Lavoro sostenuto da NIH concedere AG027297, un premio dal Kentucky Spinal Cord Injury e fiducia capo della ricerca, e un dono da parte della Fondazione Kleberg.

<ol>
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La procedura descritta in questo protocollo aiuterà ad assicurare che dissezioni del cervello sono effettuati almeno più rapidamente ed efficacemente in età, come nei ratti adulti giovani. Inoltre fornisce dettagli sufficienti per il principiante di stabilire la loro fetta propri studi su LTP e LTD. Se ulteriori esplorazioni di invecchiamento e le modifiche dC in funzione sinaptica e la plasticità è uno dei vostri obiettivi, ci sono almeno due altre questioni metodologiche, accennato sopra, che meritano ulteriore considerazione. In primo luogo, molti laboratori hanno dimostrato che il Ca 2 +: Mg2 + in rapporto registrazione ACSF può avere un effetto marcato sulla plasticità sinaptica induzione in fettine di ippocampo 2,10,24,25. Nel liquor dei mammiferi, il Ca 2 +: Mg2 + rapporto è di circa un (vedi ad esempio 26). Tuttavia, ACSF Ca 2 +: Mg2 + rapporto più vicino al 2 sono comunemente utilizzati negli studi fetta di funzione sinaptica e della plasticità. Nei primi studi, questa pratica era probabilmente adattato per ottimizzare l&#39;induzione di LTP, poi successivamente è diventato di routine per tutti gli studi plasticità. Tuttavia, questa pratica può essere problematico in studi di invecchiamento e dC a causa delle differenze ben caratterizzato in neuronale Ca 2 + regolamento. In particolare, Ca 2 + afflusso e / o Ca 2 + indotto da Ca 2 +-release è elevato in ratti anziani e / o topi modello dC durante l&#39;attivazione neuronale 3,27-31. Induzione di LTD è particolarmente sensibile ai cambiamenti sottili ACSF Ca 2 + livelli. Il nostro protocollo, che utilizza 2mM Ca 2 + e 2 mM Mg2 +, comunemente risultati LTD per anziani, ma non animali giovani adulti 2, mentre gli studi con un Ca 2 +: Mg2 rapporto + vicino a due, hanno osservato LTD robusto negli adulti in assenza di una differenza di età 2,10 o in combinazione con LTD ridotto nei ratti di 32 anni. Queste osservazioni evidenziano la necessità di considerare attentamente ACSF Ca 2 + e Mg2 + quando si confrontano livelli di Ca 2 +-dipendente plasticità in animali adulti anziani e giovani. Il secondo problema metodologico riguarda la forte dipendenza della LTP in depolarizzazione postsinaptica 33 e possibile l&#39;invecchiamento / genotipo differenze nella forza sinaptica. In un esperimento tipico LTP, al basale e l&#39;intensità di stimolazione LTP è generalmente regolata per produrre una mezza massimo (o di tre quarti massima) EPSP ampiezza. Il problema potenziale è che ratti anziani e APP/PS1 topi di solito mostrano una ridotta forza sinaptica rispetto alle loro controparti più giovani tipo e / o selvatici, il che significa che i valori basali EPSP sarà anche più piccolo in ratti anziani e APP/PS1 topi. Piccoli EPSPS può tradurre a meno depolarizzazione durante la stimolazione LTP, con un conseguente probabilità ridotta per indurre LTP 33. A causa di questo potenziale confondere, è difficile stabilire se questi animali presentano un deficit di produttività, un deficit di plasticità o entrambi. Cioè, i meccanismi di induzione LTP in età e / o APP/PS1 topi possono essere funzionalmente intatta (nessun deficit di plasticità), ma non sufficientemente stimolato (deficit di throughput) in queste condizioni. Questa distinzione è fondamentale, come i meccanismi di velocità e meccanismi di plasticità può rispondere in modo molto diverso a un determinato trattamento farmacologico. Cerchiamo di minimizzare l&#39;impatto di throughput ridotto sull&#39;induzione LTP normalizzando l&#39;ampiezza EPSP allo stesso livello (ad esempio 1 mV) in tutte le sezioni prima di stimolazione LTP. Altre strategie possono essere efficaci anche (ad esempio l&#39;uso di tensione o corrente pinza per equalizzare il potenziale di membrana tra i gruppi durante la stimolazione LTP), e deve essere considerata nell&#39;ambito delle indagini sulla LTP in questi modelli animali.

