Questo lavoro è stato finanziato dalla Neuro-Bio Ltd. Vorremmo ringraziare il Dr. Giovanni Ferrati e Dr. Sergio Rotondo (Neuro-Bio) per i loro commenti e consigli sul manoscritto.

Tutti gli studi sugli animali sono stati effettuati nell'ambito di protocolli approvati.
Nota: In questa sezione, la sequenza delle fasi principali eseguite durante la procedura sperimentale e l'intervallo di tempo consigliato è fornito (Figura 1). Inoltre, una descrizione dettagliata del protocollo è completata da un pannello illustrativo, mostrando azioni critiche che vanno dalla rimozione del cervello per omogeneizzazione del tessuto dopo il periodo di incubazione (Figura 2). I dettagli per quanto riguarda i materiali e le istruzioni per costruire l'apparato e la successiva fase per l'analisi di WB sono descritti in precedenza22.
1. chirurgici strumenti, impostazioni Vibratome e preparazione di trattamento
Nota: Eseguire le seguenti operazioni prima di avviare la procedura sperimentale:
<ol><li>Preparare due diversi artificiali liquidi cerebrospinali (aCSFs), uno usato per il taglio del cervello e l'altro per la fase di incubazione come precedentemente descritto29. Brevemente, le concentrazioni di lavoro (in mmol) dei due aCSFs sono per la aCSF "slicing": 120 NaCl, 5 KCl, 20 NaHCO3, 2,4 CaCl2, 2 MgSO4, 1,2 KH2PO4e 10 glucosio; 6.7 HEPES sale e 3,3 HEPES acido; pH: 7.1. Mentre per la "registrazione" aCSF: 124 NaCl, 3,7 KCl, 26 NaHCO3, 2 CaCl2, 1.3 MgSO4, 1,3 KH2PO4e 10 glucosio; pH: 7.1.</li>
	<li>Mescolare bene le soluzioni e poi mettere il ghiaccio e ossigenare il aCSF affettare al fine di avere freddo durante la rimozione del cervello e il sezionamento successive. Tenere a temperatura ambiente (TA) e fornitura di ossigeno per la registrazione aCSF.</li>
	<li>Posizionare gli strumenti sotto il cofano di dissezione per la rimozione di decapitazione e cervello, vale a dire, ghigliottina, kit di dissezione chirurgica (forbici, pinze, lame) e spatola.</li>
	<li>Consente di impostare i parametri del vibratome selezionando lo spessore di taglio (300 µm in questa preparazione) e la frequenza alle 7 e la velocità a 6. Inserire la camera vibratome in suo spazio adeguato e circondano con ghiaccio.</li>
	<li>Aprire la valvola di carbogen (95% O2, 5% CO2) per ossigenare il "taglio" (aCSF) durante il sezionamento del cervello. Selezionare la portata minima, circa 2 mL/min.</li>
	<li>Prendere due tubi di 15 mL per preparare le condizioni per testare. Aggiungere in una provetta 10 mL della "registrazione" aCSF da solo (gruppo di controllo) e in altro, 10 mL della "registrazione" aCSF arricchita con T30 ad una concentrazione di 2 µM (gruppo trattato). Vortice sia tubi e tenerli sul ghiaccio fino al loro utilizzo nella fase di preparazione di installazione (sezione 3).</li>
</ol>2. anestesia, estrazione di cervello e affettare
Nota: In questo studio, 9 P14 tipo selvaggio ratti maschii di Wistar sono stati usati. Estrazione di cervello e affettare successive costituiscono una parte fondamentale del protocollo poiché deve essere eseguite rapidamente (entro 10 min) al fine di prevenire o almeno rallentare i processi di degenerazione del tessuto.
<ol><li>Aggiungere 1,5 mL 100% w/w di isoflurane su uno strato di cotone nella camera di induzione, metti l'animale all'interno e chiudere il coperchio. Attendere fino a quando l'animale è anestetizzato, confermando l'assenza del riflesso del pedale e quindi decapitare l'animale utilizzando la ghigliottina.</li>
	<li>Mantenere la testa dell'animale all'interno di una pentola contenente ghiacciata affettare aCSF durante la fase di rimozione del cervello (Figura 2A -C).</li>
	<li>Incidere la pelle sopra il cranio con una lama chirurgica, poi tagliare il cranio con le forbici seguendo la linea mediana, dalla parte posteriore e procedere anteriormente fino a raggiungere l'osso sopra gli OB (Figura 2A).</li>
