The authors thank Dr. Mike Ehlers and his laboratory members for originally demonstrating the protocol for subcellular fractionation of synaptic plasma membranes. We also thank Wendy Horsfall for management of the mouse colonies. This work was supported by operating grants from CIHR (AJR and AS).

Il seguente protocollo è conforme alle linee guida del Consiglio canadese della cura degli animali ed è stato approvato dalla Facoltà di Medicina e Farmacia Comitato cura degli animali presso l&#39;Università di Toronto. 1 Preparare Obbligatorio reagenti come descritto nella Tabella 1 <ol><li> Aggiungi proteasi e fosfatasi (se necessario) inibitori a tutte le soluzioni di saccarosio, tamponi e DDH 2 O a concentrazioni indicate nella tabella 1. Importante: eseguire tutte le operazioni a 4 ° C e pre-raffreddare tutti i reagenti e apparecchiature prima dell&#39;inizio dell&#39;esperimento. Etichettare tutte le provette, compresi i tubi ultracentrifuga, con pennarello indelebile. </li></ol> 2 Dissezione della regione del cervello appropriata <ol><li> Sacrifica l&#39;animale da dislocazione cervicale o decapitazione. Casa e eutanasia degli animali in conformità con le politiche istituzionali e governativi sulla cura degli animali. </li><li> Rapidamente rimuovere the cervello dal cranio e posto su un blocco dissezione refrigerata. </li><li> Sezionare la regione del cervello appropriata dal tessuto fresco e procedere al passaggio 3. </li></ol> 3 Tissue omogeneizzazione con Motor Driven vetro-teflon Omogeneizzatore <ol><li> Posizionare 50-100 mg (per SPM), o 100-200 mg (per PSD) del tessuto sezionato in una etichetta 13 ml tubo di polipropilene contenente 4 ml di soluzione di saccarosio 0.32 M HEPES-buffered. Trasferire il campione in un 15 ml conica fibra di vetro-teflon smerigliatrice / omogeneizzatore, impostare l&#39;unità motore a 900 giri (impostazione 7) e omogeneizzare il campione con 12 colpi in un periodo di 30 sec. Utilizzare un diverso omogeneizzatore vetro-teflon tra i campioni, o sciacquare la omogeneizzatore con acqua distillata fredda tra i campioni e asciugare con un Kimwipe. NOTA: Fino a 4 g di tessuto può essere omogeneizzato in 4 ml di soluzione di saccarosio 0.32 M HEPES-buffered, tuttavia è consigliabile utilizzare meno tessuto per garantire il corretto frazionamento. </li><li> Trasferire il campione omogeneizzato posteriore to lo stesso tubo di polipropilene 13 ml. Riserva 100 ml di omogenato e conservare a -80 ° C per la successiva quantificazione di proteine ​​e analisi western blot della frazione totale di proteine. </li></ol> 4. bassa velocità di centrifugazione per eliminare completamente le frazioni nucleare (rendimenti supernatante S1) <ol><li> Centrifugare l&#39;omogeneizzato in un rotore ad angolo fisso a 900 xg per 10 min a 4 ° C. Trasferire il surnatante (S1) per un nuovo, marcato 13 ml di tubo e risospendere il pellet frazione nucleare (P1) in 500 ml di saccarosio 0,32 M HEPES-buffered. NOTA: La (P1) frazione può essere conservato a -80 ° C ed utilizzato per l&#39;analisi western blot della frazione nucleare. </li></ol> 5. arricchimento della Frazione Crude sinaptosomale (rendimenti a pellet P2) <ol><li> Centrifugare il surnatante (S1) a 10.000 xg per 15 min a 4 ° C. Rimuovere il surnatante (S2) e riserva 500 ml in una provetta per conservare a -80 ° C per la successiva proteina quantificazione e analisi western blot della frazione citosolica membrana / luce. Risospendere il pellet rimanente frazione sinaptosomale greggio (P2) in 1 ml di soluzione di saccarosio 0.32 M HEPES-buffered e poi aggiungere un&#39;altra soluzione di saccarosio 3 ml di 0,32 M HEPES-buffered. </li><li> Centrifugare a 10.000 x g per 15 min a 4 ° C. Rimuovere il surnatante (S2 &#39;) e riserva 500 ml in una provetta per conservare a -80 ° C per la successiva quantificazione di proteine ​​e analisi western blot della frazione citosolica membrana / luce. Salvare il lavato sinaptosomale greggio pellet (P2 &#39;) nel tubo di polipropilene. </li></ol> 6. lisi (ipoosmotico Shock) della Frazione Crude sinaptosomale <ol><li> Lyse il pellet sinaptosomale grezzo (P2 &#39;) nel tubo di polipropilene da risospendere in 1 ml di DDH 2 O. Aggiungi un altro 3 ml di DDH 2 O, trasferimento in un omogeneizzatore tessuto di vetro-teflon, e omogeneizzare a mano con 3 colpi. NOTA: E &#39;importanteper eseguire questo passaggio il più rapidamente possibile e rapidamente passare al punto 6.2. </li><li> Campioni di trasferimento dalla omogeneizzatore nuovamente dentro lo stesso tubo di polipropilene 13 ml e regolare rapidamente il campione torna a HEPES 4 mm con 16 ml di soluzione 1 M HEPES, miscelarlo. </li><li> Ruotare i campioni a 4 ° C per 30 minuti per garantire la completa lisi. </li></ol> 7 Arricchimento della Frazione