Questo lavoro è stato finanziato dal NIH sovvenzioni DA15169 e DA035224 di HEM

Tutti movimentazione degli animali e la raccolta dei tessuti è stata effettuata in conformità con le linee guida della University of Massachusetts Medical School Institutional Animal Care Comitato Usa (IACUC), seguendo il protocollo # approvato A1506 (Melikian, PI). Soluzioni necessarie Liquido cerebrospinale artificiale (ACSF) - Make fresco tutti i giorni 125 mM NaCl, 2,5 mM KCl, 1,2 mM NaH 2 PO 4, 1.2 mM MgCl 2, 2.4 mM CaCl 2, 26 NaHCO mm 3, e 11 mm di glucosio Nota: Preparare ACSF come una soluzione madre 10x, esclusi NaHCO 3 e glucosio. Rendere soluzioni di lavoro 1x ogni giorno dal magazzino 10x, che completa con fresco NaHCO 3 e glucosio. Saccarosio-integrato ACSF (SACSF) - Make fresco tutti i giorni 250 suc mMrosa, 2.5 mM KCl, 1,2 mM NaH 2 PO 4, 1.2 mM MgCl 2, 2.4 mM CaCl 2, 26 NaHCO mm 3, e glucosio 11 mM Nota: Preparare SACSF come una soluzione madre 10x, esclusi NaHCO 3 e glucosio. Rendere soluzioni di lavoro 1x ogni giorno dal magazzino 10x, che completa con fresco NaHCO 3 e glucosio. Solfo-N-hydroxysuccinyl-SS-biotina (solfo-NHS-SS-biotina, Pierce Chemical Company) Le soluzioni madre dovrebbero essere 200 mg / ml in DMSO e sono resistenti a molteplici cicli di congelamento / scongelamento. Aliquote vengono conservati a -20 ° C. L&#39;estere succinil viene rapidamente idrolizzato in soluzione acquosa, quindi le soluzioni di lavoro deve essere preparata immediatamente prima di applicare a fette. Slice Quench Solution ACSF integrato con la glicina 100 mM RIPA Lysis Buffer Tris 10 mM, pH 7,4, 150 mM NaCl, 1,0 mM EDTA, 1% Triton-X-100, 0,1% SDS, 1% Na desossicolato RIPA con inibitori della proteasi (RIPA / PI) - Fare fresco tutti i giorni RIPA integrato con 1 mM leupeptina, 1 mM pepstatina, 1 micron aprotinin, e 1 mm phenylmethyl fluoruro sulfonyl. 1. Preparare cervello Fette <ol><li> Fai fresco SACSF 1x e 1x ACSF </li><li> Raffreddare SACSF sul ghiaccio in un piccolo bicchiere. Questo verrà utilizzato per contenere il cervello di topo appena raccolto. </li><li> Saturare il ACSF e SACSF con l&#39;ossigeno facendo gorgogliare con il 95% / 5% O 2 / CO 2, 20 min in ghiaccio. </li><li> P30-38 topi dovrebbero essere utilizzati per la vitalità del tessuto ottimale. Sacrificare animali per dislocazione cervicale e decapitazione e rapidamente rimuovere i cervelli in prechilled, SACSF ossigenato. </li><li> Utilizzando un microtomo vibrante, fare 300 sezioni di cervello micron nella regione di interesse. </li><li> Se lo si desidera, le fette possono essere ulteriormente sezionati prima del recupero per arricchire di particolari regioni del cervello o di diritto separato e emisferi sinistro da utilizzare come controllo e fette sperimentali, rispettivamente. </li><li> Utilizzando un incendio lucidato pipetta Pasteur, trasferire fette di maglia a fondo le camere fissati in piastre da 24 pozzetti. </li><li> Lasciare le fette di recuperare per 40 min, 31 ° C in ACSF ossigenato, con continui, gentile bubbling. </li></ol> 2. Trattamento farmacologico (se del caso) e Slice biotinilazione <ol><li> Una volta recuperato, lavare fette 3x in preriscaldata (37 ° C), ossigenato ACSF gorgogliamento costantemente con il 95% / 5% O 2 / CO 2. </li><li> Aggiungere composti di prova e incubare con l&#39;ossigenazione continua. <ol><li> Per comodità, aggiungere un secolo volume di 10x farmaco concentrato 1/10, e miscelare capovolgendo delicatamente. </li><li> Agitare delicatamente le piastre in un bagno di acqua alla temperatura desiderata. </li></ol></li><li> Dopo il trattamento farmaco, rapidamentefette freddo di lavaggio 3x in ghiaccio ACSF freddo. </li></ol> 3. Proteine ​​di superficie Biotinylate <ol><li> Preparare 1,0 mg / ml solfo-NHS-SS-biotina in