Questo lavoro è stato sostenuto da NIH National Institute of Neurological Disorder and Stroke (NINDS) (NS049442).&gt;

Risospendere peptide β-amiloide Prima di iniziare sono pronti: <ul><li> Aβ 1-42 (polvere liofilizzata) </li><li> 1,1,1,3,3,3-esafluoro-2-propanolo (HFIP) </li><li> Low-binding tubi in polipropilene microcentrifuga (1,5 ml) </li><li> Hamilton siringa a tenuta di gas con stop tuffo Teflon (250 microlitri) </li><li> Dimetilsolfossido (DMSO) </li></ul><ol><li> Lasciare Aβ liofilizzato 1-42 equilibrare a temperatura ambiente per 30 minuti per evitare la condensa dopo l&#39;apertura del flacone peptide. </li><li> Sotto la cappa, risospendere peptidi Aβ 1-42 in HFIP ghiacciata per ottenere una soluzione 1 mM e vortex la soluzione per alcuni secondi. </li><li> Utilizzando una siringa di vetro con tappo a tenuta di gas Hamilton teflon, subito dividere il Aβ 1-42 / HFIP soluzione altrettanto in tre fiale in polipropilene e sigillare le fiale. </li><li> Le fiale vengono quindi incubate per 2 ore per consentire monomerization Aβ. Quindi, aprire le fiale e concentrare la soluzione Aβ 1-42 / HFIP sotto vuoto usando una centrifuga SpeedVac (800 g, temperatura ambiente) fino a quando un film peptide chiaro si osserva sul fondo dei flaconi. Controllare accuratamente la temperatura all&#39;interno della centrifuga SpeedVac per evitare il degrado peptide (max 25 ° C). </li><li> Sigillare le fiale e conservare le aliquote a -80 ° C. Pellicola Aβ può essere conservato per 6 mesi. </li><li> Risospendere un film con l&#39;aggiunta di DMSO per ottenere una concentrazione fino a 5 mm Aβ 1-42. Sonicare a bagnomaria per 10 minuti per garantire la completa risospensione. </li><li> Questa aliquota 5mM Aβ 1-42 soluzione in fiale in polipropilene. Sigillare tutti i flaconi e conservarli a -20 ° C. Aliquote vanno scongelati solo una volta. Attenzione alla condensa l&#39;acqua all&#39;interno del congelatore. Peptidi in soluzione degrada molto facilmente. </li></ol> Oligomerizzazione Prima di iniziare sono pronti: <ul><li> Aβ 1-42 / DMSO (5mm) </li><li> Tampone fosfato sterile </li><li> Low-binding tubi in polipropilene microcentrifuga (1,5 ml) </li><li> Low-binding polipropilene provette (50 ml) </li></ul><ol><li> Diluire il Aβ ottenuto 5mM 1-42 / aliquota DMSO con tampone fosfato sterile (fino a 100 l). </li><li> Vortex per 30 secondi e poi incubare per 12 ore a 4 ° C </li></ol> Fetta trattamento dell&#39;ippocampo Durante il trattamento fettine di ippocampo, è importante per evitare la mancanza di specifiche adesione peptide su bicchieri, superfici tubo per perfusione, e la camera di registrazione. Usare preferibilmente a basso legame tubi e contenitori in polipropilene. Evitare l&#39;uso di oggetti di vetro o di plastica generico-ware. Mezzi di perfusione deve essere siero o albumina-free. Perfusione Aβ Prima di iniziare sono pronti: <ul><li> Oligomerized Aβ 1-42 </li><li> Registrazione del buffer (in mm: 124 NaCl, 4.4 KCl, 1 Na 2 HPO 4, 25 NaHCO 3, 2 CaCl 2, 2 MgCl 2, e 10 di glucosio) </li><li> Registrazione elettrofisiologica set-up con camera di interfaccia </li><li> Trasversale fettine di ippocampo incubate in un tampone di registrazione ossigenato per almeno 90 minuti dopo la dissezione (T = 29 ° C; tasso di perfusione costante = 2 ml / minuto) </li></ul><ol><li> Immediatamente prima l&#39;esperimento, diluire il Aβ oligomerized 1-42 aliquota alla concentrazione di lavoro appropriati (200 nm o superiore) in un tubo in polipropilene con pre-ossigenato buffer di registrazione. </li><li> Profumato il Aβ 1-42 soluzione per 20 minuti per le fette prima dell&#39;induzione della plasticità sinaptica. </li></ol> Induzione di plasticità e di follow-up <ol><li> Tipicamente, il modello più diffuso di plasticità sinaptica è la LTP. Essa può essere indotta attraverso la stimolazione tetanica di una sinapsi dato nell&#39;ippocampo. Registriamo risposte campo da sinapsi tra le proiezioni del Shaffer collaterali e neuroni piramidali CA1 nel radiatum strato. Il nostro stimolazione tetanica è costituito da una stimolazione scoppio theta che comprende 3 treni separati da intervalli di 15 secondi. Ogni treno è composto da 10 scatti a 5 Hz dove ogni raffica comporta 5 impulsi a 100 Hz. Applicare la stimolazione tetanica subito dopo la perfusione di Aβ oligomerized è stata completata. </li><li> Subito dopo la stimolazione tetanica tornare a perfusione con buffer di registrazione normale. Mantenere una velocità costante di perfusione, temperatura della camera di registrazione e la corretta ossigenazione per tutta la durata dell&#39;esperimento. </li></ol> Risultati rappresentative e possibili problemi La Aβ oligomerized secondo il protocollo classico di Stine et coll. 1, genera monomeri Aβ e una varietà di oligomeri di diverse dimensionis (dimero, trimero, ecc) La perfusione di oligomerized Aβ 1-42 porta a LTP diminuita in Aβ trattati con fette rispetto a fette di controllo. Nelle nostre condizioni sperimentali, in cui 3-5 mesi topi vecchi C57BL/6J maschi sono utilizzati, LTP in 200 nM Aβ trattati con fette è in media del 150% dei valori di base a due ore dopo la stimolazione tetanica, mentre a fette di controllo i valori sono LTP in media 200-250% di baseline2. In alcuni casi, il trattamento con Aβ potrebbe non riuscire a ridurre la LTP ippocampale. Diversi errori critici che potrebbero verificarsi comprendono pellicola durante l&#39;asciugatura eccessiva concentrazione di Aβ e oligomerizzazione incompleta. Perfusione Aβ in fettine di ippocampo potrebbe essere un&#39;altra fonte di preoccupazione a causa delle proprietà fisico-chimiche del peptide Aβ in soluzione, che è incline a non specifica adesione alla plastica. Risoluzione dei problemi di queste questioni è discusso di seguito.