	<li>Tirare lateralmente i due lati del cranio usando un paio di pinze per avere accesso al cervello (Figura 2B). Inserire una spatola ventralmente al cervello, delicatamente scoop fuori e tenere che idratata in aCSF ghiacciata "slicing" per circa 5 min (Figura 2).</li>
	<li>Smaltire il cervello su carta da filtro e tagliare fuori il cervelletto usando una lama (Figura 2D). Incollare il cervello verticalmente sul disco vibratome e inserirlo all'interno della camera di taglio, riempirlo con gelida aCSF "affettare" e fornire ossigeno (Figura 2E).</li>
	<li>Regolare l'intervallo di taglio e procedere con il cervello di sezionamento (Figura 2F). Raccogliere le sezioni che contengono la regione di interesse, come il setto mediale (MS), banda diagonale di Broca (DBB) e substantia innominata (SI), come descritto in precedenza22. Dall'alto verso il basso, le tre fette contengono rostrale (R), intermedio (I) e caudale (C) parte del BF (Figura 2).</li>
	<li>Dividere lungo la linea mediana ogni sezione con piccole forbici, ottenendo due speculari hemislices (Figura 2 H), utilizzati singolarmente come il controllo e la condizione trattata presso sullo stesso piano anatomico.</li>
	<li>Mantenere la hemisections nella camera vibratome per 5-10 minuti, quindi trasferirli con una pipetta di vetro in una pentola di bubbling con registrazione aCSF. Conservare a temperatura ambiente per circa 30 minuti per recuperare.</li>
	<li>Riempire, nel frattempo, tre fiale di vetro con 3 mL di registrazione aCSF da solo (precedentemente preparata al punto 1.6 per il gruppo di controllo) e tre fiale con 3 mL di aCSF arricchito con T30 (precedentemente preparata al punto 1.6) per il gruppo trattato.</li>
</ol>3. preparazione di setup
<ol><li>Trasferire la hemisections nei loro corrispondenti flaconi di vetro (Figura 2I) al fine di avere tre hemislices consecutivi (compresa la regione BF rostrale, intermedia e caudale) per il gruppo di controllo (cornice verde, Figura 2J) e loro controparti per il gruppo trattato (cornice rossa, Figura 2J) di corrispondenza.</li>
	<li>Trasferire il tessuto cerebrale con due pennelli diversi, uno per il hemislices di controllo e un altro per le controparti trattate, al fine di evitare qualsiasi contaminazione tra condizioni.</li>
	<li>Ogni flaconcino di vetro con il coperchio corrispondente, che presenta l'ago ossigenante (21G) (Figura 2J) della guarnizione. Collegare il tubo principale dell'apparato all'origine di carbogen (Figura 2 K). Fornire ossigeno ai hemislices per tutto il periodo di incubazione con una portata minima di 2 mL/min per evitare qualsiasi movimento del tessuto cerebrale e possibili danni meccanici durante l'esperimento. Iniziare l'incubazione di 5 h. Eseguire i passaggi 3.1-3.3 entro 5 min. Nota: Controllare frequentemente (ogni 15-20 minuti) se l'ossigeno è fornito in tutte le fiale e che il flusso non si muove il tessuto dal basso.</li>
</ol>4. campione omogeneizzazione
<ol><li>Interrompere il flusso di ossigeno dopo l'incubazione e rimuovere tutti i coperchi dai flaconcini. Trasferire la hemislices, utilizzando i pennelli corretta, in provette da 1,5 mL contenente 250 µ l di tampone di Lisi, mantenere i tubi sul ghiaccio (Figura 2 L). Per rendere il lysis buffer, mescolare PBS 1 x con un cocktail di inibitori di proteasi e fosfatasi sia diluito a 1: 100 e omogeneizzare il tessuto con diversi pestelli per prevenire la contaminazione tra i campioni. Completare queste azioni entro 10-15 min.</li>
	<li>Centrifugare il cervello lisato a 1.000 x g per 5 min a 4 ° C. Trasferire il surnatante in nuovi tubi e mantenere i campioni a-80 ° C.</li>
</ol>5. la lettura della concentrazione nella proteina e analisi Western Blot
<ol><li>Determinare la concentrazione di proteina di ogni campione e successivamente preparare le aliquote per l'analisi Western blot come descritto in precedenza22.</li>
	<li>Effettuare analisi statistiche per valutare gli effetti del trattamento sull'espressione della proteina.</li>
</ol>