ghiaccio ACSF fredda immediatamente prima dell&#39;etichettatura. </li><li> Aggiungere 0,75 ml sulfo-NHS-SS-biotina a fette e fette di incubare in ghiaccio, 45 min. </li><li> Lavare le fette tre volte velocemente con ghiaccio freddo ACSF, poi incubare per 10 min in ghiaccio ACSF freddo sul ghiaccio. </li><li> Lavare le fette tre volte con ghiaccio freddo tampone fetta quench e incubare con 0,75 ml fetta tampone di tempra due volte, 25 min, sul ghiaccio per placare la connessione solfo-NHS-SS-biotina. </li></ol> 4. Preparare lisati di tessuto <ol><li> Lavare le fette tre volte in ghiaccio ACSF freddo e trasferire ogni fetta in una provetta con una pipetta Pasteur fuoco lucido. </li><li> Pellet delicatamente fetta mediante centrifugazione a 200 xg, 1 min e con attenzione aspirare restante ACSF. </li><li> Aggiungere 400 ml di ghiaccio freddo RIPA / PI e rompere il tessuto pipettando su e giù una volta attraverso un pip P200ette. </li><li> Trasferimento dissociato fetta / RIPA in una nuova provetta e incubare 30 min, 4 ° C, rotazione, per completare lisi. </li><li> Pellet detriti cellulari mediante centrifugazione, 18.000 xg, 15 min, 4 ° C. </li><li> Determinare concentrazioni di proteine ​​lisato usando il saggio proteico BCA, con albumina di siero bovino (BSA) come standard. </li></ol> 5. Isolare Biotinylated Proteine <ol><li> Ottimizzare Bead / totale rapporto proteine. Nota: I singoli livelli di espressione della proteina può variare notevolmente tra le diverse regioni del cervello. A meno che tutte le proteine ​​biotinilato in una data quantità di lisato tissutale è stata ripresa, non è possibile rilevare con precisione i potenziali cambiamenti nell&#39;espressione superficie. Pertanto, è imperativo determinare empiricamente la ottimale tallone / rapporto proteine ​​totali per una particolare regione proteina / cervello prima di intraprendere un nuovo studio fetta biotinylation. <ol><li> Incubare 25 microlitri streptavidina agarosio perline con crescente quantitàs di lisato tissutale (intervallo di 25-200 mg), e poi procedere come descritto di seguito per l&#39;associazione, lavaggio e eluizione passi. </li><li> Quantificare le bande immunoreattive conseguenti e scegliere un rapporto tallone / lisato nel range lineare del legame che permetterà quantificazione accurata di una aumentata o diminuita espressione di superficie proteica. </li></ol></li><li> Preparare Beads streptavidina-agarosio <ol><li> Determinare il volume totale cordone necessario per tutti i campioni. Preparare un volume di cordone sufficiente per tale importo più uno supplementare (cioè 4 campioni a 25 ml / campione = 100 perline microlitri + uno supplementare = 125 perline microlitri. </li><li> Vortex streptavidina tallone stock e pipetta fuori desiderato volume. Se si utilizza una pipetta P200, tagliare l&#39;estremità della punta per evitare il blocco o il tallone danni. </li><li> Lavare perle 3 volte in 0,5-1,0 ml RIPA / PI per rimuovere i conservanti, vortex dopo ogni aggiunta RIPA e la raccolta di perle tra un lavaggio mediante centrifugazione 18.000 xg, 1 min, a temperatura ambiente temperamentoatura. </li><li> Aspirare il tampone tra un lavaggio con una pipetta Pasteur di vetro collegato a un pallone di vuoto. Un P200 punta di plastica attaccato alla fine della pipetta Pasteur di vetro offre un controllo più preciso durante l&#39;aspirazione. Non è necessario rimuovere assolutamente tutto il buffer per i primi due lavaggi, in quanto rischia di perline di aspirazione nella pipetta. Dopo l&#39;ultimo lavaggio, rimuovere quanto più il tampone in eccesso possibile senza aspirare le perline. </li><li> Aggiungi RIPA / PI per portare perle al loro volume originario, pipettare su e giù per risospendere. Evitare vortex, a questo punto, come