<ol>
	<li>Stine, W. B., Dahlgren, K. N., Krafft, G. A., LaDu, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+characterization+of+conditions+for+amyloid-beta+peptide+oligomerization+and+fibrillogenesis.">In vitro characterization of conditions for amyloid-beta peptide oligomerization and fibrillogenesis.</a> J Biol Chem. 278, 11612-11622 (2003).</li><li>Vitolo, O. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Amyloid+beta+-peptide+inhibition+of+the+PKA/CREB+pathway+and+long-term+potentiation:+reversibility+by+drugs+that+enhance+cAMP+signaling.">Amyloid beta -peptide inhibition of the PKA/CREB pathway and long-term potentiation: reversibility by drugs that enhance cAMP signaling.</a> Proc Natl Acad Sci U S A. 99, 13217-13221 (2002).</li><li>Puzzo, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Picomolar+amyloid-beta+positively+modulates+synaptic+plasticity+and+memory+in+hippocampus.">Picomolar amyloid-beta positively modulates synaptic plasticity and memory in hippocampus.</a> J Neurosci. 28, 14537-14545 (2008).</li><li>Lewczuk, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+sample+collection+tubes+on+cerebrospinal+fluid+concentrations+of+tau+proteins+and+amyloid+beta+peptides.">Effect of sample collection tubes on cerebrospinal fluid concentrations of tau proteins and amyloid beta peptides.</a> Clin Chem. 52, 332-334 (2006).</li><li>Masliah, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanisms+of+synaptic+dysfunction+in+Alzheimer's+disease.">Mechanisms of synaptic dysfunction in Alzheimer's disease.</a> Histol Histopathol. 10, 509-519 (1995).</li><li>Cullen, W. K., Suh, Y. H., Anwyl, R., Rowan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Block+of+LTP+in+rat+hippocampus+in+vivo+by+beta-amyloid+precursor+protein+fragments.">Block of LTP in rat hippocampus in vivo by beta-amyloid precursor protein fragments.</a> Neuroreport. 8, 3213-3217 (1997).</li><li>Itoh, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impairments+of+long-term+potentiation+in+hippocampal+slices+of+beta-amyloid-infused+rats.">Impairments of long-term potentiation in hippocampal slices of beta-amyloid-infused rats.</a> Eur J Pharmacol. 382, 167-175 (1999).</li><li>Walsh, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Naturally+secreted+oligomers+of+amyloid+beta+protein+potently+inhibit+hippocampal+long-term+potentiation+in+vivo.">Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo.</a> Nature. 416, 535-539 (2002).</li><li>Lambert, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=nonfibrillar+ligands+derived+from+Abeta1-42+are+potent+central+nervous+system+neurotoxins.">nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.</a> Proc Natl Acad Sci U S A. 95, 6448-6453 (1998).</li><li>Gong, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ubiquitin+hydrolase+Uch-L1+rescues+beta-amyloid-induced+decreases+in+synaptic+function+and+contextual+memory.">Ubiquitin hydrolase Uch-L1 rescues beta-amyloid-induced decreases in synaptic function and contextual memory.</a> Cell. 126, 775-788 (2006).</li><li>Trinchese, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+calpains+improves+memory+and+synaptic+transmission+in+a+mouse+model+of+Alzheimer+disease.">Inhibition of calpains improves memory and synaptic transmission in a mouse model of Alzheimer disease.</a> J Clin Invest. 118, 2796-2807 (2008).</li></ol>