sinaptosomale membrana (P3) <ol><li> Centrifugare a 25.000 x g per 20 minuti a 4 ° C. Rimuovere il surnatante grezzo frazione vescicolare (S3) e prenotare in una provetta 5 ml di conservare a -80 ° C per la successiva quantificazione delle proteine ​​e western blot. Risospendere la frazione di membrana sinaptosomale (P3) in 1 ml di soluzione di saccarosio 0.32 M HEPES-buffered. </li></ol> 8 Preparazione del Discontinuo gradiente di saccarosio <ol><li> Dispensare esattamente 3.5 ml di soluzione di saccarosio 1,2 M HEPES-buffered ed esattamente 3.0 ml di 1,0 M e 0,8 M HEPESoluzione di saccarosio S-buffered in separati 6 ml provette con tappo a scatto. NOTA: Questo viene fatto per pre-misurare i volumi dei buffer che vengono utilizzati per rendere il gradiente di saccarosio. Misurazione esatta dei volumi è importante per garantire che i gradienti risultanti saranno bilanciati per ultracentrifugazione. </li><li> Usando una pipetta Pasteur e bulbo di vetro, trasferire 1,2 M HEPES-buffered soluzione di saccarosio a 12 ml tubo ultracentrifuge polyallomer. Strato con attenzione la soluzione di saccarosio 1.0 M HEPES-tampone sulla parte superiore della soluzione di saccarosio 1.2 M HEPES-buffered. Infine, lo strato di soluzione di saccarosio 0,8 M HEPES-tampone sulla parte superiore della soluzione di saccarosio 1.0 M HEPES-buffered. Utilizzare una pipetta Pasteur fresco per ogni soluzione di saccarosio e fare attenzione a non disturbare il gradiente di saccarosio. Vibrazioni da un vortice panchina o microcentrifuga disturberà l&#39;integrità del gradiente. </li><li> Usando una pipetta Pasteur, sovrapporre la frazione di membrana sinaptosomale risospeso (P3) sulla parte superiore del preparato discontinuo gradiente di saccarosio.Assicurarsi che i gradienti sono bilanciate tramite pesatura all&#39;interno del secchio del rotore oscillante. Se i secchi non sono equilibrati, utilizzare una pipetta P200 aggiungere attentamente saccarosio 0,32 M HEPES-buffered all&#39;inizio della sfumatura e bilanciare i secchi rotore. </li></ol> 9 Frazionamento del Synaptic membrana plasmatica (SPM) <ol><li> Ultracentrifuge in un rotore oscillante secchio a 150.000 xg per 2 ore a 4 ° C. Rimuovere con attenzione i gradienti di saccarosio dai secchi ultracentrifuga. Utilizzando un ago da 18 G e una siringa da 1 ml, forare il tubo sul fondo della soluzione 1,0 M / 1,2 M HEPES-buffered saccarosio interfase e ritirare lo strato di membrana plasmatica sinaptica (SPM). </li><li> Prendere nota del volume che ogni campione SPM occupa nella siringa da 1 ml. Posizionare lo strato SPM raccolti in 3,5 ml tubi ultracentrifuga spessa parete, aggiungere esattamente 2,5 volumi di HEPES 4 mM per regolare la concentrazione di saccarosio da 1,2 M a 0.32 M. Dopo ogni campione è stato regolareed al 0,32 M saccarosio, bilanciare i tubi con soluzione di saccarosio 0,32 M HEPES-buffered. </li><li> Ultracentrifuga in un rotore ad angolo fisso a 200.000 xg per 30 min a 4 ° C. Rimuovere e scartare il surnatante. Risospendere il pellet sinaptica membrana plasmatica (SPM) in 300 ml di soluzione di 50 mM EDTA HEPES / 2 mm. Conservare i campioni a -80 ° C fino a quando utilizzati per western blotting. </li></ol> 10 Preparazione della Frazione Postsynaptic Density (PSD) <ol><li> Scongelare i campioni SPM sul ghiaccio. </li><li> Fare una soluzione di 0,54% di Triton X-100 in 50 mM EDTA soluzione HEPES / 2 mM. </li><li> In una provetta da 5 ml polistirene, combinare 2,7 ml di Triton X-100 / soluzione / EDTA e 300 microlitri frazione SPM HEPES e ruotare il campione per 15 minuti a 4 ° C </li><li> Campioni Trasferire 3,5 ml tubi ultracentrifuga spessore-parete e Centrifugare i campioni a 32.000 xg per 20 min a 4 ° C. </li><li> Prenotate il surnatante (Triton X-100 frazione solubile [TS]) e conservare a -80; ° C. NOTA: In questa fase, il pellet rappresenta la frazione &quot;PSD-1T&quot;. Un secondo trattamento con detergente produrrà una frazione &quot;PSD-2T&quot;. Se una frazione PSD-1T si desidera, passare al punto 10.6. <ol><li> Per produrre una frazione PSD-2T, risospendere il pellet in 3 ml di 0,5% Triton X-100 in 50 mM HEPES / 2 soluzione mM EDTA. Ruotare i campioni per 15 minuti a 4 ° C. Centrifugare i campioni a 32.000 xg per 20 min a 4 ° C e passare al punto 10.6. </li></ol></li><li> Eliminare il supernatante e risospendere il pellet postsinaptica (PSD) in 50 / soluzione EDTA mM mM HEPES 2. NOTA: Il volume risospensione può dipendere dalle dimensioni del pellet; Si raccomanda un volume di 50-75 ml. Poiché la frazione PSD è una frazione detergente-insolubili, è spesso necessario aggiungere 1-2 ml di 0,5% SDS per consentire completa risospensione delle proteine. Conservare i campioni a -80 ° C fino a quando utilizzati per western blotting. </li></ol>

