Ringraziamo Irmgard Weiss per assistenza tecnica. Questo lavoro è stato supportato dal DFG tramite il Cluster di eccellenza per la microscopia a campo del nanometro e fisiologia molecolare del cervello (CNMPB, B1-7, a T.D.).

Non con gli animali vivi sono stati condotti studi. Gli esperimenti che coinvolgono animali eutanasizzati per ottenere cell culture sono state approvate dalle autorità locali di protezione animale (Tierschutzkommission der Universitätsmedizin Göttingen) sotto l'approvazione numero T10/30. Gli esperimenti sono stati condotti con i protocolli approvati.
1. primario delle cellule ippocampali cultura
<ol><li>Preparare la coltura delle cellule dissociate di ippocampo del ratto del giorno embrionale 1916,17. Le cellule sulle lamelle di 12 mm rivestite con polyethyleneimine (PEI) a 24 pozzetti piatti ad una densità di 50.000-60.000 cellule/pozzetto della piastra. Controllare la densità utilizzando una cella ottica di contrasto di fase e camera di conteggio.</li>
	<li>I neuroni della coltura per 3 giorni (giorno in vitro (DIV) 3) in una piastra a 24 pozzetti nell'incubatore a 37 ° C con 5% CO2.</li>
	<li>Valutare i coprioggetti per indicatori di salute delle cellule usando la microscopia chiara trasmessa (ad es., fase ottica contrasto ad un ingrandimento di 10-20 X). Verifica per i seguenti indicatori di buona salute: un alone di contrasto di fase chiara, neurites senza strutture in rilievo e nessun soma clustering o neurite impacchettato.</li>
</ol>2. transfezione
Nota: Il seguente protocollo si riferisce a una doppio di transfezione per 3 pozzi. Tuttavia, il protocollo funziona meglio quando gli importi sufficienti per 4 pozzetti sono preparati.
<ol><li>Preparare 500 mL di buffer di transfezione (274 mM NaCl, 10 mM KCl, 1,4 mM Na2HPO4, glucosio di 15 mM, 42 mM HEPES) in un matraccio di Erlenmeyer.<ol>
<li>Sciogliere 8,0 g di NaCl, 0,37 g di KCl, 0,095 g di Na2HPO4, 1,35 g di glucosio e 5,0 g di HEPES in 400 mL di acqua distillata in un matraccio di Erlenmeyer.</li>
		<li>Regolare il pH a 6,95 con 1 M NaOH utilizzando un pH-metro.</li>
		<li>Regolare il volume con acqua distillata a 500 mL e controllare il pH utilizzando un pH-metro.</li>
		<li>Rendere le aliquote di 20-30 mL di buffer di transfezione con i seguenti valori di pH di pipettaggio 1 M NaOH al buffer di transfezione: 6,96, 6,97, 6,98, 6,99, 7.00, 7.01, 7.02, 7,03, 7.04, 7.05, 7,06, 7,07, 7,08, 7,09, 7.11. Nota: Il pH del tampone transfezione è cruciale per l'efficacia di transfezione.</li>
		<li>Per verificare che conduce di buffer di transfezione per il maggior numero di cellule trasfettate, test ogni valore pH da 6,96 a 7.11. Utilizzare il metodo di transfezione descritto in 2.2-2.11 e un plasmide convalidato che esprimono GFP. Determinare il numero di cellule trasfettate per vetrino coprioggetto per ogni valore di pH del tampone di transfezione valutare quali buffer funziona meglio.</li>
		<li>Aliquotare il buffer con la massima efficienza di trasfezione in provette microcentrifuga 2 mL congelare e conservare i capillari a-20 ° C.</li>
	</ol>
</li>
	<li>Pre-riscaldare il siero ridotto medie, mezzo di coltura cellulare e acqua distillata a 37 ° C nel bagno d'acqua.</li>
	<li>Preparare la miscela di transfezione in una microcentrifuga da 1,5 mL. Lavorare sotto la cappa a flusso laminare per garantire condizioni di lavoro sterile.<ol>
<li>Mix 7,5 µ l di cloruro di calcio di 2 M con 4 µ g di ogni DNA privo di endotossina (Synaptophysin-mOrange ed EGFP/GFP-Rogdi). Aggiungere acqua per raggiungere un volume totale di 60 mL in una provetta da microcentrifuga da 1,5 mL.</li>
		<li>Aggiungere 60 mL di buffer di transfezione al mix. Per ottenere i migliori risultati, aggiungere goccia a goccia il buffer di transfezione agitando delicatamente il DNA-mix il vortice.</li>