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Gli autori dichiarano concorrenti interessi finanziari. Susan A. Greenfield è il fondatore e Presidente di Neuro-Bio Limited, un'azienda privatamente posseduta e detiene azioni della società. Emanuele Brai e Antonella Cogoni sono dipendenti di Neuro-Bio Ltd.

L'aspetto principale di questo protocollo, basato sulla tecnica del cervello ben consolidata ex vivo , permette di testare in modo sincrono due hemislices speculare, ottenuti sullo stesso piano anatomico, controllo della loro risposta dopo l'applicazione di uno specifico condizione (controllare o trattati); Questo offre quindi un paradigma sperimentale sotto stretto controllato quanto possibile. La possibilità di valutare in un tempo - dose- e site-specific modo diverse sostanze neurochimiche legate al deficit ...

Annuncio è che una patologia cronica caratterizzata da danno progressivo neurodegenerative che interessano aree differenti del cervello, come la corteccia di entorhinal (CE), BF, ippocampo (HC) e bulbo olfattivo (OB)1,2,3, 4,5. La fine delle fasi di sviluppo AD portare ad un declino conoscitivo progressivo, rendendo questa malattia la più comune forma di demenza, pari circa al 70% di tutti i casi6. Malgrado vasto tentativo di comprendere le fasi iniziali che causano AD, non esiste attualmente una definita indicazione sperimentale delucidamento li. Inoltre, la teoria più popolare - del "ipotesi amiloide" - è sempre più messa in discussione dal momento che non fornisce un profilo completo nello spiegare pathobiology annuncio, né una destinazione farmaceutica che si è rivelata efficace7,8 ,9.
Una teoria alternativa che sta ricevendo crescente attenzione suggerisce che i meccanismi iniziali che si verificano durante la neurodegenerazione sono correlati a un cluster di un neurone principalmente suscettibile AD3,10,11 , 12 , 13 , 14. questo hub cellulare eterogeneo rientra all'interno i progetti di BF, mesencefalo e del tronco cerebrale, più aree, quali la CE, HC e OB15,16. Nonostante la sua diversità nella morfologia neuronale e sintesi del neurotrasmettitore, questo nucleo di cellule condivide una caratteristica comune nell'esprimere il dolore, che può anche avere una funzione non-enzimatica17,18. Questo ruolo non-classica come un romanzo di segnalazione molecola media il flusso di calcio (Ca2 +) in neuroni che possono subire gli eventi trofici o tossici in relazione di Ca2 + dose, disponibilità e un neurone età17,18 , 19.
Durante la neurodegenerazione, la perdita cellulare osservata potrebbe essere quindi associata a questa funzione non enzimatici17,18,20, che è attribuibile ad un peptide 30mer (T30) spaccato da mal di C-terminale 20. in linea con i risultati precedenti, riportati su coltura cellulare e optical imaging preparazioni di18,21 , abbiamo dimostrato, attraverso un approccio innovativo basato su ex vivo il fettine di cervello di ratto contenente strutture BF, che ha indotto T30 un annuncio-come il profilo22. In particolare, questa nuova metodologia offre uno scenario più fisiologico di coltura cellulare, poiché mantiene molte delle caratteristiche di un tessuto intatto, che vanno da anatomica alla conservazione di circuiti, seppur per una finestra di tempo di ore. Abbiamo applicato questo protocollo per esplorare gli eventi che si svolgono durante le fasi iniziali della neurodegenerazione, monitoraggio della risposta acuta all'atto dell'applicazione T30.
Nonostante il grande corpo di letteratura sull'uso di cervello fette di indagare i meccanismi molecolari implicati nel danno neuronale o neurogenesi23,24, questo protocollo fornisce per la prima volta un più immediato e sensibile leggere rispetto per l'uso comune di organotipiche fette. Tuttavia, come è il caso per organotipiche sezioni del cervello, questa procedura di fetta acuta può essere adottata per diversi scopi, come ad esempio la valutazione di neuroprotective o molecole neurotossiche, scoperta di primarie cambiamenti molecolari che si verificano in un processo specifico, patologie correlate: analisi immunohistochemical e dosaggi farmacologici per il sistema nervoso centrale.