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E., Chammas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+lysosomal-associated+membrane+protein+LAMP-1+is+a+novel+differentiation+marker+for+HC11+mouse+mammary+epithelial+cells.">The lysosomal-associated membrane protein LAMP-1 is a novel differentiation marker for HC11 mouse mammary epithelial cells.</a> Differentiation. 61, 113-120 (1996).</li><li>Otera, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peroxisomal+targeting+signal+receptor+Pex5p+interacts+with+cargoes+and+import+machinery+components+in+a+spatiotemporally+differentiated+manner:+conserved+Pex5p+WXXXF/Y+motifs+are+critical+for+matrix+protein+import.">Peroxisomal targeting signal receptor Pex5p interacts with cargoes and import machinery components in a spatiotemporally differentiated manner: conserved Pex5p WXXXF/Y motifs are critical for matrix protein import.</a> Mol Cell Biol. 22, 1639-1655 (2002).</li><li>Goubaeva, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+mitochondrial+connexin+43+regulates+apoptosis.">Cardiac mitochondrial connexin 43 regulates apoptosis.</a> Biochem Biophys Res Commun. 352, 97-103 (2007).</li><li>Lamers, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+S-100B,+neuron-specific+enolase+(NSE),+myelin+basic+protein+(MBP)+and+glial+fibrillary+acidic+protein+(GFAP)+in+cerebrospinal+fluid+(CSF)+and+blood+of+neurological+patients.">Protein S-100B, neuron-specific enolase (NSE), myelin basic protein (MBP) and glial fibrillary acidic protein (GFAP) in cerebrospinal fluid (CSF) and blood of neurological patients.</a> Brain Res Bull. 61, 261-264 (2003).</li><li>Arunachalam, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Munc18-1+is+critical+for+plasma+membrane+localization+of+syntaxin1+but+not+of+SNAP-25+in+PC12+cells.">Munc18-1 is critical for plasma membrane localization of syntaxin1 but not of SNAP-25 in PC12 cells.</a> Mol Biol Cell. 19, 722-734 (2008).</li><li>Brandstatter, J. H., Fletcher, E. L., Garner, C. C., Gundelfinger, E. D., Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+of+the+presynaptic+cytomatrix+protein+bassoon+among+ribbon+synapses+in+the+mammalian+retina.">Differential expression of the presynaptic cytomatrix protein bassoon among ribbon synapses in the mammalian retina.</a> Eur J Neurosci. 11, 3683-3693 (1999).</li></ol>

