Gli autori desiderano ringraziare Matteo Firenze per la preparazione delle culture fetta dell&#39;ippocampo. Questo lavoro è stato sostenuto dalle concessioni dal National Institute on Aging e Associazione Alzheimer di NZG.

1. Fissazione Fissativi sono cancerogene, indossare guanti e gestire i fissativi in ​​una cappa aspirante. Se non diversamente specificato, tutte le incubazioni sono effettuate su tutte le soluzioni ghiaccio e deve essere filtrata prima dell&#39;uso. Utilizzare microscopia elettronica di grado reagenti. Giorno 1 <ol><li class="jove_content"> Dopo condizioni sperimentali (ad esempio iniezione virale, trattamento farmacologico), posizionare la membrana con organotipiche fettine di ippocampo in un 60 x 15 millimetri capsula di Petri in polistirolo contenente ghiacciata 0.1 M tampone fosfato (tampone fosfato Sorensen), pH 7,3. Aggiungere 1 - 2 ml di tampone fosfato 0,1 M direttamente sopra le fette per tenerli freddo. </li></ol> FASE CRITICA: utilizzare sempre freschi tamponi preparati e fissativi. Assicurarsi che il pH del tampone è nel raggio d&#39;azione desiderato. Un fallimento in tal senso potrebbe danneggiare le strutture cellulari. <ol start="2"><li> Per isolare il sottocampo CA1 della fetta, utilizzare unmonouso bisturi per tagliare delicatamente sulla fetta successiva alla DG, parallelo allo strato CA1 cella (Figura 1). Poi tagliare la fetta rimanente in verticale per rimuovere il sottocampo CA3 e subiculum. Tagliare un angolo della fetta per identificare la superficie superiore del tessuto. </li></ol> NOTA: Nel caso di consegna virale di proteina di interesse è richiesta, la sezione intero tessuto può essere fissato dopo che il supporto viene rimosso con un delicato risciacquo di tampone ghiacciato. Questo è seguito da tagliare il CA1. Passaggio critico: tenere traccia del lato superiore del tessuto in modo corretto per il sezionamento delle griglie in seguito. <ol start="3"><li> Rimuovere accuratamente il tessuto dalla membrana utilizzando il retro del bisturi. Utilizzare una pipetta Pasteur per trasferire delicatamente il tessuto ad un 12-pozzetti contenente 0,1 M tampone fosfato. </li><li> Rimuovere il tampone nel pozzo dalla piastra e aggiungere 500 &amp; mu; l - 1 ml di fissativo ghiacciato (pH 7,3) comprende il seguente: 0,1% acido picrico, 1% paraformaldeide e glutaraldeide 2,5% in tampone fosfato 0,1 M. Incubare per 2 ore a 4 ° C. </li></ol> NOTA: L&#39;acido picrico può essere esplosivo allo stato secco. Keep it bagnato con acqua in un contenitore ermeticamente chiuso. Conservare in luogo asciutto e ben ventilato. <ol start="5"><li> Rimuovere il fissativo dal pozzo e lavare campioni 3 volte (20 minuti ciascuna) in tampone fosfato 0,1 M. </li><li> Incubare per 40 min in 1% acido tannico (w / v) in tampone maleato 0,1 M, pH 6,0. </li><li> Risciacquare due volte (20 minuti ciascuna) in tampone maleato. </li><li> Incubare per 40 min in acetato di uranile 1% (w / v) in tampone maleato al buio. Acetato di uranile è sensibile alla luce ed è radioattivo. Coprire il becher con Parafilm per soluzioni e memorizzare qualsiasi buffer utilizzato al buio a 4 ° C. </li><li> Risciacquare due volte (20 minuti ciascuna) in tampone maleato. </li><li> Incubare per 20 min a cloro platino 0,5%ide (w / v) in tampone maleato. </li><li> Risciacquare due volte (20 minuti ciascuna) in tampone maleato. Conservare i campioni a 4 ° C fino a quando non sono pronti per ulteriori elaborazioni. </li></ol> 2. Disidratazione e Embedding Ossido di propilene è cancerogeno. Evitare i vapori lavorando con essa in una cappa aspirante. Utilizzare assoluto, etanolo puro al 100% che non contiene alcuna traccia di acqua. Diluire la etanolo assoluto per la produzione di diverse concentrazioni per la disidratazione. Giorno 2 <ol><li> Incubare per 5 min in 50% etanolo e poi per 5 minuti in etanolo al 70%. </li><li> Incubare per 15 min in appena preparato 1% p-fenilendiammina (PPD) in etanolo al 70%. </li><li> Risciacquare tre volte in 70% di etanolo. </li><li> Incubare per 5 min in 80% etanolo e poi per 5 minuti in etanolo al 95%. </li><li> Incubare in etanolo al 100% due volte ogni 5 min. </li><li> Preparare fiale di vetro con tappo a vite e assicurarsi che siano puliti e completamente asciutti. Trasferimento fette di fiale di vetro eetichettare ogni flacone semplice e chiaro. </li><li> Incubare per 5 min in etanolo 01:01: ossido di propilene. </li><li> Incubare in ossido di propilene al 100% due volte ogni 5 min. </li><li> Aggiungi resina (Epon) ad ogni flacone di fare una miscela 1:1 con ossido di propilene. Mescolare delicatamente per 2 ore. Non agitare le fiale troppo rigorosamente per evitare bolle che potrebbero interferire con l&#39;incorporamento. </li><li> Aggiungere resina per fare una miscela 3:1 con ossido di propilene, e mescolare per 2 h. </li><li> Trasferimento al 100% resina e incubare O / N. </li></ol> Giorno 3 <ol start="12"><li> Campioni Sandwich tra strisce di plastica ACLAR e cura per 24 ore a 60 ° C. </li></ol> 3. Sezionamento e Montaggio su griglie Giorno 4 <ol><li> Tagliare semi-sottili (0,5 micron) sezioni e macchia con l&#39;1% blu di toluidina + 1% borace per determinare il corretto orientamento dei campioni. </li><li> Tagliare sezioni ultrasottili (60 nm) e montare sulle griglie di nichel, una sezione per ogni griglia. </li></ol>4. Immunoistochimica Tutti i tamponi e l&#39;acqua deve essere filtrata prima dell&#39;uso. Giorno 5 <ol><li> Posizionare una goccia (~ 50 ml) di tampone Tween-20/phosphate 1% (T / PB), pH 7,5, su un pezzo di Parafilm pulito su una superficie piana. Delicatamente sollevare la griglia con pinza pulita dal bordo e galleggiare sul buffer, sezione giù, e incubare per 10 minuti a RT. </li><li> Drenare il liquido in eccesso dalla griglia mettendo fianco sezione sulla carta da filtro e poi galleggiare su 50 mM glicina in T / PB per 15 minuti a RT. </li></ol> PUNTO CRITICO: per evitare &#39;riporto&#39; eccessivo di soluzioni da una goccia soluzione all&#39;altra durante le incubazioni, scolare il liquido in eccesso toccando delicatamente il bordo della griglia su # 1 carta da filtro tra ogni cambio di soluzione. Rimuovere anche la soluzione intrappolato tra le braccia della EM pinza che tiene la griglia delicatamente assorbente con una scheggia di