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> NaCl </td><td> Pescatore </td><td> BP358-1 </td></tr><tr><td> KCl </td><td> Pescatore </td><td> BP366-500 </td></tr><tr><td> KH 2 PO 4 (monobasico) </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 (diidrato) </td><td> Sigma </td><td> C3306-250G </td></tr><tr><td> NaHCO 3 </td><td> Pescatore </td><td> S233-500 </td></tr><tr><td> C 6 H 12 O 6 (destrosio) </td><td> Pescatore </td><td> BP350-1 </td></tr></tbody></table> Reagenti Tabella 1. Necessaria <table border="1"><tbody><tr><td> Nome del materiale </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Beute </td><td> Pescatore </td><td> FB-500-2000 FB-500-1000 </td><td></td></tr><tr><td> Acquario Bubbler </td><td></td><td></td><td> Utilizzati per i media ossigenante. Disponibile nella maggior parte negozi di animali </td></tr><tr><td> 50 ml di vetro bicchiere </td><td> Pescatore </td><td> 02-540g </td><td> Per storarge cervello in ACSF </td></tr><tr><td> Parafilm </td><td> Pescatore </td><td> 13-374-10 </td><td></td></tr><tr><td> Animali piccola ghigliottina </td><td> Mondo Strumenti di precisione (WPI) </td><td> DCAP-M </td><td></td></tr><tr><td> Tovagliolo di carta piatto </td><td></td><td></td><td></td></tr><tr><td> # 11 Feather lama chirurgica </td><td> Pescatore </td><td> 08-916-5B </td><td></td></tr><tr><td> Beebee osso forbici </td><td> Strumenti Scienza Fine (FST) </td><td> 16044-10 </td><td></td></tr><tr><td> Lempert Rongeurs </td><td> Roboz </td><td> RS-8321 </td><td> Utilizzare per i ratti </td></tr><tr><td> Friedman-Pearson Rongeurs </td><td> FST </td><td> 16020-14 </td><td> Da utilizzare per topi o ratti </td></tr><tr><td> Hippocampus strumento </td><td> FST </td><td> 10099-15 </td><td></td></tr><tr><td> Cucchiaio </td><td></td><td></td><td> Un cucchiaino di plastica farà </td></tr><tr><td> Spatola </td><td> Pescatore </td><td> 21-401-25A </td><td> Spatola </td></tr><tr><td> Iris forbici chirurgiche </td><td> FST </td><td> 14058-09 </td><td></td></tr><tr><td> pipette di trasferimento di plastica </td><td> Pescatore </td><td> 13-711-43 </td><td></td></tr><tr><td> 110 millimetri filtro di carta Whatman </td><td> Pescatore </td><td> 09-805E </td><td> Cat Whatman. 1001-110 </td></tr><tr><td> Vetro scatola di Petri </td><td> Pescatore </td><td></td><td></td></tr><tr><td> Leica VT1000P manuale vibrante microtomo </td><td> Vibratome </td><td></td><td></td></tr><tr><td> 0,1 millimetri FA-10 Feather S lama </td><td> Ted Pella </td><td> 121-9 </td><td> 0,1 millimetri FA-10 Feather S lama </td></tr><tr><td> Pipettare Pasteur in vetro borosilicato (con gommino) </td><td> Pescatore </td><td> 13-678-20A </td><td> Per il trasferimento di fette: Tip