		<li>Incubare a temperatura ambiente (TA) per 20 minuti. Evitare di scuotere il tubo di incubazione durante il periodo di incubazione di posizionamento del tubo a fianco della cappa a flusso laminare.</li>
	</ol>
</li>
	<li>Sotto la cappa a flusso laminare, rimuovere il terreno di coltura cellulare ("medium condizionato") dai pozzi usando una pipetta da 1000 mL e conservarla in un contenitore separato nell'incubatrice.</li>
	<li>Aggiungere 500 mL di mezzo di riduttore del siero in ciascun pozzetto. Incubare le cellule a 37 ° C e 5% CO2 fino a quando il periodo di incubazione di 20 minuti (punto 2.3.3).</li>
	<li>Aggiungere 30 mL di miscela di transfezione in ciascun pozzetto pipettando alcune gocce. Scartare il residuo nella parte inferiore del tubo.</li>
	<li>Dopo tutti i pozzi sono stati forniti con mix di transfezione, agitare delicatamente la piastra a 24 pozzetti per assicurare la distribuzione della miscela transfezione in mezzo.</li>
	<li>Incubare per 60 minuti a 37 ° C e 5% CO2.</li>
	<li>Rimuovere e scartare il mix di transfezione e lavare tre volte con mezzo di coltura cellulare. Aggiungere 1 mL di mezzo di coltura cellulare in ciascun pozzetto e li Incubare per 30 secondi alla RT. rimuovere 750 mL di terreno e aggiungere la stessa quantità di terreno fresco. Ripetere l'operazione tre volte. Nota: La fase di lavaggio è critica. Tenere il tempo che ciascun pozzetto non ha nessun mezzo minimo (cioè., rimuovere e sostituire ben-di-bene) e aggiungere il supporto di lavare delicatamente.</li>
	<li>Rimuovere e scartare il mezzo di coltura cellulare e aggiungere 450 mL della condizionata media ben-di-bene.</li>
	<li>Lasciate che i neuroni maturi nell'incubatore a 37 ° C e 5% di CO2 -10 DIV.</li>
</ol>3. la stimolazione e l'assorbimento di Syt1
Nota: Il seguente protocollo riguarda l'assorbimento 3 pozzi. Per depolarizzazione di qualsiasi altro numero di pozzi, regolare di conseguenza gli importi.
<ol><li>Preparare 50 mL di tampone di depolarizzazione 10 x (640 mM NaCl, 700 mM KCl, 10mm MgCl2, 20mm CaCl2, glucosio di 300 mM, 200 mM HEPES, pH 7.4) in un matraccio di Erlenmeyer. Nota: Buffer di depolarizzazione può essere conservato a 4 ° C per diverse settimane. Se una soluzione non depolarizzante è utilizzata anche, preparare un 10 soluzione di x Tyrode composta da 1290 mM NaCl, 50 mM KCl, 10mm MgCl2, 20mm CaCl2, 300 mM glucosio, pH di 200 mM HEPES 7.4 al fine di confrontare la stimolazione indotta da riciclo con spontanea riciclaggio. Dopo la diluizione a 1 x, aggiungere 1 µM di tetrodotossina prima dell'uso per bloccare la generazione di potenziali d'azione.<ol>
<li>Sciogliere 1,87 g di NaCl, 2,61 g di KCl, 0,1 g di MgCl2-6 H20, 0,15 g di CaCl2-2 H2O e 3,0 g di glucosio-1 H2O e 2,38 g di HEPES in 50 mL di acqua distillata in un matraccio di Erlenmeyer. Regolare il pH con NaOH e filtro sterile la soluzione. Diluire il tampone 01:10 in acqua distillata per ottenere una concentrazione di 1 x.</li>
	</ol>
</li>
	<li>Preparare 4% paraformaldeide (PFA) in 1X PBS (pH 7.4) per la fissazione dopo stimolazione.<ol>
<li>Per 500 mL di 4% PFA in PBS 1X, sciogliere 20 g di paraformaldeide in 450 mL di acqua distillata H2O. Nota: La soluzione di riscaldamento può accelerare la dissoluzione, ma non riscaldare la soluzione oltre i 70 ° C, come il PFA può disintegrarsi. Attenzione: PFA è tossico, potenzialmente cancerogene e teratogene. Indossare guanti quando si lavora con PFA, lavorare sotto la cappa ed evitare l'ingestione.</li>
		<li>Lasciare raffreddare la soluzione a RT e aggiungere 50 mL della soluzione madre PBS di 10 x. Aggiustare il pH a 7.4 con NaOH/HCl utilizzando un pH-metro.</li>
	</ol>
</li>