carta da filtro tra le braccia, mentre pinze assicusuonare le sezioni non si asciughi completamente. <ol start="3"><li> Asciugare la griglia con carta da filtro e poi galleggiano sul blocco soluzione contenente 2,5% di BSA e 2,5% di siero da animali dell&#39;anticorpo secondario in T / PB per 30 minuti a RT. </li><li> Incubare con l&#39;anticorpo primario (in T / PB) a RT o O / N a 4 ° C. Il tempo ottimale e la temperatura di incubazione, nonché la concentrazione di anticorpi, devono essere determinati sperimentalmente. </li><li> Lavare la griglia di tre volte (2 min ciascuno) con T / PB. </li><li> Incubare con anticorpo secondario (1:20 anti-coniglio o anti-topo accoppiata a 10 nm oro) per 1 ora a RT. </li><li> Lavare tre volte (2 min ciascuno) con T / PB. </li><li> Postfix con 2% di glutaraldeide in T / PB per 5 min. </li><li> Lavare tre volte (2 min ciascuno) con T / PB e poi tre volte (2 min ciascuno) con acqua filtrata. </li><li> Incubare con acetato di uranile 2% in acqua per 10 min. </li></ol> Mentre la griglia è la colorazione, preparare un 2-free camera di CO mise enga pezzo di Parafilm nel centro di un piatto di vetro Petri. Quindi posizionare 4-6 pellet di NaOH nel piatto intorno Parafilm di assorbire CO 2 nell&#39;aria. Mantenere alto chiuso per alcuni minuti. Utilizzare una pipetta Pasteur e trasferire rapidamente un piccolo volume di soluzione di citrato di piombo Reynold alla Parafilm nella capsula Petri di vetro. Aprire il top appena sufficiente inserire la pipetta Pasteur per minimizzare la reintroduzione di CO 2 nella camera. NOTA: Per la soluzione di piombo citrato di Reynold, far bollire 100 ml di acqua deionizzata in un forno a microonde e lasciate raffreddare in un contenitore ermetico. In un pallone da 50 ml con tappo, nitrato di piombo mix 1,33 g, 1,76 g di citrato di sodio e 30 ml di acqua bollita agitando vigorosamente per 1 min. Poi agitare intermittenza per 30 min. A questa soluzione nuvoloso, aggiungere 8 ml di idrossido di sodio 1 M e lentamente invertire il pallone un paio di volte. Soluzione sarà chiaro. Portare la soluzione a 50 ml con il wa bollitoter. Conservare la soluzione ermeticamente chiusi. Se precipitato, scartare e fare una nuova. <ol start="11"><li> In tre bicchieri più piccoli preparare calda, acqua appena bollita deionizzata. Lavare l&#39;acetato di uranile immergendo la griglia nel bicchiere prima e delicatamente roteare intorno per 30 sec. Ripetere questa operazione per le altre due bicchieri. </li><li> Aprire la parte superiore del piatto di vetro Petri basta per inserire la griglia sulla goccia di soluzione di citrato di piombo Reynold. Se possibile, indossare una maschera mentre si fa questo per evitare di inalare il citrato di piombo e per prevenire la formazione di precipitato. Incubare per 10 min. </li><li> Aprire il top basta per eliminare la griglia. Evitare di respirare il citrato di piombo. Lavare la griglia di tre volte, immergendolo in sequenza in tre bicchieri con acqua calda e bollente distillata. Lasciare asciugare la griglia su un pezzo di carta da filtro, la sezione. Il campione è pronto per il microscopio elettronico. </li></ol>