perle si attaccano alla parete del tubo. </li><li> Perline aliquote in provette da microcentrifuga utilizzando un P200 con la punta tagliata. Pipetta su e giù parecchie volte tra il campionamento per assicurare perle rimangono uniformemente sospese e disperse tra i tubi. </li></ol></li><li> Proteine ​​biotinilate legarsi a streptavidina perline <ol><li> Distribuire lisati cellulari ai tubi contenenti le perline agarosio. Per gli esperimenti whe<html> re più campioni vengono confrontati, assicurarsi di utilizzare la stessa quantità di proteine ​​per ogni campione per confronti accurati. </li><li> Aggiungere ulteriori RIPA / PI per portare a campioni ad un volume di 200 microlitri minimo. Questo assicura che i campioni si mescoleranno in modo adeguato durante l&#39;incubazione e anche unifica le concentrazioni di proteine ​​di tutti i campioni. </li><li> Porre i tubi in un rotatore provetta e miscelare notte a 4 ° C. </li><li> In tubi separati, dispensare un equivalente del volume totale lisato utilizzato per ogni campione. In alternativa, se i volumi di campione sono alti, una frazione del volume totale lisato può essere riservato invece, per accogliere volumi carico massimo su gel SDS-PAGE. Questi saranno utilizzati per normalizzare l&#39;espressione di superficie alla quantità totale di proteine. </li><li> Aggiungere sia un uguale volume di 2x o 1/5 volume di 6x tampone campione SDS-PAGE e sia incubare a 4 ° C, in parallelo con i campioni di perline o conservare a -20 ° C fino vengono analizzati campioni. </li></ol></li></ol>ove_title &quot;&gt; 6. Elute e analizzare campioni <ol><li> Pellet perline mediante centrifugazione 18.000 xg, 2 min, a temperatura ambiente. </li><li> Aspirare il surnatante e lavare perline tre volte con 0,75 ml RIPA. Per ridurre al minimo la perdita di tallone, lasciare un piccolo volume di testa di tampone sopra perline tra i lavaggi. Dopo l&#39;ultimo lavaggio, rimuovere il più RIPA possibile, senza disturbare il pellet tallone. </li><li> Eluire le proteine ​​biotinilate da perline streptavidina riducendo il legame disolfuro. Aggiungere 25 microlitri 2x SDS-PAGE riduzione tampone del campione, vortice bene e ruotare campioni di 30 minuti, a temperatura ambiente. Nota: Molte proteine ​​di membrana hanno una forte tendenza ad aggregarsi quando bollito in tampone campione SDS-PAGE, gravemente compromettere la loro mobilità elettroforetica. Se questo è il caso per la proteina indagato, evitare riscaldamento dei campioni e, invece, eluire lentamente a temperatura ambiente. Se bollente è assolutamente necessario (ad esempio, se un&#39;altra proteina valutati in parallelo richiede bollente), sample buffer può essere completato con 2 M urea (concentrazione finale) per ridurre al minimo l&#39;aggregazione. Mentre efficace nel ridurre l&#39;aggregazione in alcuni casi, urea compromette spesso apparenze banda. </li><li> Analizzare i campioni immunoblot. </li><li> Scongelare campioni totali lisato e ruotare in parallelo con i campioni del branello, 30 min, temperatura ambiente. </li><li> Proteine ​​separate su gel SDS-PAGE. </li><li> Identificare proteine ​​(s) di interesse mediante immunoblotting. <ol><li> Essere certi che le bande vengono rilevati nell&#39;intervallo lineare di rilevamento per una corretta quantificazione. </li><li> Per assicurare che il reagente biotinilazione non ha avuto accesso alle proteine ​​intracellulari via le cellule danneggiate / compromessi. Questo si realizza meglio mediante immunoblotting in parallelo per una proteina intracellulare specifiche per il tipo di cellule in fase di studio. </li><li> Quantificare densità band e calcolare la proteina relativa densità di superficie come percentuale del livello di espressione della proteina totale. </li></ol></li></ol>