	<li>Pre-riscaldare 600 mL di tampone di depolarizzazione 1x e 10 mL di terreno di coltura cellulare a 37 ° C nel bagno d'acqua.</li>
	<li>Aggiungere 1 mL di anticorpo di topo anti-Syt1 (clone 604.2) a 1x buffer di depolarizzazione e vortex per 10 secondi.</li>
	<li>Rimuovere e gettare il mezzo di coltura cellulare dalle cellule. Aggiungere 200 mL di miscela depolarizzazione-anticorpo di ogni bene e incubare per 5 minuti a 37 ° C e 5% di CO2 nell'incubatrice.</li>
	<li>Rimuovere e scartare il mix di depolarizzazione-anticorpo e lavare tre volte con mezzo di coltura cellulare. Aggiungere 1 mL di mezzo di coltura cellulare ad ogni pozzetto e incubare per 30 secondi alla RT. rimuovere 750 mL di terreno e aggiungere la stessa quantità di terreno fresco. Ripetere tre volte.</li>
	<li>Rimuovere e scartare il mezzo di coltura cellulare e aggiungere 300 mL di 4% PFA in PBS 1X. Incubare per 20 minuti a 4 ° C.</li>
	<li>Lavare tre volte per 5 minuti ciascuno con PBS 1X. Nota: Il protocollo può essere messo in pausa qui.</li>
</ol>4. immunocitochimica
<ol><li>Preparare 50 mL di tampone bloccante. Nota: Blocco buffer può essere mantenuto a-20 ° C per diversi mesi.<ol>
<li>Sciogliere 2,5 g di saccarosio e 1 g di albumina di siero bovino (BSA) in 5 mL di soluzione di riserva di PBS x 10. Aggiungere 1,5 mL di soluzione detergente 10%. Mescolare la soluzione fino a quando tutti i componenti siano correttamente sciolti e aggiungere acqua distillata H2O fino a raggiungere un volume finale di 50 mL. Aliquota della soluzione e congelare le aliquote per l'archiviazione.</li>
	</ol>
</li>
	<li>Diluire l'anticorpo secondario, accoppiati a fluorophore (diretto contro le specie di Syt1-anticorpo primarie) in 200 mL di tampone bloccante in ciascun pozzetto ad una diluizione di 1: 1000.</li>
	<li>Rimuovere e gettare il PBS 1x da ogni bene contenente un vetrino coprioggetti.</li>
	<li>Aggiungere 200 mL di blocco buffer-anticorpo mix ad ogni pozzetto e incubare per 60 minuti a TA. Attenzione: Poiché gli anticorpi secondari sono sensibili alla luce, tutti i passi avanti in movimento devono essere eseguiti nel buio.</li>
	<li>Dopo l'incubazione, lavare le cellule tre volte per 5 minuti con 1 mL di PBS 1X.</li>
	<li>Incorporare i coprioggetti su vetrini da microscopio con il mezzo di inclusione.<ol>
<li>Aggiungere una goccia di 7 mL di mezzo il vetrino del microscopio di inclusione. Rimuovere il vetrino coprioggetto dalla piastra a 24 pozzetti sollevandolo con una siringa e afferra con una pinza. Attenzione: Le cellule sulla superficie del coprivetrino sono facilmente danneggiate, pinze devono essere maneggiate con cura.</li>
		<li>Immergere il vetrino coprioggetto in acqua distillata per rimuovere il PBS ed asciugarla accuratamente toccando un bordo ad un tessuto morbido.</li>
		<li>Capovolgere il coprioggetto sull'incorporamento gocciolina medio, in modo che affronta la superficie portando le cellule al microscopio, quindi incorporare le cellule nel mezzo di incorporamento.</li>
	</ol>
</li>
	<li>Lasciare le diapositive ad asciugare sotto il cofano per 1-2 h (coprirli per evitare l'esposizione alla luce) e memorizzarli in una casella di scorrimento microscopio a 4 ° C. Nota: Il protocollo può essere messo in pausa qui.</li>
</ol>5. analisi microscopica
<ol><li>Dopo asciugare i vetrini coprioggetti, metterli sotto il microscopio con l'obiettivo e la fotocamera.</li>
	<li>Regolare il tempo di esposizione per ogni canale in modo che alcuni pixel sono sovraesposte per assicurare la massima distribuzione dei valori di grigio. Nota: Mentre il tempo di esposizione può variare tra i canali, dovrebbe essere costante per un unico canale garantire la comparabilità tra i coprioggetti.</li>
