. Using these methods, we discovered that in symptomatic HD mice ~40% of PT-type corticostriatal synapses exhibited insufficient glutamate clearance, suggesting that these synapses might be at risk to excitotoxic damage. The results underline the usefulness of TauD as a biomarker of dysfunctional synapses in Huntington mice with a hypokinetic phenotype.

We present a protocol to evaluate the balance between glutamate release and clearance at single corticostriatal glutamatergic synapses in acute slices from adult mice. This protocol uses the fluorescent sensor iGluu for glutamate detection, a sCMOS camera for signal acquisition and a device for focal laser illumination.

Indicateurs synapses uniques de la libération et de l’absorption des glutamates dans les tranches cérébrales aigues des souris normales et huntingtones

Synapses are highly compartmentalized functional units that operate independently on each other. In Huntington&#39;s disease (HD) and other ...

Preparation of Acute Human Hippocampal Slices for Electrophysiological Recordings

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for sudden death. Despite an abundance of available treatment options, about 30% of patients are drug-resistant. Several novel therapeutics have been developed using animal models, though the rate of drug-resistant patients remains unaltered. One of probable reasons is the lack of translation between rodent models and humans, such as a weak representation of human pharmacoresistance in animal models. Resected human brain tissue as a preclinical evaluation tool has the advantage to bridge this translational gap. Described here is a method for high quality preparation of human hippocampal brain slices and subsequent stable induction of epileptiform activity. The protocol describes the induction of burst activity during application of 8 mM KCl and 4-aminopyridin. This activity is sensitive to established AED lacosamide or novel antiepileptic candidates, such as dimethylethanolamine (DMEA). In addition, the method describes induction of seizure-like events in CA1 of human hippocampal brain slices by reduction of extracellular Mg2+ and application of bicuculline, a GABAA receptor blocker. The experimental set-up can be used to screen potential antiepileptic substances for their effects on epileptiform activity. Furthermore, mechanisms of action postulated for specific compounds can be validated using this approach in human tissue (e.g., using patch-clamp recordings). To conclude, investigation of vital human brain tissue ex vivo (here, resected hippocampus from patients suffering from temporal lobe epilepsy) will improve the current knowledge of physiological and pathological mechanisms in the human brain.

The presented protocol describes the transport and preparation of resected human hippocampal tissue with the ultimate goal to use vital brain slices as a preclinical evaluation tool for potential antiepileptic substances.

Préparation de tranches d’hippocampe humaine aiguë pour les enregistrements électrophysiologiques

Epilepsy affects about 1% of the world population and leads to a severe decrease in quality of life due to ongoing seizures as well as high risk for ...