The authors have no conflicts of interest to disclose.

Ci sono diversi passaggi della procedura che sono fondamentali per un esito positivo. Nel passaggio 3, è importante che un grado costante di omogeneizzazione è ottenuta per ogni campione. Quando omogeneizzando tessuto con l&#39;omogeneizzatore a motore, una velocità costante viene utilizzata non solo per la rotazione del pestello, ma anche con il numero di colpi. Il tempo di incubazione durante lo shock osmotico deve essere preciso, dal momento omogeneizzazione estesa o incubazione nella soluzione ipotonica si lisare...

I neuroni comunicano attraverso le sinapsi, e la qualità di questa comunicazione è regolata in gran parte da alterazioni nella composizione delle proteine ​​alla sinapsi. In particolare, le proteine ​​ubicata nella densità postsinaptica partecipa nella comunicazione neuronale recettori dei neurotrasmettitori intimamente ponteggi con i loro sistemi di trasduzione del segnale 1. Inoltre, durevoli cambiamenti nella forza di efficacia sinaptica sono controllate mediante l&#39;aggiunta o la rimozione di recettori alla densità post-sinaptica 1-6. Pertanto, l&#39;isolamento e la quantificazione delle proteine ​​sinaptiche è una tecnica necessaria ed utile al fine di conoscere i modi in cui i neuroni rispondono a stimoli e alterare l&#39;efficacia sinaptica 7. Questo articolo descrive una tecnica comune per isolare le proteine ​​sinaptiche da tessuto cerebrale dei roditori da ultracentrifugazione su gradiente discontinuo di saccarosio. La frazione di membrana plasmatica sinaptica può essere arricchita e isolato basa susua densità di saccarosio, che è stato determinato empiricamente essere simile a 1,2 M saccarosio. A seconda della domanda biologica, frazioni subcellulari possono essere separati da gradienti continui o discontinui di entrambi saccarosio o Percoll. Gradienti continui consentono la separazione di proteine ​​in più frazioni; questo può essere particolarmente utile per dimostrare la co-localizzazione delle proteine ​​all&#39;interno di una data frazione 8. Tuttavia, la preparazione di gradienti continui è più laborioso e non è necessaria per molte applicazioni. Gradienti discontinui sono relativamente facili da preparare e possono essere usate per separare le proteine ​​in poche frazioni, generalmente definiti. Gradienti discontinui che sono costituiti da tre strati di saccarosio degli crescente molarità sono stati ampiamente utilizzati per isolare le proteine ​​associate alla membrana plasmatica sinaptica (SPM). Questa frazione membrana plasmatica sinaptica può essere ulteriormente elaborato per la fractio densità post-sinaptican (PSD) per trattamento detergente e l&#39;isolamento della frazione detergente-insolubili. Quando questo processo è stato descritto nel 1960 del 9,10, microscopia elettronica è stato utilizzato per dimostrare gli organelli e membrane che circa definiscono la membrana plasmatica sinaptica e frazioni di densità post-sinaptici 9-14. Questi studi hanno dimostrato l&#39;inclusione delle membrane pre e post-sinaptici e vescicole sinaptiche nella frazione SPM; dopo il trattamento detergente principalmente l&#39;elettrone-densi, densità post-sinaptica erano visibili. Nella procedura, uno shock ipotonico viene utilizzato per pizzicare fuori i processi sinaptici dal corpo cellulare 10. Questo passaggio sfrutta il fatto che i mitocondri sono più resistenti agli shock osmotico e rimangono intatte, e quindi sedimentare sul fondo del gradiente di saccarosio (Figura 1). Usando la stessa tecnica di arricchimento, le frazioni SPM e PSD erano di prima biochimicamentedefinito mediante elettroforesi su gel di poliacrilammide e sequenziamento dei principali componenti proteiche 15-17. Blot Successivamente occidentale è stato utilizzato per rilevare e quantificare i livelli di proteine ​​sinaptiche e definire ulteriormente queste frazioni (Figura 2). Abbiamo utilizzato questa tecnica nei nostri laboratori di quantificare i cambiamenti nei livelli sinaptici del trasportatore della dopamina che si verificano quando il locus Slc6a3 viene duplicato nei topi 18. Abbiamo anche usato questa tecnica in recettori NMDA topi deficienti per scoprire riduzioni specifiche per sinapsi in proteine ​​che fanno parte della DISC1 interattoma 19. E &#39;evidente da analisi western blot che le frazioni SPM contengono proteine ​​di membrana delle vescicole sinaptiche, marcatori degli endosomi, proteine ​​mitocondriali, membrana associata enzimi sintetici e molecole di trasduzione del segnale, così come componenti integrali della densità post-sinaptica e membrane plasmatiche sinaptiche 20-23. Efrazioni PSD Ven possono avere contaminazione con abbondanti proteine ​​mitocondriali e può essere necessario eseguire una seconda sedimentazione sfumatura o fasi di purificazione aggiuntivi per rimuoverli 13. Recentemente, spettrometria di massa quantitativa ha fornito un elenco di oltre 100 proteine ​​nei soli densità post-sinaptica, così come un&#39;indicazione della relativa abbondanza di questi componenti 24,25.