	<li>Acquisizione di immagini multi-canale per 10 regioni di interesse (ROI) per vetrino coprioggetti. Controllare che il ROI contiene axonal processi da un neurone trasfettato controllando la fluorescenza di GFP, che dovrebbe essere punctate.</li>
</ol>6. elaborazione statistica
<ol><li>Esportare le immagini come file. TIF. Caricare le immagini in OpenView18 facendo clic su File | Carica il file immagine.</li>
	<li>Scegliere l'immagine di Synaptophysin-mOrange come canale 1, l'immagine di Rogdi-GFP/EGFP come canale 2 e l'immagine di fluorescenza Alexa647 come canale 3.</li>
	<li>Soglia la ROIs.<ol>
<li>Fare clic su analisi | Zona posto sopra puncta. Scegliere i valori di soglia e Delta intensità in modo che all'ispezione visiva, fluorescenza diffusa è esclusa, lasciando solo punctati segnali nell'immagine del canale 1 (che rappresentano lo Synaptophysin-mOrange fluorescenza). Mantenere la stessa soglia di tutte le immagini.</li>
	</ol>
</li>
	<li>Facendo clic su Esegui ora, trasferire questi ROIs al corrispondente canale 2 (fluorescenza GFP-Rogdi/EGFP) e 3 (Syt1-fluorescenza) di ciascuna area del vetrino coprioggetti. Nota: Il ROIs deve essere considerato solo se la cella è doppio-transfettate. In questo metodo, mOrange - e GFP-fluorescenza dovrebbero essere chiaramente visibile.</li>
	<li>Salvare i dati nell'editor del registro di analisi facendo clic su dati di Log.</li>
	<li>Aprire l'editor del registro di analisi sotto la scheda di Windows e copiare i valori per ciascun canale. Incollare i valori per i canali separati in un foglio di calcolo.</li>
	<li>Determinare l'intensità di fluorescenza media del Syt1-canale presso il ROIs in entrambe le condizioni di transfezione (GFP-Rogdi ed EGFP).</li>
	<li>Applicare appropriati test statistici quali di Student t-test per determinare le differenze significative.</li>
</ol>

<ol>
	<li>Koh, T., Bellen, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin+I,+a+Ca2++sensor+for+neurotransmitter+release.">Synaptotagmin I, a Ca2+ sensor for neurotransmitter release.</a> Trends in Neurosciences. 26, 413-422 (2003).</li><li>Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+Does+Synaptotagmin+Trigger+Neurotransmitter+Release.">How Does Synaptotagmin Trigger Neurotransmitter Release.</a> Annual Review of Biochemistry. 77, 615-641 (2008).</li><li>Perin, M. S., 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=Structural+and+Functional+Conservation+of+Synaptotagmin+(p65)+in+Drosophila+and+Humans.">Structural and Functional Conservation of Synaptotagmin (p65) in Drosophila and Humans.</a> Journal of Biological Chemistry. 266, 615-622 (1991).</li><li>Matteoli, M., Takei, K., Perrin, M. S., S&#252;dhof, T. C., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exo-endocytotic+Recycling+of+Synaptic+Vesicles+in+Developing+Processes+of+Cultured+Hippocampal+Neurons.">Exo-endocytotic Recycling of Synaptic Vesicles in Developing Processes of Cultured Hippocampal Neurons.</a> Journal of Cell Biology. 117, 849-861 (1992).</li><li>Ko, 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=Neuroligin-1+performs+neurexin-dependent+and+neurexin-independent+functions+in+synapse+validation.">Neuroligin-1 performs neurexin-dependent and neurexin-independent functions in synapse validation.</a> The EMBO Journal. 28, 3244-3255 (2009).</li><li>Shinoda, Y., 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=BDNF+enhances+spontaneous+and+activity-dependent+neurotransmitter+release+at+excitatory+terminals+but+not+at+inhibitory+terminals+in+hippocampal+neurons.">BDNF enhances spontaneous and activity-dependent neurotransmitter release at excitatory terminals but not at inhibitory terminals in hippocampal neurons.</a> Frontiers in Synaptic Neuroscience. 6, 27 (2014).