Gli esperimenti sugli animali sono stati eseguiti secondo le linee guida e regolamenti stabiliti dalla Columbia University IACUC.

Come descritto in precedenza, diversi problemi nella preparazione di Aβ oligomerici può provocare LTP inalterata. Un modo per valutare se il degrado Aβ o la mancanza di oligomerizzazione Aβ potrebbe essersi verificato è quello di valutare la preparazione Aβ con TRIS-Tricine PAGE / analisi Western Blotting e ultracentrifugazione analitica (non descritte in questo protocollo). Quando Western Blotting campioni sono preparati in condizioni non-denaturing/non-reducing, una preparazione di successo dovrebbe generare una...

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		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>A&beta; 1-42 (lyophilized) </td>
<td>&nbsp;</td>
<td>American Peptide Co.</td>
<td>62-0-80</td>
<td>&nbsp;</td>
<tr>
<td>1,1,1,3,3,3-Hexafluoro-2-Propanol (HFIP)</td>
<td>&nbsp;</td>
<td>Fluka</td>
<td>52517</td>
<td>&nbsp;</td>
<tr>
<td>Dimethylsulfoxide (DMSO)</td>
<td>&nbsp;</td>
<td>Biotium</td>
<td>90082</td>
<td>&nbsp;</td>
<tr>
<td>50mL Polypropylene Centrifuge Tubes</td>
<td>&nbsp;</td>
<td>Corning</td>
<td>05-538-67</td>
<td>&nbsp;</td>
<tr>
<td>1.5ml MaxyClear microtubes Maximum recovery</td>
<td>&nbsp;</td>
<td>Axygen</td>
<td>MCT-150-L-C</td>
<td>&nbsp;</td>
<tr>
<td>Phosphate-Buffered Saline pH 7.4</td>
<td>&nbsp;</td>
<td>Invitrogen-Gibco</td>
<td>10010-023</td>
<td>&nbsp;</td>
<tr>
<td>GASTIGHT Syringes 250 &mu;L</td>
<td>&nbsp;</td>
<td>Hamilton</td>
<td>1725</td>
<td>&nbsp;</td>
<tr>
<td>Bath sonicator</td>
<td>&nbsp;</td>
<td>Branson</td>
<td>3510</td>
<td>&nbsp;</td>
<tr>
<td>Savant SpeedVac Concentrator System</td>
<td>&nbsp;</td>
<td>Various distributors</td>
<td>SC 110A</td>
<td>&nbsp;</td>		</tbody>
		</table>
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preparation of oligomeric &beta;-amyloid1-42 and induction of synaptic plasticity impairment on hippocampal slices

Riduzione di valore delle connessioni sinaptiche è probabile che alla base della amnesico sottili cambiamenti che si verificano nelle prime fasi del morbo di Alzheimer s (AD). β-amiloide (Aβ), un peptide prodotto in grandi quantità in AD, è noto per ridurre potenziamento a lungo termine (LTP), un cellulare correlato di apprendimento e memoria. Infatti, compromissione LTP causata da Aβ è un utile paradigma sperimentale per lo studio delle disfunzioni sinaptiche nei modelli dC e lo screening per i farmaci in grado di mitigare tali menomazioni o ritorno sinaptica. Studi hanno dimostrato che Aβ produce l&#39;interruzione LTP preferenzialmente attraverso la sua forma oligomerici. Qui forniamo un protocollo dettagliato per LTP da compromettere la perfusione di oligomerized sintetico Aβ1-42 peptide su fettine di ippocampo acuta. In questo video, delineiamo un passo-passo procedura per la preparazione di Aβ oligomerici<sub&gt; 1-42</sub&gt;. Poi, seguiamo un esperimento individuale in cui è ridotta LTP in fettine di ippocampo esposti a Aβ oligomerized<sub&gt; 1-42</sub&gt; Rispetto a fette in un esperimento di controllo dove non Aβ<sub&gt; 1-42</subEsposizione&gt; era accaduto.