è rotto e il calore-lucido per le più grandi di apertura </td></tr><tr><td> Piatto 35 millimetri Cultura Polysterine </td><td> Corning </td><td> 430588 </td><td> Utilizzati per la raccolta fette dopo la dissezione </td></tr></tbody></table> Strumenti tabella 2. E materiali per la dissezione <table border="1"><tbody><tr><td> Attrezzature / Materiali </td><td> Azienda </td><td> Commenti (opzionale) </td></tr><tr><td> Tenendo camera </td><td> Custom Built </td><td></td></tr><tr><td> P-97 orizzontale pipette Puller </td><td> Sutter Instrument Co. </td><td></td></tr><tr><td> Isolamento dalle vibrazioni tavolo </td><td> Tecnico Manufacturing Corporation (TMC) </td><td></td></tr><tr><td> Gabbia di Faraday </td><td> Su misura </td><td></td></tr><tr><td> Pirex Bottiglia Aspiratore con Sidearm inferiore (Product # 1220-1L) </td><td> Corning </td><td></td></tr><tr><td> Gravità contolled serie IV con regolatore (Product # 2C8891) </td><td> Baxter </td><td></td></tr><tr><td> Vuoto Central Line </td><td></td><td> Disponibile nella maggior parte dei laboratori moderni </td></tr><tr><td> 95% O 2 / 5% di CO 2 Mix Gas </td><td> Scozzeset-Gross Co. </td><td></td></tr><tr><td> TygonTM Lab tubi Per O 2 / CO 2 di consegna </td><td> Fisher Scientific </td><td> Non tossico, non ossidanti, è disponibile in una varietà di formati. </td></tr><tr><td> Eclipse E600FN Microscopio </td><td> Nikon </td><td> con obiettivi 10x e 40x, vicino infared filtro, e GFP, DS-Rosso2 filtri </td></tr><tr><td> Raffreddare ES Snap Digital Camera </td><td> Fotometrici </td><td> Raffreddare ES Snap Digital Camera </td></tr><tr><td> X-Cite Illuminatore fluorescente </td><td> EXFO </td><td> X-Cite Illuminatore fluorescente </td></tr><tr><td> Microscopio Piattaforma </td><td> Siskiyou </td><td> Assemblati su misura </td></tr><tr><td> RC-22 sommergibili camera di registrazione (Product # 64-0228) </td><td> Warner Instruments (WI) </td><td> Richiede P-1 adattatore piattaforma e lo stadio (Product # 64-0277 da Warner) </td></tr><tr><td> TC2BIP 2/3Ch Regolatore di temperatura </td><td> Cellula Microcontrols </td><td> TC2BIP 2/3Ch Regolatore di temperatura </td></tr><tr><td> 4 assi manuale manipolatore in miniatura </td><td> Siskiyou </td><td></td></tr><tr><td> Filo di platino iridio (0,002 in) (Articolo # PTT0203) </td><td> WPI </td><td></td></tr><tr><td> A365 Stimolo isolatore </td><td> WPI </td><td> A365 Stimolo isolatore </td></tr><tr><td> Multiclamp Amplificatore 700B </td><td> Axon Instruments </td><td></td></tr><tr><td> Digidata 1322A convertitore A / D </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> Conca </td><td></td></tr></tbody></table> Tabella 3. Elettrofisiologia attrezzature e materiali