</li><li>Ivanova, D., 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=Synaptic+activity+controls+localization+and+function+of+CtBP+1+via+binding+to+Bassoon+and+Piccolo.">Synaptic activity controls localization and function of CtBP 1 via binding to Bassoon and Piccolo.</a> The EMBO Journal. 34, 1056-1077 (2015).</li><li>Crawford, D. C., Ramirez, D. M. O., Trauterman, B., Monteggia, L. M., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selective+molecular+impairment+of+spontaneous+neurotransmission+modulates+synaptic+efficacy.">Selective molecular impairment of spontaneous neurotransmission modulates synaptic efficacy.</a> Nature Communications. 8, 1-14 (2017).</li><li>Riemann, D., Wallrafen, R., Dresbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Kohlsch&#252;tter-T&#246;nz+syndrome+associated+gene+Rogdi+encodes+a+novel+presynaptic+protein.">The Kohlsch&#252;tter-T&#246;nz syndrome associated gene Rogdi encodes a novel presynaptic protein.</a> Scientific Reports. 7, (2017).</li><li>Kim, M., 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=Rogdi+Defines+GABAergic+Control+of+a+Wake-promoting+Dopaminergic+Pathway+to+Sustain+Sleep+in+Drosophila.">Rogdi Defines GABAergic Control of a Wake-promoting Dopaminergic Pathway to Sustain Sleep in Drosophila.</a> Scientific Reports. 7, 1-14 (2017).</li><li>Schossig, A., Wolf, N. I., Kapferer, I., Kohlsch&#252;tter, A., Zschocke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epileptic+encephalopathy+and+amelogenesis+imperfecta:+Kohlsch&#252;tter-T&#246;nz+syndrome.">Epileptic encephalopathy and amelogenesis imperfecta: Kohlsch&#252;tter-T&#246;nz syndrome.</a> Euopean Journal of Medical Genetics. 55, 319-322 (2012).</li><li>Kraszewski, K., 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=Synaptic+Vesicle+Dynamics+in+Living+Cultured+Hippocampal+Neurons+Visualized+with+CY3-Conjugated+Antibodies+Directed+against+the+Lumenal+Domain+of+Synaptotagmin.">Synaptic Vesicle Dynamics in Living Cultured Hippocampal Neurons Visualized with CY3-Conjugated Antibodies Directed against the Lumenal Domain of Synaptotagmin.</a> Journal of Neuroscience. 15, 4328-4342 (1995).</li><li>Willig, K. I., Rizzoli, S. O., Westphal, V., Jahn, R., Hell, S. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=STED+microscopy+reveals+that+synaptotagmin+remains+clustered+after+synaptic+vesicle+exocytosis.">STED microscopy reveals that synaptotagmin remains clustered after synaptic vesicle exocytosis.</a> Nature. 440, 935-939 (2006).</li><li>Petkova, A., Goedecke, N., Korte, M., Dresbach, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuroligins+mediate+presynaptic+maturation+through+brain-derived+neurotrophic+factor+signaling.">Neuroligins mediate presynaptic maturation through brain-derived neurotrophic factor signaling.</a> bioRxiv. , 262246 (2018).</li><li>Fuchs, C., 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=GABA(A)+receptors+can+initiate+the+formation+of+functional+inhibitory+GABAergic+synapses.">GABA(A) receptors can initiate the formation of functional inhibitory GABAergic synapses.</a> European Journal of Neuroscience. 38, 3146-3158 (2013).</li><li>Dresbach, T., 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=Functional+regions+of+the+presynaptic+cytomatrix+protein+Bassoon:+Significance+for+synaptic+targeting+and+cytomatrix+anchoring.">Functional regions of the presynaptic cytomatrix protein Bassoon: Significance for synaptic targeting and cytomatrix anchoring.</a> Molecular and Cellular Neuroscience. 23, 279-291 (2003).</li><li>Seibenhener, M. L., Wooten, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+Culture+of+Hippocampal+Neurons+from+Prenatal+Mice.">Isolation and Culture of Hippocampal Neurons from Prenatal Mice.</a> Journal of Visualized Experiments. (65), 4-9 (2012).