<ol>
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Non abbiamo nulla da rivelare.

In questo protocollo, abbiamo adottato il metodo Phend e Weinberg per il cervello e del midollo spinale per studiare spine dendritiche nelle culture fetta dell&#39;ippocampo di ratto. Spine dendritiche in ippocampo CA3-CA1 zona sono strutture delicate che contengono una grande varietà di proteine ​​che svolgono un ruolo importante nel regolare le funzioni neuronali. Il metodo proposto fornisce un approccio equilibrato per raggiungere antigenicità maggiore pur mantenendo una buona conservazione ultrastrutturale <st...

<table border="1">
 <tbody>
 <tr>
 <td>NAME OF REAGENT</td>
 <td>COMPANY</td>
 <td>CATALOG NUMBER</td>
 <td></td>
 </tr>
 <tr>
 <td>60 x 15 mm polystyrene Petri dish</td>
 <td>Falcon</td>
 <td>351007</td>
 <td></td>
 </tr>
 <tr>
 <td>Disposable scalpel</td>
 <td>EXELINT</td>
 <td>29552</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell culture inserts</td>
 <td>Millipore</td>
 <td>PICM03050</td>
 <td></td>
 </tr>
 <tr>
 <td>10 nm Goat-anti-rabbit gold</td>
 <td>Electron Microscopy Sciences</td>
 <td>25108</td>
 <td></td>
 </tr>
 <tr>
 <td>Anti-Neurogranin antibody</td>
 <td>Millipore</td>
 <td>AB5620</td>
 <td></td>
 </tr>
 <tr>
 <td>100% Picric acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>19550</td>
 <td></td>
 </tr>
 <tr>
 <td>96% Paraformaldehyde</td>
 <td>Acros Organics</td>
 <td>AC41678-0030</td>
 <td></td>
 </tr>
 <tr>
 <td>25% Glutaraldehyde (EM grade)</td>
 <td>Sigma</td>
 <td>G5882</td>
 <td></td>
 </tr>
 <tr>
 <td>Uranyl acetate</td>
 <td>Electron Microscopy Sciences</td>
 <td>22400</td>
 <td></td>
 </tr>
 <tr>
 <td>p-Phenylenediamine</td>
 <td>Sigma</td>
 <td>P6001</td>
 <td></td>
 </tr>
 <tr>
 <td>Platinum (IV) chloride</td>
 <td>Sigma</td>
 <td>379840</td>
 <td></td>
 </tr>
 <tr>
 <td>Tannic acid</td>
 <td>Electron Microscopy Sciences</td>
 <td>21710</td>
 <td></td>
 </tr>
 </tbody>
</table>

post-embedding immunogold labeling of synaptic proteins in hippocampal slice cultures

Microscopia Immunoelectron è un potente strumento per studiare molecole biologiche a livello subcellulare. Anticorpi accoppiati elettroni dense marcatori come oro colloidale può rivelare la localizzazione e la distribuzione di antigeni specifici in vari tessuti 1. Le due tecniche più utilizzate sono pre-embedding e post-embedding tecniche. In pre-embedding immuno-microscopia elettronica (EM) tecniche, il tessuto deve essere permeabilizzate per consentire la penetrazione di anticorpi prima di essere incorporato. Queste tecniche sono l&#39;ideale per conservare le strutture, ma scarsa penetrazione dell&#39;anticorpo (spesso solo i primi micrometri) è uno svantaggio notevole 2. I post-embedding metodi di etichettatura possibile evitare questo problema perché l&#39;etichettatura avviene su sezioni di tessuti fissati in cui gli antigeni sono più facilmente accessibili. Nel corso degli anni, una serie di modifiche hanno migliorato le post-embedding metodi per migliorare e preservare immunoreattività ultrastruttura <sup&gt; 3-5. Fissazione dei tessuti è una parte cruciale di studi EM. Fissativi chimicamente reticolare le macromolecole per bloccare le strutture di tessuto in posizione. La scelta di fissativo influenza non solo la conservazione strutturale ma anche antigenicità e contrasto. Tetrossido di osmio (OsO 4), formaldeide e glutaraldeide sono stati i fissativi standard per decenni, anche per il sistema nervoso centrale (SNC), tessuti che sono particolarmente inclini a danni strutturali durante la lavorazione chimica e fisica. Purtroppo, OsO 4 è altamente reattivo e ha dimostrato di mascherare antigeni 6, con conseguente etichettatura povero e insufficiente. Approcci alternativi per evitare la fissazione chimica comprendono il congelamento dei tessuti. Ma queste tecniche sono difficili da eseguire e richiedono strumentazione costosa. Per risolvere alcuni di questi problemi e di migliorare tessuto del SNC etichettatura, Phend et al. sostituito OsO 4 con acetato di uranile (UA) e tannico acid (TA), e introdotto con successo ulteriori modifiche per migliorare la sensibilità della rilevazione dell&#39;antigene e conservazione strutturale nel cervello e del midollo spinale 7. Abbiamo adottato questo osmio senza post-embedding metodo al tessuto del cervello di ratto e ottimizzato la tecnica immunogold etichettatura per rilevare e studiare le proteine ​​sinaptiche. Vi presentiamo qui un metodo per determinare la localizzazione ultrastrutturale delle proteine ​​sinaptiche nel ratto CA1 dell&#39;ippocampo neuroni piramidali. Usiamo organotipiche fettine di ippocampo in coltura. Queste fette mantenere la circuiteria trisynaptic dell&#39;ippocampo, e quindi sono particolarmente utili per studiare la plasticità sinaptica, un meccanismo ampiamente pensato alla base dell&#39;apprendimento e della memoria. Organotipiche fettine ippocampali di postnatali giorni 5 e 6 di topo / ratto cuccioli possono essere preparati come descritto in precedenza 8, e sono particolarmente utili per acutamente knockdown o esprimono molti proteine ​​esogene. Abbiamo già usato questo protocollo per characterize neurogranin (Ng), un neurone-specifica proteina con un ruolo critico nella regolazione della funzione sinaptica 8,9. Abbiamo anche usato per caratterizzare la localizzazione ultrastrutturale di calmodulina (CaM) e Ca 2 + / CaM-dipendente proteina chinasi II (CaMKII) 10. Come illustrato nei risultati, questo protocollo permette una buona conservazione ultrastrutturale delle spine dendritiche e l&#39;etichettatura efficiente del Ng per caratterizzare la sua distribuzione nella colonna vertebrale 8. Inoltre, la procedura qui descritta può avere un&#39;ampia applicabilità in studio molte altre proteine ​​coinvolte in funzioni neuronali.

