Arbeit unterstützt durch NIH AG027297, eine Auszeichnung von der Kentucky Spinal Cord und Head Injury Research Trust, und ein Geschenk der Kleberg Foundation.

<ol>
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Die Schritte in diesem Protokoll beschriebenen hilft sicherzustellen, dass Gehirn Dissektionen out sind mindestens so schnell und effizient in Jahren durchgeführt, wie in jungen erwachsenen Ratten. Wir bieten auch ausreichend detailliert für den Anfänger für die Errichtung ihrer eigenen Slice Studien LTP und LTD. Wenn die weitere Erforschung des Alterns und AD Veränderungen der synaptischen Funktion und Plastizität ist eines Ihrer Ziele, gibt es mindestens zwei weitere methodische Fragen, oben angedeutet, dass weitere Beachtung verdienen. Zunächst in verschiedenen Laboren haben gezeigt, dass die Ca 2 +: Mg2 +-Verhältnis in der Aufnahme ACSF eine deutliche Wirkung auf die Induktion synaptischer Plastizität im Hippocampus Scheiben 2,10,24,25 können. In Säugetierzellen CSF, die Ca 2 +: Mg2 +-Verhältnis von rund eins (siehe zB 26). Allerdings ACSF Ca 2 +: Mg2 +-Verhältnisse näher zu 2 werden in der Regel in Scheiben Studien der synaptischen Funktion und Plastizität eingesetzt. In frühen Studien, diese Praxis wahrscheinlich wurde angepasst, um die Induktion von LTP zu optimieren, anschließend wurden zur Routine für alle Plastizität Studien. Allerdings kann diese Praxis in Alterungs-und AD-Studien problematisch, weil der gut charakterisierten Unterschiede in der neuronalen Ca 2 +-Regulation. Genauer gesagt, Ca 2 +-Einstrom und / oder Ca 2 +-induzierte Ca 2 +-Version ist in alten Ratten und / oder AD-Modell-Mäusen während der neuronalen Aktivierung 3,27-31 erhöht. Induktion von LTD ist besonders empfindlich auf subtile Veränderungen in ACSF Ca 2 +-Spiegel. Unser Protokoll, das 2 mM Ca 2 + und 2 mM Mg2 +, führt häufig zu LTD für ältere, nicht aber junge Tiere 2, während Studien mit einem Ca 2 + verwendet: Mg2 + Verhältnis näher bei zwei, haben robuste LTD bei Erwachsenen beobachtet in der Abwesenheit von einem Altersunterschied 2,10 oder in Verbindung mit reduzierten LTD in alten Ratten 32. Diese Beobachtungen unterstreichen die Notwendigkeit, sorgfältig zu prüfen, ACSF Ca 2 + und Mg2 +-Konzentrationen beim Vergleich Ca 2 +-abhängige Plastizität in alten und jungen erwachsenen Tieren. Das zweite methodische Problem betrifft die starke Abhängigkeit von LTP auf postsynaptische Depolarisation 33 und die Alterung / Genotyp Unterschiede in der synaptischen Stärke. In einem typischen Experiment LTP, Baseline und LTP Stimulationsintensität ist in der Regel eingestellt, um eine halbmaximale (oder drei Viertel maximal) EPSP Amplitude zu erzeugen. Das mögliche Problem ist, dass im Alter von Ratten und Mäusen APP/PS1 zeigen meist synaptischen Stärke im Verhältnis zu ihren jüngeren und / oder Wildtyp-Gegenstücke reduziert, so dass Baseline EPSP Werte werden auch kleinere in alten Ratten und Mäusen APP/PS1. Kleinere EPSPs kann auf weniger Depolarisation während LTP Stimulation zu übersetzen, was zu einer verringerten Wahrscheinlichkeit für die Induktion LTP 33. Aufgrund dieser potenziellen verwechseln, ist es schwierig, festzustellen, ob diese Tiere einen Durchsatz Defizit, eine Plastizität Defizit oder beides aufweisen. Das heißt, LTP Induktion Mechanismen in Alter und / oder APP/PS1 Mäusen funktionell intakt (keine Plastizität Defizit), aber nicht ausreichend stimuliert (Durchsatz Defizit) unter diesen Bedingungen. Diese Unterscheidung ist wichtig, da die Mechanismen für den Durchsatz und Mechanismen für die Plastizität sehr unterschiedlich reagieren können, um eine spezifische pharmakologische Behandlung. Wir versuchen, die Auswirkungen eines geringeren Durchsatz auf LTP-Induktion durch die Normalisierung der EPSP Amplitude auf der gleichen Ebene (z. B. 1 mV) über alle Scheiben vor der LTP Stimulation zu minimieren. Andere Strategien können wirksam sein, als auch (zB Verwendung von Spannung oder Strom Klemme an das Membranpotential zwischen Gruppen auszugleichen während LTP-Stimulation), und sollte bei der Untersuchung von LTP in diesen Tiermodellen werden.