Watch this Scientific Journal Video about Preparation of Oligomeric Î²-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices at JoVE.com

Preparation of Oligomeric &amp;#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

Una caratteristica della malattia di Alzheimer è l&#39;elevazione di Aβ<sub&gt; 1-42</sub&gt; Peptide. Qui forniamo un protocollo per la preparazione di Aβ sintetico<sub&gt; 1-42</sub&gt; Oligomeri, che danneggia dell&#39;ippocampo potenziamento a lungo termine, un cellulare correlato di memoria. Questa procedura è utile per lo studio dei meccanismi di Aβ indotta lo screening della patologia e della droga.

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). ...

preparation-oligomeric-amyloid1-42-induction-synaptic-plasticity

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Neuroscience

Preparation of Oligomeric &beta;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

23.0K Views.  Columbia University. Una caratteristica della malattia di Alzheimer è l'elevazione di Aβ 1-42 Peptide. Qui forniamo un protocollo per la preparazione di Aβ sintetico 1-42 Oligomeri, che danneggia dell'ippocampo potenziamento a lungo termine, un cellulare correlato di memoria. Questa procedura è utile per lo studio dei meccanismi di Aβ indotta lo screening della patologia e della droga.

Video: Preparation of Oligomeric &#946;-amyloid1-42 and Induction of Synaptic Plasticity Impairment on Hippocampal Slices

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 ...

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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 ...

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 ...

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

Immunoelectron microscopy is a powerful tool to study biological molecules at the subcellular level. Antibodies coupled to electron-dense markers such as colloidal gold can reveal the localization and distribution of specific antigens in various tissues1. The two most widely used techniques are pre-embedding and post-embedding techniques. In pre-embedding immunogold-electron microscopy (EM) techniques, the tissue must be permeabilized to allow antibody penetration before it is embedded. These techniques are ideal for preserving structures but poor penetration of the antibody (often only the first few micrometers) is a considerable drawback2. The post-embedding labeling methods can avoid this problem because labeling takes place on sections of fixed tissues where antigens are more easily accessible. Over the years, a number of modifications have improved the post-embedding methods to enhance immunoreactivity and to preserve ultrastructure3-5.
Tissue fixation is a crucial part of EM studies. Fixatives chemically crosslink the macromolecules to lock the tissue structures in place. The choice of fixative affects not only structural preservation but also antigenicity and contrast. Osmium tetroxide (OsO4), formaldehyde, and glutaraldehyde have been the standard fixatives for decades, including for central nervous system (CNS) tissues that are especially prone to structural damage during chemical and physical processing. Unfortunately, OsO4 is highly reactive and has been shown to mask antigens6, resulting in poor and insufficient labeling. Alternative approaches to avoid chemical fixation include freezing the tissues. But these techniques are difficult to perform and require expensive instrumentation. To address some of these problems and to improve CNS tissue labeling, Phend et al. replaced OsO4 with uranyl acetate (UA) and tannic acid (TA), and successfully introduced additional modifications to improve the sensitivity of antigen detection and structural preservation in brain and spinal cord tissues7. We have adopted this osmium-free post-embedding method to rat brain tissue and optimized the immunogold labeling technique to detect and study synaptic proteins.
We present here a method to determine the ultrastructural localization of synaptic proteins in rat hippocampal CA1 pyramidal neurons. We use organotypic hippocampal cultured slices. These slices maintain the trisynaptic circuitry of the hippocampus, and thus are especially useful for studying synaptic plasticity, a mechanism widely thought to underlie learning and memory. Organotypic hippocampal slices from postnatal day 5 and 6 mouse/rat pups can be prepared as described previously8, and are especially useful to acutely knockdown or overexpress exogenous proteins. We have previously used this protocol to characterize neurogranin (Ng), a neuron-specific protein with a critical role in regulating synaptic function8,9 . We have also used it to characterize the ultrastructural localization of calmodulin (CaM) and Ca2+/CaM-dependent protein kinase II (CaMKII)10. As illustrated in the results, this protocol allows good ultrastructural preservation of dendritic spines and efficient labeling of Ng to help characterize its distribution in the spine8. Furthermore, the procedure described here can have wide applicability in studying many other proteins involved in neuronal functions.