<table>
	<tbody>
		<tr>
			<td>Sodium chloride (NaCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S7653</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium chloride (KCl)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P9333</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Sodium bicarbonate (NaHCO3)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>S5761</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Magnesium sulphate heptahydrate (MgSO4 (7H2O))</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>63138</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Potassium phosphate monobasic (KH2PO4)</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>P5655</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes salt</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H7006</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Hepes acid</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>H3375</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>G7528</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>Calcium chloride dehydrate</td>
			<td>Sigma-Aldrich, Germany</td>
			<td>223506</td>
			<td>Reagent for aCSF preparation</td>
		</tr>
		<tr>
			<td>T30 peptide</td>
			<td>Genosphere Biotechnologies, France</td>
			<td></td>
			<td>AChE-derived peptide tested</td>
		</tr>
		<tr>
			<td>Surgical dissecting kit</td>
			<td>World Precision Instruments, USA</td>
			<td>Item #: MOUSEKIT</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Surgical blades</td>
			<td>Swann-Morton, UK</td>
			<td>BS 2982</td>
			<td>Brain removal step</td>
		</tr>
		<tr>
			<td>Filter paper</td>
			<td>Fisher Scientific, USA</td>
			<td>11566873</td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Glue</td>
			<td></td>
			<td></td>
			<td>Brain preparation for slicing</td>
		</tr>
		<tr>
			<td>Vibratome</td>
			<td>Leica, Germany</td>
			<td>VT1000 S</td>
			<td>Slicing</td>
		</tr>
		<tr>
			<td>Brushes</td>
			<td></td>
			<td></td>
			<td>Tissue handling</td>
		</tr>
		<tr>
			<td>Oxygen canister</td>
			<td></td>
			<td></td>
			<td>Sectioning and incubation phase</td>
		</tr>
		<tr>
			<td>1x Phosphate buffer saline (PBS)</td>
			<td>Fisher Scientific, USA</td>
			<td>BP2438-4</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Phosphatase inhibitors</td>
			<td>Fisher Scientific, USA</td>
			<td>1284-1650</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Protease inhibitors</td>
			<td>Roche complete PIC, USA</td>
			<td>4693116001</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Pestles</td>
			<td>Starlab, UK</td>
			<td>I1415-5390</td>
			<td>Homogenization step</td>
		</tr>
		<tr>
			<td>Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce 660 nm Protein Assay</td>
			<td>Thermo Scientific, USA</td>
			<td>22660</td>
			<td>Protein concentration</td>
		</tr>
	</tbody>
</table>

an alternative approach to study primary events in neurodegeneration using ex vivo rat brain slices