<table border="1"><tbody><tr><td> Name des Reagenzes </td><td> Firma </td><td> Katalog-Nummer </td></tr><tr><td> NaCl </td><td> Fischer </td><td> BP358-1 </td></tr><tr><td> KCl </td><td> Fischer </td><td> BP366-500 </td></tr><tr><td> KH 2 PO 4 (einbasischen) </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 (Dihydrat) </td><td> Sigma </td><td> C3306-250G </td></tr><tr><td> NaHCO 3 </td><td> Fischer </td><td> S233-500 </td></tr><tr><td> C 6 H 12 O 6 (Dextrose) </td><td> Fischer </td><td> BP350-1 </td></tr></tbody></table> Tabelle 1. Reagenzien erforderlich <table border="1"><tbody><tr><td> Bezeichnung des Geräts </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> Erlenmeyerkolben </td><td> Fischer </td><td> FB-500-2000 FB-500-1000 </td><td></td></tr><tr><td> Aquarium Bubbler </td><td></td><td></td><td> Wird für sauerstoffartig Medien. Erhältlich in den meisten Zoohandlungen </td></tr><tr><td> 50 ml Becherglas </td><td> Fischer </td><td> 02-540G </td><td> Für Gehirn storarge in ACSF </td></tr><tr><td> Parafilm </td><td> Fischer </td><td> 13-374-10 </td><td></td></tr><tr><td> Small Animal Guillotine </td><td> World Precision Instruments (WPI) </td><td> DCAP-M </td><td></td></tr><tr><td> Flache Papiertuch </td><td></td><td></td><td></td></tr><tr><td> Nr. 11 Feather Skalpell </td><td> Fischer </td><td> 08 bis 916-5B </td><td></td></tr><tr><td> Beebee Knochen Schere </td><td> Feine Science Tools (FST) </td><td> 16044-10 </td><td></td></tr><tr><td> Lempert Rongeure </td><td> Roboz </td><td> RS-8321 </td><td> Verwenden Sie für Ratten </td></tr><tr><td> Friedman-Pearson Rongeure </td><td> FST </td><td> 16020-14 </td><td> Verwenden Sie für Mäuse oder Ratten </td></tr><tr><td> Hippocampus-Tool </td><td> FST </td><td> 10099-15 </td><td></td></tr><tr><td> Löffel </td><td></td><td></td><td> Ein Kunststoff Teelöffel tun </td></tr><tr><td> Spachtel </td><td> Fischer </td><td> 21 bis 401-25A </td><td> Spachtel </td></tr><tr><td> Chirurgische Iris Schere </td><td> FST </td><td> 14058-09 </td><td></td></tr><tr><td> Kunststoff-Transfer Pipetten </td><td> Fischer </td><td> 13-711-43 </td><td></td></tr><tr><td> 110mm Whatman Filterpapier </td><td> Fischer </td><td> 09-805E </td><td> Whatman Katze. 1001-110 </td></tr><tr><td> Glas-Petrischale </td><td> Fischer </td><td></td><td></td></tr><tr><td> Leica VT1000P Manuelle Vibrating Mikrotom </td><td> Vibratom </td><td></td><td></td></tr><tr><td> 0.1mm FA-10 Feather S Klinge </td><td> Ted Pella </td><td> 121-9 </td><td> 0.1mm FA-10 Feather S Klinge </td></tr><tr><td> Borosilikatglas Pasteur-Pipette (mit Gummiball) </td><td> Fischer </td><td> 13 bis 678-20A </td><td> Für die Übertragung von Scheiben: Tip abgebrochen und