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Gli autori non hanno interessi economici in conflitto con questo lavoro.

Nonostante la conoscenza di lunga data che il traffico endocytic criticamente l&#39;impatto segnalazione sinaptica nel cervello, si è dimostrato impegnativo per misurare quantitativamente cambiamenti nell&#39;espressione superficie della proteina nei neuroni adulti. In questo lavoro, riportiamo un metodo affidabile per etichettare proteina di superficie ex vivo in fettine cerebrali acute. Preparazioni fetta cervello hanno una lunga storia di utilità per le registrazioni elettrofisiologiche, in quanto mantengo...

Traffico endocitico è un meccanismo cellulare ubiquitario che perfeziona la presentazione membrana plasmatica di una varietà di proteine ​​integrali di membrana. Endocitosi fornisce nutrienti vitali per l&#39;ambiente intracellulare 1 e desensibilizza segnalazione dei recettori in risposta all&#39;attivazione del recettore 2. Riciclo indietro alla membrana plasmatica endocitosi può inoltre migliorare segnalazione cellulare aumentando i livelli di espressione della proteina sulla superficie cellulare 3. Inoltre, perturbazioni traffico di membrana sono implicati in numerose malattie e condizioni patologiche 4,5, sottolineando la necessità di studiare i meccanismi molecolari che governano la proteina traffico endocitico. Mentre molte proteine ​​utilizzano meccanismi di internalizzazione classici clatrina-dipendente, prova di montaggio nel corso degli ultimi anni dimostra che più i meccanismi di endocitosi clatrina-indipendente governano il potenziale endocitica di una serie crescente diproteine ​​6,7. Quindi, la necessità di indagare i meccanismi endocitiche agevolare il traffico nei sistemi pertinenti fisiologici è cresciuta notevolmente. Nel cervello, il traffico endocitico dei recettori, canali ionici e trasportatori di neurotrasmettitori ha un ruolo primario nella creazione di plasticità sinaptica 8-11 e risposta ai farmaci d&#39;abuso 12-15, in ultima analisi, un impatto eccitabilità neuronale e risposte sinaptiche. Ad oggi la maggior parte degli studi di traffico neuronali contare su entrambi i sistemi di espressione eterologa o neuroni primari in coltura, nessuno dei quali può riflettere in maniera affidabile i meccanismi in gioco in neuroni adulti. Qui riportiamo un approccio che utilizza biotinilazione superficie per misurare quantitativamente i livelli di proteina di superficie in fettine cerebrali acute derivanti da roditori adulti. Usando questo approccio, presentiamo i dati che dimostrano che il topo striato trasportatore della dopamina interiorizza rapidamente in rerisposta a forbolo estere mediata proteina chinasi C (PKC) attivazione.

<table border="0" cellpadding="0" cellspacing="0" width="682">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">sulfo NHS-SS-biotin</td>
			<td style="width:83px;">Pierce</td>
			<td style="width:119px;">21331</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Streptavidin agarose</td>
			<td style="width:83px;">Pierce</td>
			<td>20347</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">IgG-free, Protease-free Bovine serum albumin</td>
			<td style="width:83px;">Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Vibrating microtome sectioner</td>
			<td style="width:83px;">Various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:315px;">Shaking water bath</td>
			<td style="width:83px;">various</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:315px;">Milli-cell mesh-bottomed inserts (8&#181;m pore size)</td>
			<td style="width:83px;">Millipore</td>
			<td style="width:119px;">PI8P 012 50</td>
			<td>These can be washed by hand and re-used</td>
		</tr>
	</tbody>
</table>

brain slice biotinylation: an ex vivo approach to measure region-specific plasma membrane protein trafficking in adult neurons

Traffico endocytic regolamentata è il meccanismo centrale facilitando una serie di eventi neuromodulatori, controllando dinamicamente recettore, canale ionico, e la cella presentazione superficie trasportatore su una scala minuti di tempo. C&#39;è una vasta diversità di meccanismi che controllano il traffico endocitico delle singole proteine. Gli studi che indagano le basi molecolari del traffico hanno fatto affidamento principalmente sulla biotinilazione superficie per misurare quantitativamente cambiamenti nell&#39;espressione superficiale proteina di membrana in risposta a stimoli esogeni e la manipolazione genetica. Tuttavia, questo approccio è stato limitato principalmente alle cellule coltivate, che potrebbero non riflettere fedelmente i meccanismi fisiologicamente rilevanti in gioco in neuroni adulti. Inoltre, gli approcci di cellule in coltura possono sottovalutare le differenze regionali specifici meccanismi di traffico. Qui, descriviamo un approccio che si estende biotinylation superficie cellulare per la preparazione fetta acuta cervello. Noidimostrano che questo metodo fornisce un approccio ad alta fedeltà per misurare rapidi cambiamenti nei livelli superficiali delle proteine ​​di membrana in neuroni adulti. Questo approccio rischia di avere un ampio programma di utilità nel campo del traffico endocytic neuronale.

Il trasportatore della dopamina neuronale è internalizzato in risposta all&#39;attivazione PKC in linee cellulari 16-20. Nonostante le molte relazioni che dimostrino la PKC-indotte perdite di superficie DAT in una varietà di linee cellulari e sistemi di espressione, è stato impegnativo per confermare questo dato nei neuroni dopaminergici in coltura 21-23. Abbiamo usato fettine striatali mouse per testare direttamente se DAT interiorizza in risposta all&#39;attivazione PKC nei neuroni dopaminergi...