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preparation of synaptic plasma membrane and postsynaptic density proteins using a discontinuous sucrose gradient

Neuronali tecniche di frazionamento subcellulare consentono la quantificazione di proteine ​​che sono vittime della tratta da e per la sinapsi. Come originariamente descritto nel tardo 1960, proteine ​​associate alla membrana plasmatica sinaptica possono essere isolati da ultracentrifugazione su gradiente di densità di saccarosio. Una volta membrane sinaptiche sono isolati, il complesso macromolecolare nota come la densità post-sinaptica può essere successivamente isolato per la sua insolubilità detersivo. Le tecniche utilizzate per isolare le membrane plasmatiche sinaptiche e proteine ​​densità post-sinaptica rimane essenzialmente la stessa dopo 40 anni, e sono ampiamente utilizzati nella ricerca neuroscientifica attuale. Questo articolo descrive il frazionamento delle proteine ​​associate alla membrana plasmatica sinaptica e la densità post-sinaptica utilizzando un gradiente di saccarosio discontinuo. Risultanti preparati proteici sono adatti per western blotting o l&#39;analisi 2D DIGE.

La preparazione del gradiente di densità di saccarosio dovrebbe tradursi in una netta separazione delle tre soluzioni molari di saccarosio (0.8, 1.0, e 1.2 M saccarosio). Vedere Figura 3A per un esempio del gradiente prima di aggiungere il campione di proteine. Se il gradiente è preparato troppo in anticipo, o se è disposta su una superficie panchina con vibrazioni da altri apparecchi, il gradiente sarà compromessa e separazione corretta non sarà raggiunto. Se una netta separazione delle tre soluzi...

Watch this Scientific Journal Video about Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient at JoVE.com

Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Questo articolo descrive l&#39;arricchimento di proteine ​​associate con la membrana plasmatica sinaptica da ultracentrifugazione su gradiente di saccarosio discontinuo. La successiva preparazione di proteine ​​di densità post-sinaptica è anche descritto. Preparati proteici sono adatti per western blotting o l&#39;analisi 2D DIGE.