</li><li>Tsuriel, S., 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=Local+sharing+as+a+predominant+determinant+of+synaptic+matrix+molecular+dynamics.">Local sharing as a predominant determinant of synaptic matrix molecular dynamics.</a> PLOS Biology. 4, 1572-1587 (2006).</li><li>Iwabuchi, S., Kakazu, Y., Koh, J., Goodman, K. M., Harata, N. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+Synaptic+Vesicle+Recycling+Using+FM+Dyes+During+Evoked,+Spontaneous,+and+Miniature+Synaptic+Activities.">Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities.</a> Journal of Visualized Experiments. (85), 1-10 (2014).</li><li>Villarreal, S., Lee, S. H., Wu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+Synaptic+Vesicle+Endocytosis+in+Cultured+Hippocampal+Neurons.">Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons.</a> Journal of Visualized Experiments. (127), 1-8 (2017).</li><li>Kavalali, E. T., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualizing+presynaptic+function.">Visualizing presynaptic function.</a> Nature Neuroscience. 17, 10-16 (2014).</li><li>Opazo, 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=Limited+Intermixing+of+Synaptic+Vesicle+Components+upon+Vesicle+Recycling.">Limited Intermixing of Synaptic Vesicle Components upon Vesicle Recycling.</a> Traffic. 11, 800-812 (2010).</li><li>Wollebo, H. S., Woldemichaele, B., White, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lentiviral+transduction+of+neuronal+cells.">Lentiviral transduction of neuronal cells.</a> Methods in Molecular Biology. 1078, 141-146 (2013).</li><li>Yang, X., Kaeser-Woo, Y. J., Pang, Z. P., Xu, W., S&#252;dhof, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complexin+Clamps+Asynchronous+Release+by+Blocking+a+Secondary+Ca2++Sensor+via+Its+Accessory+&#945;+Helix.">Complexin Clamps Asynchronous Release by Blocking a Secondary Ca2+ Sensor via Its Accessory &#945; Helix.</a> Neuron. 68, 907-920 (2010).</li><li>Wittenmayer, N., 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=Postsynaptic+Neuroligin1+regulates+presynaptic+maturation.">Postsynaptic Neuroligin1 regulates presynaptic maturation.</a> Proceedings of the National Academy of Sciences of the United States of America. 106, 13564-13569 (2009).</li><li>Lazarevic, V., Schone, C., Heine, M., Gundelfinger, E. D., Fejtova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+Remodeling+of+the+Presynaptic+Cytomatrix+upon+Homeostatic+Adaptation+to+Network+Activity+Silencing.">Extensive Remodeling of the Presynaptic Cytomatrix upon Homeostatic Adaptation to Network Activity Silencing.</a> Journal of Neuroscience. 31, 10189-10200 (2011).</li><li>Kraszewski, K., Daniell, L., Mundigl, O., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mobility+of+synaptic+vesicles+in+nerve+endings+monitored+by+recovery+from+photobleaching+of+synaptic+vesicle-associated+fluorescence.">Mobility of synaptic vesicles in nerve endings monitored by recovery from photobleaching of synaptic vesicle-associated fluorescence.</a> Journal of Neuroscience. 16, 5905-5913 (1996).</li><li>Hua, Y., Sinha, R., Martineau, M., Kahms, M., Klingauf, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+common+origin+of+synaptic+vesicles+undergoing+evoked+and+spontaneous+fusion.">A common origin of synaptic vesicles undergoing evoked and spontaneous fusion.</a> Nature Neuroscience. 13, 1451-1453 (2010).</li><li>Han, W., 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=N-Glycosylation+Is+Essential+for+Vesicular+Targeting+of+Synaptotagmin+1.">N-Glycosylation Is Essential for Vesicular Targeting of Synaptotagmin 1.</a> Neuron. 41, 85-99 (2004).