La Figura 2B mostra un esempio di distribuzione di molecole endogene Ng in spine dendritiche dei neuroni dell&#39;ippocampo CA1 piramidale. Griglie nichel con ultrasottili (60 nm) tessuti contenenti regione CA1 dell&#39;ippocampo (come visto in Figura 2A) sono stati coperti in 1% T / PB, 50 mM glicina, poi bloccate con BSA 2,5% e 2,5% di siero prima dell&#39;incubazione con anticorpi anti Ng-anticorpo. Dopo lavaggio con T / PB, griglie sono state poi coperto in anti-coniglio anticorpo s...

Watch this Scientific Journal Video about Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures at JoVE.com

Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

La localizzazione e la distribuzione delle proteine ​​forniscono informazioni importanti per la comprensione delle loro funzioni cellulari. La superiore risoluzione spaziale di microscopia elettronica (EM) può essere utilizzato per determinare la localizzazione subcellulare di un dato antigene immunoistochimica seguito. Per tessuti del sistema nervoso centrale (CNS), preservando l&#39;integrità strutturale mentre antigenicità mantenendo è stato particolarmente difficile in studi EM. Qui, si adotta una procedura che è stata usata per conservare le strutture e gli antigeni del sistema nervoso centrale per studiare e caratterizzare le proteine ​​sinaptiche nel ratto CA1 dell&#39;ippocampo neuroni piramidali.

Post-embedding Etichettatura immuno di proteine ​​sinaptiche in Culture Slice ippocampali

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as ...

post-embedding-immunogold-labeling-synaptic-proteins-hippocampal

Research

JoVE Journal

Neuroscience

17.0K Views.  Medical College of Wisconsin. La localizzazione e la distribuzione delle proteine ​​forniscono informazioni importanti per la comprensione delle loro funzioni cellulari. La superiore risoluzione spaziale di microscopia elettronica (EM) può essere utilizzato per determinare la localizzazione subcellulare di un dato antigene immunoistochimica seguito. Per tessuti del sistema nervoso centrale (CNS), preservando l'integrità strutturale mentre antigenicità mantenendo è stato particolarmente dif...

Video: Post-embedding Immunogold Labeling of Synaptic Proteins in Hippocampal Slice Cultures

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). ...