Wärme-poliert für größere Öffnung </td></tr><tr><td> 35 mm Polysterine Kulturschale </td><td> Corning </td><td> 430588 </td><td> Wird für das Sammeln Scheiben nach der Sektion </td></tr></tbody></table> Tabelle 2. Werkzeuge und Materialien für die Präparation <table border="1"><tbody><tr><td> Ausstattung / Werkstoffe </td><td> Firma </td><td> Kommentare (optional) </td></tr><tr><td> Aufnahmekammer </td><td> Custom Built </td><td></td></tr><tr><td> P-97 Horizontal Pipette Puller </td><td> Sutter Instrument Co. </td><td></td></tr><tr><td> Schwingungsisolierung Tabelle </td><td> Technische Manufacturing Corporation (TMC) </td><td></td></tr><tr><td> Faraday-Käfig </td><td> Maßarbeit </td><td></td></tr><tr><td> Pyrex Aspirator Flasche mit Bottom Sidearm (Product # 1220-1L) </td><td> Corning </td><td></td></tr><tr><td> Gravity-kontrollierter IV Set mit Regler (Product # 2C8891) </td><td> Baxter </td><td></td></tr><tr><td> Zentrale Vakuum-Leitung </td><td></td><td> Erhältlich in den meisten modernen Labors </td></tr><tr><td> 95% O 2 / 5% CO 2 Gas Mix </td><td> Schottet-Gross Co. </td><td></td></tr><tr><td> TygonTM Lab Schlauch Für O 2 / CO 2 Lieferung </td><td> Fisher Scientific </td><td> Nicht giftig, nicht oxidierenden, kommt in einer Vielzahl von Größen. </td></tr><tr><td> Eclipse-E600FN Mikroskop </td><td> Nikon </td><td> mit 10x und 40x-Objektive, in der Nähe infared Filter und GFP, DS-Red2 Filter </td></tr><tr><td> Coole Snap-ES Digital Camera </td><td> Photometrics </td><td> Coole Snap-ES Digital Camera </td></tr><tr><td> X-Cite Fluorescent Illuminator </td><td> EXFO </td><td> X-Cite Fluorescent Illuminator </td></tr><tr><td> Mikroskop-Plattform </td><td> Siskiyou </td><td> Benutzerdefinierte montiert </td></tr><tr><td> RC-22 Submersible Aufnahme Kammer (Product # 64-0228) </td><td> Warner Instruments (WI) </td><td> Benötigt P-1-Plattform und Bühne Adapter (Product # 64-0277 Von Warner) </td></tr><tr><td> TC2BIP 2/3Ch Temperaturregler </td><td> Zell Microcontrols </td><td> TC2BIP 2/3Ch Temperaturregler </td></tr><tr><td> 4-Achsen-Handbuch Miniature Manipulator </td><td> Siskiyou </td><td></td></tr><tr><td> Platinum Iridium-Draht (0,002 in) (Teil # PTT0203) </td><td> WPI </td><td></td></tr><tr><td> A365 Stimulus Isolator </td><td> WPI </td><td> A365 Stimulus Isolator </td></tr><tr><td> Multiclamp 700b Verstärker </td><td> Axon Instruments </td><td></td></tr><tr><td> Digidata 1322a A / D-Wandler </td><td> Axon Instruments </td><td></td></tr><tr><td> PClamp Software </td><td> Axon Instruments </td><td></td></tr><tr><td> Personal Computer (Pentium 4) </td><td> Dell </td><td></td></tr></tbody></table> Tabelle 3. Elektrophysiologie Ausrüstung und Materialien