Watch this Scientific Journal Video about Brain Slice Biotinylation: An Ex Vivo Approach to Measure Region-specific Plasma Membrane Protein Trafficking in Adult Neurons at JoVE.com

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

Traffico di membrana neuronale controlla dinamicamente la membrana plasmatica disponibilità di proteine ​​e gli impatti in modo significativo la neurotrasmissione. Ad oggi, è stato impegnativo per misurare neuronale traffico endocitico in neuroni adulti. Qui, descriviamo un metodo quantitativo altamente efficace per misurare rapidi cambiamenti nell&#39;espressione della proteina superficie ex vivo in fettine cerebrali acute.

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 ...

brain-slice-biotinylation-an-ex-vivo-approach-to-measure-region

Research

JoVE Journal

Neuroscience

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

12.8K Views.  University of Massachusetts Medical School.  Traffico di membrana neuronale controlla dinamicamente la membrana plasmatica disponibilità di proteine ​​e gli impatti in modo significativo la neurotrasmissione. Ad oggi, è stato impegnativo per misurare neuronale traffico endocitico in neuroni adulti. Qui, descriviamo un metodo quantitativo altamente efficace per misurare rapidi cambiamenti nell'espressione della proteina superficie ex vivo in fettine cerebrali acute. 

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

Quantitative Analysis of Synaptic Vesicle Pool Replenishment in Cultured Cerebellar Granule Neurons using FM Dyes

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis. Retrieved SVs are then refilled with neurotransmitter and rejoin the recycling pool, defined as SVs that are available for exocytosis1,2. The recycling pool can generally be subdivided into two distinct pools - the readily releasable pool (RRP) and the reserve pool (RP). As their names imply, the RRP consists of SVs that are immediately available for fusion while RP SVs are released only during intense stimulation1,2. It is important to have a reliable assay that reports the differential replenishment of these SV pools in order to understand 1) how SVs traffic after different modes of endocytosis (such as clathrin-dependent endocytosis and activity-dependent bulk endocytosis) and 2) the mechanisms controlling the mobilisation of both the RRP and RP in response to different stimuli.
FM dyes are routinely employed to quantitatively report SV turnover in central nerve terminals3-8. They have a hydrophobic hydrocarbon tail that allows reversible partitioning in the lipid bilayer, and a hydrophilic head group that blocks passage across membranes. The dyes have little fluorescence in aqueous solution, but their quantum yield increases dramatically when partitioned in membrane9. Thus FM dyes are ideal fluorescent probes for tracking actively recycling SVs. The standard protocol for use of FM dye is as follows. First they are applied to neurons and are taken up during endocytosis (Figure 1). After non-internalised dye is washed away from the plasma membrane, recycled SVs redistribute within the recycling pool. These SVs are then depleted using unloading stimuli (Figure 1). Since FM dye labelling of SVs is quantal10, the resulting fluorescence drop is proportional to the amount of vesicles released. Thus, the recycling and fusion of SVs generated from the previous round of endocytosis can be reliably quantified.
Here, we present a protocol that has been modified to obtain two additional elements of information. Firstly, sequential unloading stimuli are used to differentially unload the RRP and the RP, to allow quantification of the replenishment of specific SV pools. Secondly, each nerve terminal undergoes the protocol twice. Thus, the response of the same nerve terminal at S1 can be compared against the presence of a test substance at phase S2 (Figure 2), providing an internal control. This is important, since the extent of SV recycling across different nerve terminals is highly variable11.
Any adherent primary neuronal cultures may be used for this protocol, however the plating density, solutions and stimulation conditions are optimised for cerebellar granule neurons (CGNs)12,13.

A live fluorescence imaging technique to quantify the replenishment and mobilisation of specific synaptic vesicle (SV) pools in central nerve terminals is described. Two rounds of SV recycling are monitored in the same nerve terminals providing an internal control.

Analisi quantitativa di Replenishment pool delle vescicole sinaptiche in colture di neuroni granuli cerebellari utilizzando coloranti FM

After neurotransmitter release in central nerve terminals, SVs are rapidly retrieved by endocytosis.  Retrieved SVs are then refilled with ...