Ricerca

Neuroscienze

The Organotypic Hippocampal Slice Culture Model for Examining Neuronal Injury

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of the hippocampus is relatively preserved 1. The organotypic culture system allows for the examination of neuronal, astrocytic and microglial effects, but as an ex vivo preparation, does not address effects of blood flow, or recruitment of peripheral inflammatory cells. To that end, this culture method is frequently used to examine excitotoxic and hypoxic injury to pyramidal neurons of the hippocampus, but has also been used to examine the inflammatory response. Herein we describe the methods for generating hippocampal slice cultures from postnatal rodent brain, administering toxic stimuli to induce neuronal injury, and assaying and quantifying hippocampal neuronal death.

The organoptypic hippocampal slice culture model is an in vitro model used to examine neuronal injury in a variety of paradigms. In this article, we describe the methods for generating slice cultures and quantifying neuronal injury.

Il organotipica ippocampale Cultura Modello Slice per l&#39;esame di danno neuronale

Organotypic hippocampal slice culture is an in vitro method to examine mechanisms of neuronal injury in which the basic architecture and composition of ...

Organotypic Hippocampal Slice Cultures

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the strength of their connections after brief periods of strong activation. This phenomenon, known as long-term potentiation (LTP) can last for hours or days and has become the best candidate mechanism for learning and memory 2. In addition, the well defined anatomy and connectivity of the hippocampus 3 has made it a classical model system to study synaptic transmission and synaptic plasticity4.
As our understanding of the physiology of hippocampal synapses grew and molecular players became identified, a need to manipulate synaptic proteins became imperative. Organotypic hippocampal cultures offer the possibility for easy gene manipulation and precise pharmacological intervention but maintain synaptic organization that is critical to understanding synapse function in a more naturalistic context than routine culture dissociated neurons methods.
Here we present a method to prepare and culture hippocampal slices that can be easily adapted to other brain regions. This method allows easy access to the slices for genetic manipulation using different approaches like viral infection 5,6 or biolistics 7. In addition, slices can be easily recovered for biochemical assays 8, or transferred to microscopes for imaging 9 or electrophysiological experiments 10.

We describe a method to prepare organotypic hippocampal slices that can be easily adapted to other brain regions. Brain slices are laid on porous membranes and culture media is allowed to form an interface. This method preserves the gross architecture of the hippocampus for up to 2 weeks in culture.

Organotipica Culture Slice ippocampale

The hippocampus, a component of the limbic system, plays important roles in long-term memory and spatial navigation 1. Hippocampal neurons can modify the ...

The Analysis of Purkinje Cell Dendritic Morphology in Organotypic Slice Cultures

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly oriented in the sagittal plane and develops mostly in the postnatal period in small rodents 3. Furthermore, several antibodies are available which selectively and intensively label Purkinje cells including all processes, with anti-Calbindin D28K being the most widely used. For viewing of dendrites in living cells, mice expressing EGFP selectively in Purkinje cells 11 are available through Jackson labs. Organotypic cerebellar slice cultures cells allow easy experimental manipulation of Purkinje cell dendritic development because most of the dendritic expansion of the Purkinje cell dendritic tree is actually taking place during the culture period 4. We present here a short, reliable and easy protocol for viewing and analyzing the dendritic morphology of Purkinje cells grown in organotypic cerebellar slice cultures. For many purposes, a quantitative evaluation of the Purkinje cell dendritic tree is desirable. We focus here on two parameters, dendritic tree size and branch point numbers, which can be rapidly and easily determined from anti-calbindin stained cerebellar slice cultures. These two parameters yield a reliable and sensitive measure of changes of the Purkinje cell dendritic tree. Using the example of treatments with the protein kinase C (PKC) activator PMA and the metabotropic glutamate receptor 1 (mGluR1) we demonstrate how differences in the dendritic development are visualized and quantitatively assessed. The combination of the presence of an extensive dendritic tree, selective and intense immunostaining methods, organotypic slice cultures which cover the period of dendritic growth and a mouse model with Purkinje cell specific EGFP expression make Purkinje cells a powerful model system for revealing the mechanisms of dendritic development.