Nonostante i numerosi studi che tentano di sviluppare modelli animali affidabile che riflettendo il primario i processi di neurodegenerazione sottostante, molto pochi sono stati ampiamente accettati. Qui, vi proponiamo una nuova procedura adattata dal ben noto ex vivo fetta tecnica del cervello, che offre una più stretta in vivo-come scenario di in vitro i preparativi, per indagare gli eventi precoci innescando la degenerazione delle cellule, come osservato nel morbo di Alzheimer (annuncio). Questa variante è costituita da passi semplici e facilmente riproducibili, che consentono la conservazione della citoarchitettura anatomica della regione del cervello selezionato e la sua funzionalità locali in un ambiente fisiologico. Diverse aree anatomiche possono essere ottenuti dal cervello stesso, offrendo la possibilità di eseguire esperimenti multipli con i trattamenti in questione in un sito - dose- e tempo-dipendente. Limitazioni potenziali che potrebbero influenzare i risultati relazionati a questa metodologia sono legate alla conservazione del tessuto, vale a dire, il mantenimento della sua integrità anatomica durante le operazioni di affettamento e incubazione e lo spessore di sezione, che possono influenzare l'analisi biochimica e immunohistochemical. Questo metodo può essere impiegato per scopi diversi, come esplorare i meccanismi molecolari coinvolti in condizioni fisiologiche o patologiche, screening di farmaci o dosaggi di dose-risposta. Infine, questo protocollo potrebbe anche ridurre il numero di animali impiegati in studi comportamentali. L'applicazione segnalata qui recentemente descritto e testato per la prima volta su ex vivo il fettine di cervello di ratto contenenti proencefalo basale (BF), che è una delle regioni cerebrali colpite principalmente in annuncio. In particolare, è stato dimostrato che la somministrazione di un peptide tossico derivato dal C-terminale dell'acetilcolinesterasi (AChE) potrebbe richiedere un profilo AD-come, innescando, lungo l'asse antero-posteriore del BF, un'espressione differenziale di proteine alterate in annuncio, ad esempio il recettore nicotinico alfa7 (α7-nAChR), fosforilato Tau (p-Tau) e beta amiloide (Aβ).

Il protocollo presentato qui indica che la somministrazione di un peptide tossico, T30, modula in modo sito-dipendente l'espressione di α7-nAChR, p-Tau e Aβ in sezioni contenenti BF (Figura 3A). Il recettore nicotinico Mostra un aumento significativo nella hemislice rostrale-trattati rispetto alla sua controparte di controllo (fetta 1, p = 0.0310) (Figura 3B), mentre la fetta intermedia non rivela alcun cambiamento tra...

Watch this Scientific Journal Video about An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices at JoVE.com

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Presentiamo un metodo che può fornire ulteriori approfondimenti i primi eventi sottostante neurodegenerazione e basato sulla tecnica del cervello stabilito ex vivo , unendo i vantaggi degli esperimenti in vivo e in vitro . Inoltre, rappresenta un'opportunità unica per un confronto diretto del gruppo trattata e non trattata sullo stesso piano anatomico.

Un approccio alternativo per studiare eventi primari nella neurodegenerazione utilizzando fettine di cervello di ratto Ex Vivo

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, ...

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Research

JoVE Journal

Neuroscience

An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

6.8K Views.  Culham Science Centre. Presentiamo un metodo che può fornire ulteriori approfondimenti i primi eventi sottostante neurodegenerazione e basato sulla tecnica del cervello stabilito ex vivo , unendo i vantaggi degli esperimenti in vivo e in vitro . Inoltre, rappresenta un'opportunità unica per un confronto diretto del gruppo trattata e non trattata sullo stesso piano anatomico.

Video: An Alternative Approach to Study Primary Events in Neurodegeneration Using Ex Vivo Rat Brain Slices

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This method maintains the characteristic topographical morphology of the hippocampus while allowing direct application of pharmacological agents to the developing hippocampal dentate gyrus. Additionally, slice cultures can be maintained for up to 4 weeks and thus, allow one to study the maturation process of newborn granule neurons. Slice cultures allow for efficient pharmacological manipulation of hippocampal slices while excluding complex variables such as uncertainties related to the deep anatomic location of the hippocampus as well as the blood brain barrier. For these reasons, we sought to optimize organotypic slice cultures specifically for postnatal neurogenesis research.

Here we describe a technique for studying hippocampal postnatal neurogenesis using the organotypic slice culture technique. This method allows for in vitro manipulation of adult neurogenesis and allows for the direct application of pharmacological agents to the cultured hippocampus.