Ricerca

Neuroscienze

Imaging pHluorin-tagged Receptor Insertion to the Plasma Membrane in Primary Cultured Mouse Neurons

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an experimental approach with excellent spatial and temporal resolutions. Moreover, such an approach must also have the ability to distinguish receptors localized on the PM from those in intracellular compartments. Most importantly, detecting receptors in a single vesicle requires outstanding detection sensitivity, since each vesicle carries only a small number of receptors. Standard approaches for examining receptor trafficking include surface biotinylation followed by biochemical detection, which lacks both the necessary spatial and temporal resolutions; and fluorescence microscopy examination of immunolabeled surface receptors, which requires chemical fixation of cells and therefore lacks sufficient temporal resolution1-6 . To overcome these limitations, we and others have developed and employed a new strategy that enables visualization of the dynamic insertion of receptors into the PM with excellent spatial and temporal resolutions 7-17 . The approach includes tagging of a pH-sensitive GFP, the superecliptic pHluorin 18, to the N-terminal extracellular domain of the receptors. Superecliptic pHluorin has the unique property of being fluorescent at neutral pH and non-fluorescent at acidic pH (pH &lt; 6.0). Therefore, the tagged receptors are non-fluorescent when within the acidic lumen of intracellular trafficking vesicles or endosomal compartments, and they become readily visualized only when exposed to the extracellular neutral pH environment, on the outer surface of the PM. Our strategy consequently allows us to distinguish PM surface receptors from those within intracellular trafficking vesicles. To attain sufficient spatial and temporal resolutions, as well as the sensitivity required to study dynamic trafficking of receptors, we employed total internal reflection fluorescent microscopy (TIRFM), which enabled us to achieve the optimal spatial resolution of optical imaging (~170 nm), the temporal resolution of video-rate microscopy (30 frames/sec), and the sensitivity to detect fluorescence of a single GFP molecule. By imaging pHluorin-tagged receptors under TIRFM, we were able to directly visualize individual receptor insertion events into the PM in cultured neurons. This imaging approach can potentially be applied to any membrane protein with an extracellular domain that could be labeled with superecliptic pHluorin, and will allow dissection of the key detailed mechanisms governing insertion of different membrane proteins (receptors, ion channels, transporters, etc.) to the PM.

By tagging the extracellular domains of membrane receptors with superecliptic pHluorin, and by imaging these fusion receptors in cultured mouse neurons, we can directly visualize individual vesicular insertion events of the receptors to the plasma membrane. This technique will be instrumental in elucidating the molecular mechanisms governing receptor insertion to the plasma membrane.

Imaging pHluorin-tagged inserimento recettore alla membrana plasmatica nella Primaria neuroni mouse coltivate

A better understanding of the mechanisms governing receptor trafficking between the plasma membrane (PM) and intracellular compartments requires an ...

Using an &#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent &#945;-bungarotoxin with cells expressing constructs containing an &#945;-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the &#945; subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3&#39; of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an &amp;#945;-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

Here we demonstrate the use of fluorescent Alexa dye coupled to &#945;-bungarotoxin to measure GABA A receptor surface localization and endocytosis in hippocampal neurons. Through the use of constructs bearing a short extracellular tag that binds &#945;-bungarotoxin, analysis of plasma membrane protein endocytic trafficking can be achieved.

L&#39;utilizzo di un α-bungarotossina Binding Tag sito per studiare GABA un recettore di membrana localizzazione e il traffico

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell ...

Slice It Hot: Acute Adult Brain Slicing in Physiological Temperature

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments that largely determines the quality of results. It has been shown that omitting the cooling step during cutting procedure is beneficial in obtaining healthy slices and cells, especially when dealing with highly myelinated brain structures from mature animals. Even though the precise mechanism whereby elevated temperature supports neural health can only be speculated upon, it stands to reason that, whenever possible, the temperature in which the slicing is performed should be close to physiological conditions to prevent temperature related artifacts. Another important advantage of this method is the simplicity of the procedure and therefore the short preparation time. In the demonstrated method adult mice are used but the same procedure can be applied with younger mice as well as rats. Also, the following patch clamp experiment is performed on horizontal cerebellar slices, but the same procedure can also be used in other planes as well as other posterior areas of the brain.

In this paper we show a method for preparing acute brain slices in physiological temperature, using a conventional physiological solution without special modifications for the cutting (such as adding sucrose) and without intracardial perfusion of the animal before slice preparation.

Slice It Hot: acuta cervello adulto affettare in fisiologica Temperatura

Here we present a protocol for preparation of acute brain slices. This procedure is a critical element for electrophysiological patch-clamp experiments ...