Preparazione di Synaptic membrana plasmatica e Proteine ​​densità postsinaptica Utilizzo di un gradiente di saccarosio discontinuo

Neuronal subcellular fractionation techniques allow the quantification of proteins that are trafficked to and from the synapse. As originally described in ...

preparation-synaptic-plasma-membrane-postsynaptic-density-proteins

Research

JoVE Journal

Neuroscience

31.0K Views.  University of Toronto.  Questo articolo descrive l'arricchimento di proteine ​​associate con la membrana plasmatica sinaptica da ultracentrifugazione su gradiente di saccarosio discontinuo. La successiva preparazione di proteine ​​di densità post-sinaptica è anche descritto. Preparati proteici sono adatti per western blotting o l'analisi 2D DIGE. 

Video: Preparation of Synaptic Plasma Membrane and Postsynaptic Density Proteins Using a Discontinuous Sucrose Gradient

Studying Synaptic Vesicle Pools using Photoconversion of Styryl Dyes

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The vesicles are then retrieved from the plasma membrane (endocytosis) and grouped together with the general pool of vesicles within the nerve terminal, until they undergo a new exo- and endocytosis cycle (vesicle recycling). These processes have been studied using a variety of techniques such as electron microscopy, electrophysiology recordings, amperometry and capacitance measurements. Importantly, during the last two decades a number of fluorescently labeled markers emerged, allowing optical techniques to track vesicles in their recycling dynamics. One of the most commonly used markers is the styryl or FM dye 1; structurally, all FM dyes contain a hydrophilic head and a lipophilic tail connected through an aromatic ring and one or more double bonds (Fig. 1B). A classical FM dye experiment to label a pool of vesicles consists in bathing the preparation (Fig. 1Ai) with the dye during the stimulation of the nerve (electrically or with high K+). This induces vesicle recycling and the subsequent loading of the dye into recently endocytosed vesicles (Fig. 1Ai-iii). After loading the vesicles with dye, a second round of stimulation in a dye-free bath would trigger the FM release through exocytosis (Fig. 1Aiv-v), process that can be followed by monitoring the fluorescence intensity decrease (destaining).

Although FM dyes have contributed greatly to the field of vesicle recycling, it is not possible to determine the exact localization or morphology of individual vesicles by using conventional fluorescence microscopy. For that reason, we explain here how FM dyes can also be used as endocytic markers using electron microscopy, through photoconversion. The photoconversion technique exploits the property of fluorescent dyes to generate reactive oxygen species under intense illumination. Fluorescently labeled preparations are submerged in a solution containing diaminobenzidine (DAB) and illuminated. Reactive species generated by the dye molecules oxidize the DAB, which forms a stable, insoluble precipitate that has a dark appearance and can be easily distinguished in electron microscopy 2,3. As DAB is only oxidized in the immediate vicinity of fluorescent molecules (as the reactive oxygen species are short-lived), the technique ensures that only fluorescently labeled structures are going to contain the electron-dense precipitate. The technique thus allows the study of the exact location and morphology of actively recycling organelles.

FM dyes have been of invaluable help in the understanding of synaptic dynamics. FMs are normally followed under the fluorescent microscope during different stimulation conditions. However, photoconversion of FM dyes combined with electron microscopy allows the visualization of distinct synaptic vesicle pools, among other ultrastructure components, in synaptic boutons.

Studiare Piscine vescicole sinaptiche con fotoconversione di coloranti Styryl

The fusion of synaptic vesicles with the plasma membrane (exocytosis) is a required step in neurotransmitter release and neuronal communication. The ...