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

L&#39;ippocampo preparazione fetta roditore è forse lo strumento più largamente utilizzato per lo studio dei mammiferi funzione sinaptica e la plasticità. L&#39;ippocampo può essere estratto rapidamente e facilmente da ratti e topi e fette rimangono vitali per ore in ossigenato artificiale liquido cerebrospinale. Inoltre, le tecniche di base electrophysisologic sono facilmente applicati allo studio di funzione sinaptica in fettine di ippocampo e hanno fornito alcuni dei migliori marcatori di disturbi cognitivi. La fetta ippocampo è particolarmente popolare per lo studio dei meccanismi di plasticità sinaptica coinvolti nell&#39;apprendimento e nella memoria. Cambiamenti nella induzione di potenziamento a lungo termine e la depressione (LTP e LTD) di efficacia sinaptica in fettine di ippocampo (o la loro mancanza) sono spesso usate per descrivere il fenotipo neurologico di animali con problemi cognitivo e / o per valutare il meccanismo d&#39;azione di composti nootropica. Questo articolo descrive le procedure che utilizziamo per la preparazione di fettine di ippocampo di ratti e topi transgenici per lo studio delle alterazioni sinaptiche del cervello associata con l&#39;invecchiamento e la malattia di Alzheimer (AD) 1-3. Uso dei ratti anziani e topi modello AD può presentare una serie di sfide per i ricercatori abituati ad usare i ratti più giovani e / o topi nelle loro ricerche. Ratti anziani hanno spesso teschi e più dura del tessuto connettivo più giovani ratti e topi, che può ritardare l&#39;estrazione del cervello e / o di dissezione e di conseguenza negare o esagerare reali differenze età-correlate in funzione sinaptica e la plasticità. Invecchiamento e patologia amiloide può anche aggravare i danni subiti dell&#39;ippocampo durante la procedura di dissezione, di nuovo complicando ogni deduzioni tratte dalla valutazione fisiologica. Qui, discutiamo i passi compiuti durante la procedura di dissezione per minimizzare questi problemi. Esempi di risposte sinaptiche acquisite in &quot;buona salute&quot; e &quot;malsana&quot; fette di ratti e topi vengono forniti, oltre che rappresentante esperimenti di plasticità sinaptica. L&#39;eventuale impatto di altri fattori metodologici sulla funzione sinaptica in questi modelli animali (ad esempio i componenti della soluzione di registrazione, i parametri di stimolazione) vengono anche discussi. Mentre l&#39;attenzione di questo articolo è l&#39;utilizzo di ratti anziani e topi transgenici, i novizi di tagliare fisiologia dovrebbe trovare abbastanza dettagli qui per iniziare i propri studi, utilizzando una varietà di modelli di roditori.

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

Questo articolo descrive le procedure per la preparazione di fettine di ippocampo di ratti e topi transgenici per lo studio delle alterazioni sinaptiche associate con l&#39;invecchiamento cerebrale e le malattie neurodegenerative legate all&#39;età, come la malattia di Alzheimer.

Preparazione di acuta fettine di ippocampo di ratti e topi transgenici per lo studio delle alterazioni sinaptiche durante l&#39;invecchiamento e Amiloide Patologia

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 Views.  University of Kentucky College of Public Health. Questo articolo descrive le procedure per la preparazione di fettine di ippocampo di ratti e topi transgenici per lo studio delle alterazioni sinaptiche associate con l'invecchiamento cerebrale e le malattie neurodegenerative legate all'età, come la malattia di Alzheimer.

Video: 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.

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). ...

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Neuroscienze

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 di neuroni da fettine di cervello di roditore studio di trasmissione sinaptica e Immagine terminali presinaptici

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.

Preparazione di acuta fette zona subventricolare for Imaging di calcio

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.

Due registrazioni elettrofisiologiche di sinapticamente-evocati Risposte astrogliale e neuronale in acute fettine di ippocampo

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.

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 ...

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.

Indagine di Synaptic Tagging / Cattura e Cross-cattura utilizzando acuta fettine di ippocampo da Roditore

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.

Preparazione di fettine di cervello acuto utilizzando un' ottimizzata N-metilico-D-metodo di ripristino protettivo glucamine

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.

Registrazione di plasticità sinaptica in fettine ippocampali Acute mantenuti in un piccolo volume riciclaggio - aspersione- e sistema di immersione-tipo camera

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.

Indicatori di sinapsi singola di Soldamento Release e Uptake in Acute Brain Slices da Topi Normali e Huntington

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.

Preparazione di fette acute di ippocampale umano per registrazioni elettrofisiologiche

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 ...