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal degeneration. Lack of growth cone formation, regeneration, and loss of additional myelinated axonal projections within the spinal cord greatly limits neurological recovery following injury. To assess how central myelinated axons of the spinal cord respond to injury, we developed an ex vivo living spinal cord model utilizing transgenic mice that express yellow fluorescent protein in axons and a focal and highly reproducible laser-induced spinal cord injury to document the fate of axons and myelin (lipophilic fluorescent dye Nile Red) over time using two-photon excitation time-lapse microscopy. Dynamic processes such as acute axonal injury, axonal retraction, and myelin degeneration are best studied in real-time. However, the non-focal nature of contusion-based injuries and movement artifacts encountered during in vivo spinal cord imaging make differentiating primary and secondary axonal injury responses using high resolution microscopy challenging. The ex vivo spinal cord model described here mimics several aspects of clinically relevant contusion/compression-induced axonal pathologies including axonal swelling, spheroid formation, axonal transection, and peri-axonal swelling providing a useful model to study these dynamic processes in real-time. Major advantages of this model are excellent spatiotemporal resolution that allows differentiation between the primary insult that directly injures axons and secondary injury mechanisms; controlled infusion of reagents directly to the perfusate bathing the cord; precise alterations of the environmental milieu (e.g., calcium, sodium ions, known contributors to axonal injury, but near impossible to manipulate in vivo); and murine models also offer an advantage as they provide an opportunity to visualize and manipulate genetically identified cell populations and subcellular structures. Here, we describe how to isolate and image the living spinal cord from mice to capture dynamics of acute axonal injury.

An Ex Vivo Laser-induced Spinal Cord Injury Model to Assess Mechanisms of Axonal Degeneration in Real-time

We present a protocol utilizing two-photon excitation time-lapse microscopy to simultaneously visualize the dynamics of axon and myelin injuries in real time. This proposed protocol permits studies of both intrinsic and extrinsic factors which can influence central myelinated axon fate after injury and contribute to permanent clinical disability.

Un<em&gt; Ex Vivo</em&gt; Laser-indotta Spinal Cord Injury modello per valutare i meccanismi di degenerazione assonale in tempo reale

Injured CNS axons fail to regenerate and often retract away from the injury site. Axons spared from the initial injury may later undergo secondary axonal ...

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated proteins and subsequent manipulation of neural activity by light. Channelrhodopsins (ChRs) are light-gated cation-channels and when fused to a fluorescent protein their expression allows for visualization and concurrent activation of specific cell types and their axonal projections in defined areas of the brain. Via stereotactic injection of viral vectors, ChR fusion proteins can be constitutively or conditionally expressed in specific cells of a defined brain region, and their axonal projections can subsequently be studied anatomically and functionally via ex vivo optogenetic activation in brain slices. This is of particular importance when aiming to understand synaptic properties of connections that could not be addressed with conventional electrical stimulation approaches, or in identifying novel afferent and efferent connectivity that was previously poorly understood. Here, a few examples illustrate how this technique can be applied to investigate these questions to elucidating fear-related circuits in the amygdala. The amygdala is a key region for acquisition and expression of fear, and storage of fear and emotional memories. Many lines of evidence suggest that the medial prefrontal cortex (mPFC) participates in different aspects of fear acquisition and extinction, but its precise connectivity with the amygdala is just starting to be understood. First, it is shown how ex vivo optogenetic activation can be used to study aspects of synaptic communication between mPFC afferents and target cells in the basolateral amygdala (BLA). Furthermore, it is illustrated how this ex vivo optogenetic approach can be applied to assess novel connectivity patterns using a group of GABAergic neurons in the amygdala, the paracapsular intercalated cell cluster (mpITC), as an example.

Ex Vivo Optogenetic Dissection of Fear Circuits in Brain Slices

Optogenetic approaches are widely used to manipulate neural activity and assess the consequences for brain function. Here, a technique is outlined that&#160;upon in vivo expression of the optical activator Channelrhodopsin, allows for ex vivo analysis of synaptic properties of specific long range and local neural connections in fear-related circuits.

Ex Vivo optogenetic dissezione di circuiti paura nel cervello fette

Optogenetic approaches are now widely used to study the function of neural populations and circuits by combining targeted expression of light-activated ...

Live Images of GLUT4 Protein Trafficking in Mouse Primary Hypothalamic Neurons Using Deconvolution Microscopy

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNF&#945;, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear.
In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.

This protocol describes a technique for observation of real-time Green Fluorescence Protein (GFP) tagged Glucose Transporter 4 (GLUT4) protein trafficking upon insulin stimulation and characterization of the biological role of CCR5 in the insulin&#8211;GLUT4 signaling pathway with Deconvolution Microscopy.

Immagini in diretta della proteina GLUT4 tratta di neuroni ipotalamici primario del Mouse usando la microscopia di Deconvolution

Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral ...