Colture organotipiche Slice di studi di post-natale neurogenesi

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

Ricerca

Biologia dello sviluppo

Organotypic Slice Culture of E18 Rat Brains

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, organotypic slice cultures allow one to perform a number of manipulations that are not presently feasible in-vivo. To date, organtotypic embryonic brain slice cultures have been used to follow individual cells using time-lapse microscopy, manipulate the expression of genes in the ganglionic emanances (a region that is hard to target by in-utero electroporation), as well as for pharmacological studies. In this video protocol we demonstrate how to make organotypic slice cultures from rat embryonic day 18 embryos. The protocol involves dissecting the embryos, embedding them on ice in low melt agarose, slicing the embedded brains on the vibratome, and finally plating the slices onto filters in culture dishes. This protocol is also applicable in its present form to making organotypic slice cultures from different embryonic ages for both rats and mice. 

Slice Cultura organotipica di E18 cervello dei topi

Organotypic slice cultures from embryonic rodent brains are widely used to study brain development. While there are often advantages to an in-vivo system, ...

Biologia

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

The rodent hippocampal slice preparation is perhaps the most broadly used tool for investigating mammalian synaptic function and plasticity. The hippocampus can be extracted quickly and easily from rats and mice and slices remain viable for hours in oxygenated artificial cerebrospinal fluid. Moreover, basic electrophysisologic techniques are easily applied to the investigation of synaptic function in hippocampal slices and have provided some of the best biomarkers for cognitive impairments. The hippocampal slice is especially popular for the study of synaptic plasticity mechanisms involved in learning and memory. Changes in the induction of long-term potentiation and depression (LTP and LTD) of synaptic efficacy in hippocampal slices (or lack thereof) are frequently used to describe the neurologic phenotype of cognitively-impaired animals and/or to evaluate the mechanism of action of nootropic compounds. This article outlines the procedures we use for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and Alzheimer's disease (AD)1-3. Use of aged rats and AD model mice can present a unique set of challenges to researchers accustomed to using younger rats and/or mice in their research. Aged rats have thicker skulls and tougher connective tissue than younger rats and mice, which can delay brain extraction and/or dissection and consequently negate or exaggerate real age-differences in synaptic function and plasticity. Aging and amyloid pathology may also exacerbate hippocampal damage sustained during the dissection procedure, again complicating any inferences drawn from physiologic assessment. Here, we discuss the steps taken during the dissection procedure to minimize these problems. Examples of synaptic responses acquired in "healthy" and "unhealthy" slices from rats and mice are provided, as well as representative synaptic plasticity experiments. The possible impact of other methodological factors on synaptic function in these animal models (e.g. recording solution components, stimulation parameters) are also discussed. While the focus of this article is on the use of aged rats and transgenic mice, novices to slice physiology should find enough detail here to get started on their own studies, using a variety of rodent models.

This article outlines procedures for preparing hippocampal slices from rats and transgenic mice for the study of synaptic alterations associated with brain aging and age-related neurodegenerative diseases, such as Alzheimer&#x2019;s disease.

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 ...

Neuroscienze

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As such, primary or &#39;neuronal-like&#39; cell culture systems are commonly utilized. While&#160;these systems are relatively easy to work with, and are useful model systems in which various functional outcomes (such as cell death) can be readily quantified, the examined outcomes and pathways in cultured immature neurons (such as excitotoxicity-mediated cell death pathways) are not necessarily the same as those observed in mature brain, or in intact tissue. Therefore, there is the need to develop models in which cellular mechanisms in mature neural tissue can be examined. We have developed an in vitro technique that can be used to investigate a variety of molecular pathways in intact nervous tissue. The technique described herein utilizes rat cortical tissue, but this technique can be adapted to use tissue from a variety of species (such as mouse, rabbit, guinea pig, and chicken) or brain regions (for example, hippocampus, striatum, etc.). Additionally, a variety of stimulations/treatments can be used (for example, excitotoxic, administration of inhibitors, etc.). In conclusion, the brain slice model described herein can be used to examine a variety of molecular mechanisms involved in excitotoxicity-mediated brain injury.