We present a protocol that permits to view and to quantitatively asses the morphology of the dendritic tree of individual Purkinje cells grown in organotypic cerebellar slice cultures. This protocol is intended to promote studies on the mechanisms of Purkinje cell dendritic development.

L&#39;analisi di morfologia dendritica delle cellule di Purkinje in Culture Slice organotipiche

Purkinje cells are an attractive model system for studying dendritic development, because they have an impressive dendritic tree which is strictly ...

Lateral Diffusion and Exocytosis of Membrane Proteins in Cultured Neurons Assessed using Fluorescence Recovery and Fluorescence-loss Photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This directed trafficking is of fundamental importance to deliver, maintain or remove synaptic proteins.
Super-ecliptic pHluorin (SEP) is a pH-sensitive derivative of eGFP that has been extensively used for live cell imaging of plasma membrane proteins1-2. At low pH, protonation of SEP decreases photon absorption and eliminates fluorescence emission. As most intracellular trafficking events occur in compartments with low pH, where SEP fluorescence is eclipsed, the fluorescence signal from SEP-tagged proteins is predominantly from the plasma membrane where the SEP is exposed to a neutral pH extracellular environment. When illuminated at high intensity SEP, like every fluorescent dye, is irreversibly photodamaged (photobleached)3-5. Importantly, because low pH quenches photon absorption, only surface expressed SEP can be photobleached whereas intracellular SEP is unaffected by the high intensity illumination6-10. 
FRAP (fluorescence recovery after photobleaching) of SEP-tagged proteins is a convenient and powerful technique for assessing protein dynamics at the plasma membrane. When fluorescently tagged proteins are photobleached in a region of interest (ROI) the recovery in fluorescence occurs due to the movement of unbleached SEP-tagged proteins into the bleached region. This can occur via lateral diffusion and/or from exocytosis of non-photobleached receptors supplied either by de novo synthesis or recycling (see Fig. 1). The fraction of immobile and mobile protein can be determined and the mobility and kinetics of the diffusible fraction can be interrogated under basal and stimulated conditions such as agonist application or neuronal activation stimuli such as NMDA or KCl application8,10. 
We describe photobleaching techniques designed to selectively visualize the recovery of fluorescence attributable to exocytosis. Briefly, an ROI is photobleached once as with standard FRAP protocols, followed, after a brief recovery, by repetitive bleaching of the flanking regions. This 'FRAP-FLIP' protocol, developed in our lab, has been used to characterize AMPA receptor trafficking at dendritic spines10, and is applicable to a wide range of trafficking studies to evaluate the intracellular trafficking and exocytosis.

This report describes the use of live cell imaging and photobleach techniques to determine the surface expression, transport pathways and trafficking kinetics of exogenously expressed, pH-sensitive GFP-tagged proteins at the plasma membrane of neurons.

Diffusione laterale e esocitosi delle proteine ​​di membrana in neuroni in coltura valutata utilizzando Recovery fluorescenza e fluorescenza-loss photobleaching

Membrane proteins such as receptors and ion channels undergo active trafficking in neurons, which are highly polarised and morphologically complex. This ...

Organotypic Slice Cultures to Study Oligodendrocyte Dynamics and Myelination

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During embryonic and postnatal development they actively proliferate and generate myelinating oligodendrocytes. These cells have commonly been studied in primary dissociated cultures, neuron cocultures, and in fixed tissue. Using newly available transgenic mouse lines slice culture systems can be used to investigate proliferation and differentiation of oligodendrocyte lineage cells in both gray and white matter regions of the forebrain and cerebellum. Slice cultures are prepared from early postnatal mice and are kept in culture for up to 1 month. These slices can be imaged multiple times over the culture period to investigate cellular behavior and interactions. This method allows visualization of NG2 cell division and the steps leading to oligodendrocyte differentiation while enabling detailed analysis of region-dependent NG2 cell and oligodendrocyte functional heterogeneity. This is a powerful technique that can be used to investigate the intrinsic and extrinsic signals influencing these cells over time in a cellular environment that closely resembles that found in vivo.