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

Das Nagetier Hippocampus Scheibe Vorbereitung ist vielleicht die am weitesten verwendete Werkzeug zur Untersuchung von Säugetieren synaptische Funktion und Plastizität. Der Hippocampus kann schnell und einfach von Ratten und Mäusen gewonnen werden und Scheiben bleiben für Stunden in Sauerstoff künstlichen Liquor lebensfähig. Darüber hinaus werden grundlegende Techniken electrophysisologic leicht auf die Untersuchung der synaptischen Funktion im Hippocampus Scheiben aufgebracht und haben lieferte einige der besten Biomarker für kognitive Beeinträchtigungen. Der Hippocampus Scheibe ist besonders beliebt für das Studium der synaptischen Plastizität Mechanismen für Lernen und Gedächtnis beteiligt. Änderungen in der Induktion der Langzeit-Potenzierung und Depression (LTP und LTD) der synaptischen Wirksamkeit in Schnitten des Hippocampus (oder deren Fehlen) werden häufig verwendet, um den neurologischen Phänotyp der kognitiv-beeinträchtigten Tieren und / oder beschreiben die Wirkungsweise von bewerten nootropische Verbindungen. Dieser Artikel beschreibt die Verfahren verwenden wir für die Vorbereitung Schnitten des Hippocampus von Ratten und transgenen Mäusen für die Untersuchung der synaptischen Veränderungen mit Gehirn Altern und Alzheimer-Krankheit (AD) 1-3 verbunden. Verwenden von gealterten Ratten und AD-Modell Mäuse bieten eine einzigartige Reihe von Herausforderungen für Forscher daran gewöhnt, mit jüngeren Ratten und / oder Mäuse in ihrer Forschung. Aged Ratten haben dickere Schädel und härter Bindegewebe als jüngere Ratten und Mäusen, die Hirn-Extraktion und / oder Dissektion Verzögerung und damit zu negieren oder zu übertreiben wirkliche Alter-Unterschiede in der synaptischen Funktion und Plastizität können. Altern und Amyloid-Pathologie kann auch verschärfen Hippocampus Schaden während der Dissektion Verfahren nachhaltig, wieder verkompliziert keine Rückschlüsse aus physiologischen Beurteilung erstellt. Hier diskutieren wir die Schritte während der Dissektion Verfahren, um diese Probleme zu minimieren. Beispiele der synaptischen Antworten in &quot;gesund&quot; und &quot;ungesund&quot; Scheiben von Ratten und Mäusen gewonnen vorgesehen sind, sowie Vertreter der synaptischen Plastizität Experimente. Die möglichen Auswirkungen der anderen methodologischen Faktoren auf die synaptische Funktion in diesen Tiermodellen (zB Aufnahme-Lösungskomponenten, Stimulationsparameter) werden ebenfalls diskutiert. Während der Schwerpunkt dieses Artikels basiert auf der Verwendung von alten Ratten und transgenen Mäusen, Novizen auf die Physiologie Scheibe finden sollten detailliert genug hier, um auf ihre eigenen Studien zu beginnen, mit einer Vielzahl von Tiermodellen.

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

Dieser Artikel beschreibt Verfahren zur Herstellung von Schnitten des Hippocampus von Ratten und transgenen Mäusen für die Untersuchung der synaptischen Veränderungen mit Gehirn Altern und altersbedingte neurodegenerative Erkrankungen wie Alzheimer-Krankheit.

Vorbereitung der akuten Schnitten des Hippocampus von Ratten und transgenen Mäusen für das Studium der Synaptic Veränderungen während des Alterns und Amyloid-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

93.8K Aufrufe.  University of Kentucky College of Public Health. Dieser Artikel beschreibt Verfahren zur Herstellung von Schnitten des Hippocampus von Ratten und transgenen Mäusen für die Untersuchung der synaptischen Veränderungen mit Gehirn Altern und altersbedingte neurodegenerative Erkrankungen wie Alzheimer-Krankheit.

Serie: 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.

Herstellung von oligomeren β-Amyloid 1-42 Und Induktion der synaptischen Plastizität Impairment auf Schnitten des Hippocampus

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 von Neuronen aus Nager Hirnschnitten zu Study Synaptic Transmission and Image präsynaptischen

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.

Vorbereitung der akuten Subventrikulärzone Slices für 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 ...

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.

Dual-Elektrophysiologische Recordings synaptisch-evozierte astrogliale und neuronale Antworten in Acute Hippocampusschnitte

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.

Stereotaktische Infusion von oligomeren Amyloid-beta in die Maus-Hippocampus

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.

Untersuchung von Synaptic Tagging / Erfassung und Lang Capture mit akuten Schnitten des Hippocampus von Nagetiere

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.

Vorbereitung der akuten Gehirnscheiben mit einem optimierten N-Methyl-D-Glucamine-schützende Recovery-Methode

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.

Aufnahme synaptische Plastizität in akuten Hippocampal Scheiben gepflegt in einem kleinvolumigen Recycling - Perfusions- und untertauchen-Typ-Kammer-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 ...

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.

Einzelne Synapsenindikatoren für Glutamatfreisetzung und Aufnahme in akuten Gehirnscheiben von normalen und Huntington-Mäusen

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.

Vorbereitung von akuten menschlichen Hippocampusscheiben für elektrophysiologische Aufnahmen

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 ...