The localization and distribution of proteins provide important information for understanding their cellular functions. The superior spatial resolution of electron microscopy (EM) can be used to determine the subcellular localization of a given antigen following immunohistochemistry. For tissues of the central nervous system (CNS), preserving structural integrity while maintaining antigenicity has been especially difficult in EM studies. Here, we adopt a procedure that has been used to preserve structures and antigens in the CNS to study and characterize synaptic proteins in rat hippocampal CA1 pyramidal neurons.

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 ...

Improved Preparation and Preservation of Hippocampal Mouse Slices for a Very Stable and Reproducible Recording of Long-term Potentiation

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory encoding. LTP elicited in the CA1 region of acute hippocampal slices has been extensively studied. However the molecular mechanisms underlying the maintenance phase of this phenomenon are still poorly understood. This could be partly due to the various experimental conditions used by different laboratories. Indeed, the maintenance phase of LTP is strongly dependent on external parameters like oxygenation, temperature and humidity. It is also dependent on internal parameters like orientation of the slicing plane and slice viability after dissection.
The optimization of all these parameters enables the induction of a very reproducible and very stable long-term potentiation. This methodology offers the possibility to further explore the molecular mechanisms involved in the stable increase in synaptic strength in hippocampal slices. It also highlights the importance of experimental conditions in in vitro investigation of neurophysiological phenomena.

This paper presents a complete methodology to prepare and preserve in vitro acute hippocampal slices from adult mice. This protocol allows the recording of very stable long-lasting long-term potentiation (LTP) for more than 8 hours with a success rate of 95%.

Miglioramento della preparazione e la conservazione di fette mouse ippocampo per una registrazione molto stabile e riproducibile di potenziamento a lungo termine

Long-term potentiation (LTP) is a type of synaptic plasticity characterized by an increase in synaptic strength and believed to be involved in memory ...

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 ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic transmission during repetitive nerve firing. Impaired endocytosis in pathological conditions leads to decreases in synaptic strength and brain functions. Here, we describe methods used to measure synaptic vesicle endocytosis at the mammalian hippocampal synapse in neuronal culture. We monitored synaptic vesicle protein endocytosis by fusing a synaptic vesicular membrane protein, including synaptophysin and VAMP2/synaptobrevin, at the vesicular lumenal side, with pHluorin, a pH-sensitive green fluorescent protein that increases its fluorescence intensity as the pH increases. During exocytosis, vesicular lumen pH increases, whereas during endocytosis vesicular lumen pH is re-acidified. Thus, an increase of pHluorin fluorescence intensity indicates fusion, whereas a decrease indicates endocytosis of the labelled synaptic vesicle protein. In addition to using the pHluorin imaging method to record endocytosis, we monitored vesicular membrane endocytosis by electron microscopy (EM) measurements of Horseradish peroxidase (HRP) uptake by vesicles. Finally, we monitored the formation of nerve terminal membrane pits at various times after high potassium-induced depolarization. The time course of HRP uptake and membrane pit formation indicates the time course of endocytosis.

Synaptic vesicle endocytosis is detected by light microscopy of pHluorin fused with synaptic vesicle protein and by electron microscopy of vesicle uptake.

Endocitosi delle vescicole sinaptiche in colture di neuroni ippocampali di misura

During endocytosis, fused synaptic vesicles are retrieved at nerve terminals, allowing for vesicle recycling and thus the maintenance of synaptic ...

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 ...

Evaluation of Synapse Density in Hippocampal Rodent Brain Slices

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is tightly regulated during development and neuronal maturation. Importantly, deficits in synapse number can lead to cognitive dysfunction. Therefore, the evaluation of synapse number is an integral part of neurobiology. However, as synapses are small and highly compact in the intact brain, the assessment of absolute number is challenging. This protocol describes a method to easily identify and evaluate synapses in hippocampal rodent slices using immunofluorescence microscopy. It includes a three-step procedure to evaluate synapses in high-quality confocal microscopy images by analyzing the co-localization of pre- and postsynaptic proteins in hippocampal slices. It also explains how the analysis is performed and gives representative examples from both excitatory and inhibitory synapses. This protocol provides a solid foundation for the analysis of synapses and can be applied to any research investigating the structure and function of the brain.

A protocol for accurately identifying and analyzing synapses in hippocampal slices using immunofluorescence is outlined in this article.

Valutazione di densità di sinapsi nelle fette Hippocampal del cervello del roditore

In the brain, synapses are specialized junctions between neurons, determining the strength and spread of neuronal signaling. The number of synapses is ...