Ricerca

Neuroscienze

Membrane Potentials, Synaptic Responses, Neuronal Circuitry, Neuromodulation and Muscle Histology Using the Crayfish: Student Laboratory Exercises

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that influence a cell's membrane potential and which we address in these experiments are: (1) Ion concentration of K+ on the outside of the membrane, and (2) the permeability of the membrane to specific ions. The crayfish abdominal extensor muscles are in groupings with some being tonic (slow) and others phasic (fast) in their biochemical and physiological phenotypes, as well as in their structure; the motor neurons that innervate these muscles are correspondingly different in functional characteristics. We use these muscles as well as the superficial, tonic abdominal flexor muscle to demonstrate properties in synaptic transmission. In addition, we introduce a sensory-CNS-motor neuron-muscle circuit to demonstrate the effect of cuticular sensory stimulation as well as the influence of neuromodulators on certain aspects of the circuit. With the techniques obtained in this exercise, one can begin to answer many questions remaining in other experimental preparations as well as in physiological applications related to medicine and health. We have demonstrated the usefulness of model invertebrate preparations to address fundamental questions pertinent to all animals.

The experiments demonstrate an easy approach for students to gain experience in examining muscle structure, synaptic responses, the effects of ion gradients and permeability on membrane potentials. Also, a sensory-CNS-motor-muscle circuit is presented to show a means to test effects of compounds on a neuronal circuit.

Potenziali di membrana, Risposte Synaptic, circuiti neuronali, neuromodulazione e Istologia muscolare Utilizzando la Gamberi: Esercizi di laboratorio per studenti

The purpose of this report is to help develop an understanding of the effects caused by ion gradients across a biological membrane. Two aspects that ...

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 ...

Preparation of Synaptoneurosomes from Mouse Cortex using a Discontinuous Percoll-Sucrose Density Gradient

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters.
The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.

A method to prepare translationally active, intact synaptoneurosomes (SNs) from mouse brain cortex is described. The method uses a discontinuous Percoll-sucrose density gradient allowing for the quick preparation of active SNs.

Preparazione di Synaptoneurosomes da mouse Cortex con un discontinuo di Percoll-Saccarosio gradiente di densità

Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that ...

A General Method for Evaluating Incubation of Sucrose Craving in Rats

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and relapse to food taking 1. Food craving conceptualized in this way is akin to drug craving in response to drug-paired cues. A rich literature has been developed around understanding the behavioral and neurobiological determinants of drug craving; we and others have been focusing recently on translating techniques from basic addiction research to better understand addiction-like behaviors related to food 2-4.
As done in previous studies of drug craving, we examine sucrose craving behavior by utilizing a rat model of relapse. In this model, rats self-administer either drug or food in sessions over several days. In a session, lever responding delivers the reward along with a tone+light stimulus. Craving behavior is then operationally defined as responding in a subsequent session where the reward is not available. Rats will reliably respond for the tone+light stimulus, likely due to its acquired conditioned reinforcing properties 5. This behavior is sometimes referred to as sucrose seeking or cue reactivity. In the present discussion we will use the term "sucrose craving" to subsume both of these constructs.
In the past decade, we have focused on how the length of time following reward self-administration influences reward craving. Interestingly, rats increase responding for the reward-paired cue over the course of several weeks of a period of forced-abstinence. This "incubation of craving" is observed in rats that have self-administered either food or drugs of abuse 4,6. This time-dependent increase in craving we have identified in the animal model may have great potential relevance to human drug and food addiction behaviors.
Here we present a protocol for assessing incubation of sucrose craving in rats. Variants of the procedure will be indicated where craving is assessed as responding for a discrete sucrose-paired cue following extinction of lever pressing within the sucrose self-administration context (Extinction without cues) or as responding for sucrose-paired cues in a general extinction context (Extinction with cues).

Responding for food or drug-paired cues increases over the course of a period of abstinence and this may relate to an increased susceptibility to relapse behaviors. Here we detail a procedure for evaluating this "incubation of craving" in rats that have self-administered sucrose.

Un metodo generale per la valutazione di incubazione della brama di saccarosio in ratti

For someone on a food-restricted diet, food craving in response to food-paired cues may serve as a key behavioral transition point between abstinence and ...

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 ...