Excitotoxic Stimulation of Brain Microslices as an In vitro Model of Stroke

We have developed a brain slice model which can be used to examine molecular mechanisms involved in excitotoxicity-mediated brain injury.&#160;This technique generates viable mature brain tissue and reduces animal numbers required for experimentation, whilst keeping the neuronal circuitry, cellular interactions, and postsynaptic compartments partly intact.

Eccitotossica stimolazione del cervello Microslices come<em&gt; In vitro</em&gt; Modello di Corsa

Examining molecular mechanisms involved in neuropathological conditions, such as ischemic stroke, can be difficult when using whole animal systems. As ...

Medicina

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion channel, and transporter cell surface presentation on a minutes time scale.&#160;There is a broad diversity of mechanisms that control endocytic trafficking of individual proteins. Studies investigating the molecular underpinnings of trafficking have primarily relied upon surface biotinylation to quantitatively measure changes in membrane protein surface expression in response to exogenous stimuli and gene manipulation.&#160;However, this approach has been mainly limited to cultured cells, which may not faithfully reflect the physiologically relevant mechanisms at play in adult neurons.&#160;Moreover, cultured cell approaches may underestimate region-specific differences in trafficking mechanisms.&#160;Here, we describe an approach that extends cell surface biotinylation to the acute brain slice preparation.&#160;We demonstrate that this method provides a high-fidelity approach to measure rapid changes in membrane protein surface levels in adult neurons.&#160;This approach is likely to have broad utility in the field of neuronal endocytic trafficking.

Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons

Neuronal membrane trafficking dynamically controls plasma membrane protein availability and significantly impacts neurotransmission.&#160;To date, it has been challenging to measure neuronal endocytic trafficking in adult neurons.&#160;Here, we describe a highly effective, quantitative method to measure rapid changes in surface protein expression ex vivo in acute brain slices.

Slice Cervello biotinilazione: An<em&gt; Ex Vivo</em&gt; Approccio alla Misura Regione-specifica proteina Tratta plasma membrana in neuroni adulti

Regulated endocytic trafficking is the central mechanism facilitating a variety of neuromodulatory events, by dynamically controlling receptor, ion ...

The Analysis of Neurovascular Remodeling in Entorhino-hippocampal Organotypic Slice Cultures

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional deficits in the affected patients. Despite intensive research efforts, there is still no effective treatment option available that reduces neuronal injury and protects neurons in the ischemic areas from delayed secondary death. Research in this area typically involves the use of elaborate and problematic animal models. Entorhino-hippocampal organotypic slice cultures challenged with oxygen and glucose deprivation (OGD) are established in&#160;vitro models which mimic cerebral ischemia. The novel aspect of this study is that changes of the brain blood vessels are studied in addition to neuronal changes and the reaction of both the neuronal compartment and the vascular compartment can be compared and correlated. The methods presented in this protocol substantially broaden the potential applications of the organotypic slice culture approach. The induction of OGD or hypoxia alone can be applied by rather simple means in organotypic slice cultures and leads to reliable and reproducible damage in the neural tissue. This is in stark contrast to the complicated and problematic animal experiments inducing stroke and ischemia in vivo. By broadening the analysis to include the study of the reaction of the vasculature could provide new ways on how to preserve and restore brain functions. The slice culture approach presented here might develop into an attractive and important tool for the study of ischemic brain injury and might be useful for testing potential therapeutic measures aimed at neuroprotection.

A protocol for entorhino-hippocampal organotypic slice cultures, which allows reproducing many aspects of ischemic brain injury, is presented. By studying changes of the neurovasculature in addition to changes in the neurons, this protocol is a versatile tool to study plastic changes in neural tissue after injury.

L&#39;analisi di neurovascolare ritocca in Entorhino-ippocampali Culture Organotipica Slice

Ischemic brain injury is among the most common and devastating conditions compromising proper brain function and often leads to persisting functional ...

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 ...