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

Despite&#160;numerous studies that attempt to develop reliable animal models&#160;which reflecting the primary processes underlying neurodegeneration, very few have been widely accepted. Here, we propose&#160;a new procedure&#160;adapted from the well-known ex vivo brain slice technique, which offers a closer in vivo-like scenario than in vitro preparations, for investigating the early events triggering cell degeneration, as observed in Alzheimer&#39;s disease (AD). This variation consists of simple and easily reproducible steps, which enable preservation of the anatomical cytoarchitecture of the selected brain region and its local functionality in a physiological milieu. Different anatomical areas can be obtained from the same brain, providing the opportunity to perform multiple experiments with the treatments in question in a site-, dose-, and time-dependent manner. Potential limitations&#160;which could affect the outcomes related to this methodology are related to the conservation of the tissue, i.e., the maintenance of its anatomical integrity during the slicing and incubation steps and the section thickness, which can influence the biochemical and immunohistochemical analysis. This approach can be employed for different purposes, such as exploring molecular mechanisms involved in physiological or pathological conditions, drug screening, or dose-response assays. Finally, this protocol could also reduce the number of animals employed in behavioral studies. The application reported here has been recently described and tested for the first time on ex vivo rat brain slices containing the basal forebrain (BF), which is one of the cerebral regions primarily affected in AD. Specifically, it has been demonstrated that the administration of a toxic peptide derived from the C-terminus of acetylcholinesterase (AChE) could prompt an AD-like profile, triggering, along the antero-posterior axis of the BF, a differential expression of proteins altered in AD, such as the alpha7 nicotinic receptor (&#945;7-nAChR), phosphorylated Tau (p-Tau), and amyloid beta (A&#946;).

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

We present a method which can&#160;provide further insights into the early events underlying neurodegeneration and based on the established ex-vivo brain technique, combining the advantages of in vivo and in vitro experiments. Moreover, it represents a unique opportunity for direct comparison of treated and untreated group in the same anatomical plane.

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, ...

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model animal for endocrine research, Drosophila melanogaster, which has sophisticated genetic tools and genome information, is being increasingly used. This article describes a method for the calcium imaging of Drosophila brain explants. This method enables the detection of the direct signaling of a hormone to the brain. It is well known that many peptide hormones act through G-protein-coupled receptors (GPCRs), whose activation causes an increase in the intracellular Ca2+concentration. Neural activation also elevates intracellular Ca2+ levels, from both Ca2+ influx and the release of Ca2+ stored in the endoplasmic reticulum (ER). A calcium sensor, GCaMP, can monitor these Ca2+ changes. In this method, GCaMP is expressed in the neurons of interest, and the GCaMP-expressing larval brain is dissected and cultured ex vivo. The test peptide is then applied to the brain explant, and the fluorescent changes in GCaMP are detected using a spinning disc confocal microscope equipped with a CCD camera. Using this method, any water-soluble molecule can be tested, and various cellular events associated with neural activation can be imaged using the appropriate fluorescent indicators. Moreover, by modifying the imaging chamber, this method can be used to image other Drosophila organs or the organs of other animals.

Ex Vivo Calcium Imaging for Visualizing Brain Responses to Endocrine Signaling in Drosophila

This paper describes a protocol for ex vivo calcium imaging of the Drosophila brain. In this method, natural or synthetic compounds can be applied to the buffer to test their ability to activate particular neurons in the brain.

Ex Vivo Calcio Imaging per visualizzare le risposte del cervello alla segnalazione endocrina in Drosophila

Organ-to-organ communication by endocrine signaling, for example, from the periphery to the brain, is essential for maintaining homeostasis. As a model ...

Using Near-infrared Fluorescence and High-resolution Scanning to Measure Protein Expression in the Rodent Brain

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study of neuroscience as cellular function is determined by the composition and activity of cellular proteins. In this article, we describe how immunocytochemistry can be combined with near-infrared high-resolution scanning to provide a semi-quantitative measure of protein expression in distinct brain regions. This technique can be used for single or double protein expression in the same brain region. Measuring proteins in this fashion can be used to obtain a relative change in protein expression with an experimental manipulation, molecular signature of learning and memory, activity in molecular pathways, and neural activity in multiple brain regions. Using the correct proteins and statistical analysis, functional connectivity among brain regions can be determined as well. Given the ease of implementing immunocytochemistry in a laboratory, using immunocytochemistry with near-infrared high-resolution scanning can expand the ability of the neuroscientist to examine neurobiological processes at a systems level.

Here, we present a protocol that uses near-infrared dyes in conjunction with immunohistochemistry and high-resolution scanning to assay proteins in brain regions.

Utilizzo della fluorescenza Near-Infrared e scansione ad alta risoluzione per misurare l'espressione proteica nel cervello del roditore

Neuroscience is the study of how cells in the brain mediate various functions. Measuring protein expression in neurons and glia is critical for the study ...