Deriving the Time Course of Glutamate Clearance with a Deconvolution Analysis of Astrocytic Transporter Currents

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the membrane with the co-transport of 3 Na+ and 1 H+ and the counter-transport of 1 K+. The stoichiometric current generated by the transport process can be monitored with whole-cell patch-clamp recordings from astrocytes. The time course of the recorded current is shaped by the time course of the glutamate concentration profile to which astrocytes are exposed, the kinetics of glutamate transporters, and the passive electrotonic properties of astrocytic membranes. Here we describe the experimental and analytical methods that can be used to record glutamate transporter currents in astrocytes and isolate the time course of glutamate clearance from all other factors that shape the waveform of astrocytic transporter currents. The methods described here can be used to estimate the lifetime of flash-uncaged and synaptically-released glutamate at astrocytic membranes in any region of the central nervous system during health and disease.

We describe an analytical method to estimate the lifetime of glutamate at astrocytic membranes from electrophysiological recordings of glutamate transporter currents in astrocytes.

Derivando La durata di Liquidazione glutammato con una analisi Deconvoluzione delle Correnti Transporter astrociti

The highest density of glutamate transporters in the brain is found in astrocytes. Glutamate transporters couple the movement of glutamate across the ...

The Neuromuscular Junction: Measuring Synapse Size, Fragmentation and Changes in Synaptic Protein Density Using Confocal Fluorescence Microscopy

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. Structural changes at the NMJ can result in neurotransmission failure, resulting in weakness, atrophy and even death of the muscle fiber. Many studies have investigated how genetic modifications or disease can alter the structure of the mouse NMJ. Unfortunately, it can be difficult to directly compare findings from these studies because they often employed different parameters and analytical methods. Three protocols are described here. The first uses maximum intensity projection confocal images to measure the area of acetylcholine receptor (AChR)-rich postsynaptic membrane domains at the endplate and the area of synaptic vesicle staining in the overlying presynaptic nerve terminal. The second protocol compares the relative intensities of immunostaining for synaptic proteins in the postsynaptic membrane. The third protocol uses Fluorescence Resonance Energy Transfer (FRET) to detect changes in the packing of postsynaptic AChRs at the endplate. The protocols have been developed and refined over a series of studies. Factors that influence the quality and consistency of results are discussed and normative data are provided for NMJs in healthy young adult mice.

The neuromuscular junction (NMJ) is altered in a variety of conditions that can sometimes culminate in synaptic failure. This report describes fluorescence microscope-based methods to quantify such structural changes.

La giunzione neuromuscolare: Synapse Misurazione circonferenza, la frammentazione e Variazioni Synaptic Proteine ​​Densità Uso confocale microscopia a fluorescenza

The neuromuscular junction (NMJ) is the large, cholinergic relay synapse through which mammalian motor neurons control voluntary muscle contraction. ...

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in the mouse retrosplenial cortex (RSC) using in vivo two-photon microscopy in Thy1-GFP transgenic mouse line. This approach creates a possibility to investigate the correlation of behavioural manipulations with changes in neuronal morphology in vivo.
The cranial window implantation procedure was considered to be limited only to the easily accessible cortex regions such as the barrel field. Our approach allows visualization of neurons in the highly vascularized RSC. RSC is an important element of the brain circuit responsible for spatial memory, previously deemed to be problematic for in vivo two-photon imaging.
The cranial window implantation over the RSC is combined with an injection of mCherry-expressing recombinant adeno-associated virus (rAAVmCherry) into the dorsal hippocampus. The expressed mCherry spreads out to axonal projections from the hippocampus to RSC, enabling the visualization of changes in both presynaptic axonal boutons and postsynaptic dendritic spines in the cortex. 
This technique allows long-term monitoring of experience-dependent structural plasticity in RSC.

Simultaneous Two-photon In Vivo Imaging of Synaptic Inputs and Postsynaptic Targets in the Mouse Retrosplenial Cortex

This video shows the craniotomy procedure that allows chronic imaging of neurons in mouse retrosplenial cortex using in vivo two photon microscopy in Thy1-GFP transgenic line. This approach is combined with injection of mCherry-expressing adeno-associated virus into dorsal hippocampus. These techniques allow long-term monitoring of experience-dependent structural plasticity in RSC.

Preparazione di Synaptic membrana plasmatica e Proteine ​​densità postsinaptica Utilizzo di un gradiente di saccarosio discontinuo

Preparazione di Synaptic membrana plasmatica e Proteine densità postsinaptica Utilizzo di un gradiente di saccarosio discontinuo