Preparing Acute Brain Slices from the Dorsal Pole of the Hippocampus from Adult Rodents

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of cellular and synaptic properties. Nevertheless, there is an ever increasing need to perform correlated analyses between different experimental modes in addition to slice electrophysiology, for example: immunohistochemistry, molecular biology, in vivo imaging or electrophysiological recording; to answer evermore complex questions of brain function. However, making meaningful conclusions from these various experimental approaches is not straightforward, as even within relatively well described brain structures, a high degree of sub-regional variation of cellular function exists. Nowhere is this better exemplified than in the CA1 of the hippocampus, which has well-defined dorso-ventral properties, based on cellular and molecular properties. Nevertheless, many published studies examine protein expression patterns or behaviorally correlated in vivo activity in the dorsal extent of the hippocampus; and explain findings mechanistically with cellular electrophysiology from the ventro-medial region. This is further confounded by the fact that many acute slice electrophysiological experiments are performed in juvenile animals, when other experimental modes are performed in more mature animals. To address these issues, this method incorporates transcardial perfusion of mature (&#62;60 day old rodents) with artificial cerebrospinal fluid followed by preparation of modified coronal slices including the septal pole of the dorsal hippocampus to record from CA1 pyramidal cells. This process leads to the generation of healthy acute slices of dorsal hippocampus allowing for slice-based cellular electrophysiological interrogation matched to other measures.

The purpose of this protocol is to describe a method to produce slices of the dorsal hippocampus for electrophysiological examination. This procedure employs perfusion with chilled ACSF prior to slice preparation with a near-coronal slicing angle which allows for preservation of healthy principal neurons.

Whole-cell patch-clamp recordings from acute rodent brain slices are a mainstay of modern neurophysiological research, allowing precise measurement of ...

Acute Mouse Brain Slicing to Investigate Spontaneous Hippocampal Network Activity

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell resolution using electrophysiology, microscopy, and pharmacology. However, a major consideration in the design of in vitro experiments is the extent to which different slice preparations recapitulate naturalistic patterns of neural activity as observed in vivo. In the intact brain, the hippocampal network generates highly synchronized population activity reflective of the behavioral state of the animal, as exemplified by the sharp-wave ripple complexes (SWRs) that occur during waking consummatory states or non-REM sleep. SWRs and other forms of network activity can emerge spontaneously in isolated hippocampal slices under appropriate conditions. In order to apply the powerful brain slice toolkit to the investigation of hippocampal network activity, it is necessary to utilize an approach that optimizes tissue health and the preservation of functional connectivity within the hippocampal network. Mice are transcardially perfused with cold sucrose-based artificial cerebrospinal fluid. Horizontal slices containing the hippocampus are cut at a thickness of 450 &#956;m to preserve synaptic connectivity. Slices recover in an interface-style chamber and are transferred to a submerged chamber for recordings. The recording chamber is designed for dual surface superfusion of artificial cerebrospinal fluid at a high flow rate to improve oxygenation of the slice. This protocol yields healthy tissue suitable for the investigation of complex and spontaneous network activity in vitro.

This protocol describes the preparation of horizontal hippocampal-entorhinal cortex (HEC) slices from mice exhibiting spontaneous sharp-wave ripple activity. Slices are incubated in a simplified interface holding chamber and recordings are performed under submerged conditions with fast-flowing artificial cerebrospinal fluid to promote tissue oxygenation and the spontaneous emergence of network-level activity.

Affezione cerebrale acuta del topo per indagare sull'attività spontanea della rete ippocampale

Acute rodent brain slicing offers a tractable experimental approach to gain insight into the organization and function of neural circuits with single-cell ...

Neuronal Death Assay to Evaluate Cell Death in Ex Vivo Epilepsy Model

Source: Carvalho, M., et al., <a href="https://app.jove.com/v/61330">A Model of Epileptogenesis in Rhinal Cortex-Hippocampus Organotypic Slice Cultures.</a> J. Vis. Exp. (2021)
This video demonstrates the technique of assessing neuronal death in rhinal cortex-hippocampus organotypic slices. Neuronal death occurs via evolving epileptic seizures &#8212; resembling in vivo epilepsy &#8212; and is detected through dual-staining of the dead cells using propidium iodide and immunofluorescence.