A technique to study NG2 cells and oligodendrocytes using a slice culture system of the forebrain and cerebellum is described. This method allows examination of the dynamics of proliferation and differentiation of cells within the oligodendrocyte lineage where the extracellular environment can be easily manipulated while maintaining tissue cytoarchitecture.

Culture Organotipica Slice per studiare Oligodendrocyte Dynamics e mielinizzazione

NG2 expressing cells (polydendrocytes, oligodendrocyte precursor cells) are the fourth major glial cell population in the central nervous system. During ...

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 ...

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 ...

High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA receptors (NMDARs) and AMPA receptors (AMPARs). Physiological data have indicated that glutamate receptors at RGCs are expressed not only in postsynaptic but also in perisynaptic or extrasynaptic membrane compartments. However, precise anatomical locations for glutamate receptors at RGC synapses have not been determined. Although a high-resolution quantitative analysis of glutamate receptors at central synapses is widely employed, this approach has had only limited success in the retina. We developed a postembedding immunogold method for analysis of membrane receptors, making it possible to estimate the number, density and variability of these receptors at retinal ribbon synapses. Here we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of retinal fixation, 2) freeze-substitution, 3) postembedding immunogold electron microscope (EM) immunocytochemistry and, 4) quantitative visualization of glutamate receptors at ribbon synapses.

The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

Risoluzione alta analisi quantitativa Immunogold di Recettori di membrana a retina nastro Sinapsi

Retinal ganglion cells (RGCs) receive excitatory glutamatergic input from bipolar cells. Synaptic excitation of RGCs is mediated postsynaptically by NMDA ...

Organotypic Hippocampal Slice Cultures As a Model to Study Neuroprotection and Invasiveness of Tumor Cells

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. This model is suitable for addressing different research questions that involve studies on neuroprotection, electrophysiological experiments on neurons, neuronal networks or tumor invasion. The hippocampal architecture and neuronal activity in multisynaptic circuits are well conserved in OHSC, even though the slicing procedure itself initially lesions and leads to formation of a glial scar. The scar formation alters presumably the mechanical properties and diffusive behavior of small molecules, etc. Slices allow the monitoring of time dependent processes after brain injury without animal surgery, and studies on interactions between various brain-derived cell types, namely astrocytes, microglia and neurons under both physiological and pathological conditions. An ambivalent aspect of this model is the absence of blood flow and immune blood cells. During the progression of the neuronal injury, migrating immune cells from the blood play an important role. As those cells are missing in slices, the intrinsic processes in the culture may be observed without external interference. Moreover, in OHSC the composition of the medium-external environment is precisely controlled. A further advantage of this method is the lower number of sacrificed animals compared to standard preparations. Several OHSC can be obtained from one animal making simultaneous studies with multiple treatments in one animal possible. For these reasons, OHSC are well suited to analyze the effects of new protective therapeutics after tissue damage or during tumor invasion. 
The protocol presented here describes a preparation method of OHSC that allows generating highly reproducible, well preserved slices that can be used for a variety of experimental research, like neuroprotection or tumor invasion studies.

Organotypic hippocampal slice cultures (OHSC) represent an in vitro model that simulates the in vivo situation very well. Here we describe a vibratome-based improved slicing protocol to obtain high quality slices for use in assessing the neuroprotective potential of novel substances or the biological behavior of tumor cells.

Colture Hippocampal Fetta organotipiche come modello di studio Neuroprotection e l'invasività delle cellule del tumore

In organotypic hippocampal slice cultures (OHSC), the morphological and functional characteristics of both neurons and glial cells are well preserved. ...

Post-embedding Etichettatura immuno di proteine ​​sinaptiche in Culture Slice ippocampali

Post-embedding Etichettatura immuno di proteine sinaptiche in Culture Slice ippocampali