</li><li>Kwon, S. E., Chapman, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+is+dispensable+for+sorting+of+synaptotagmin+1+but+is+critical+for+targeting+of+SV2+and+synaptophysin+to+recycling+synaptic+vesicles.">Glycosylation is dispensable for sorting of synaptotagmin 1 but is critical for targeting of SV2 and synaptophysin to recycling synaptic vesicles.</a> Journal of Biological Chemistry. 287, 35658-35668 (2012).</li><li>Afuwape, O. A. T., Wasser, C. R., Schikorski, T., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+vesicle+pool-specific+modification+of+neurotransmitter+release+by+intravesicular+free+radical+generation.">Synaptic vesicle pool-specific modification of neurotransmitter release by intravesicular free radical generation.</a> The Journal of Physiology. 595, 1223-1238 (2017).</li><li>Sara, Y., Virmani, T., De&#225;k, F., Liu, X., Kavalali, E. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+isolated+pool+of+vesicles+recycles+at+rest+and+drives+spontaneous+neurotransmission.">An isolated pool of vesicles recycles at rest and drives spontaneous neurotransmission.</a> Neuron. 45, 563-573 (2005).</li><li>Wilhelm, B. G., Groemer, T. W., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+same+synaptic+vesicles+drive+active+and+spontaneous+release.">The same synaptic vesicles drive active and spontaneous release.</a> Nature Neuroscience. 13, 1454-1456 (2010).</li><li>Bacci, A., 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=Chronic+blockade+of+glutamate+receptors+enhances+presynaptic+release+and+downregulates+the+interaction+between+synaptophysin-synaptobrevin-vesicle-associated+membrane+protein+2.">Chronic blockade of glutamate receptors enhances presynaptic release and downregulates the interaction between synaptophysin-synaptobrevin-vesicle-associated membrane protein 2.</a> Journal of Neuroscience. 21, 6588-6596 (2001).</li><li>Piccoli, G., 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=LRRK2+Controls+Synaptic+Vesicle+Storage+and+Mobilization+within+the+Recycling+Pool.">LRRK2 Controls Synaptic Vesicle Storage and Mobilization within the Recycling Pool.</a> Journal of Neuroscience. 31, 2225-2237 (2011).</li><li>Tracy, T. E., Yan, J. J., Chen, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acute+knockdown+of+AMPA+receptors+reveals+a+trans-synaptic+signal+for+presynaptic+maturation.">Acute knockdown of AMPA receptors reveals a trans-synaptic signal for presynaptic maturation.</a> The EMBO Journal. 30, 1577-1592 (2011).</li><li>Truckenbrdot, S., Viplav, A., Jaehne, S., Vogts, A., Denker, A., Wildhagen, H., Fornasiero, E. F., Rizzoli, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ageing+synaptic+vesicles+are+inactivated+by+contamination+with+SNAP25.">Ageing synaptic vesicles are inactivated by contamination with SNAP25.</a> bioRxiv. , 172239 (2017).</li></ol>

Gli autori non hanno nulla a rivelare.

Ci sono tre saggi abitualmente utilizzati per lo studio delle vescicole sinaptiche (SV) riciclaggio. I primi due includono l'uso di un) styryl fluorescente tinture ad esempio FM1-43 (che incorporano nelle membrane, sono presi in organelli durante endocitosi e vengono rilasciati dopo l'esocitosi); e b) etichetta fluorescente proteine ricombinanti SV (che, al momento sovraespressione, incorporano i macchinari riciclaggio proteinico). Se i fluorophores allegata cambiare loro fluorescenza a seconda del pH, essi utilizzabile ...

Lo studio di riciclo delle vescicole sinaptiche è importante nel determinare come modificare proprietà presinaptici, durante plasticità sinaptica o in risposta alla perturbazione della funzione sinaptica. L'assorbimento dell'anticorpo studiando Synaptotagmin-1 (Syt1) fornisce il metodo di misurazione della quantità di riciclaggio SV. Syt1 è una SV-collegata della proteina che agisce come un sensore di Ca2 + ed è necessario per il rilascio esocitotico di neurotrasmettitore1,2. È una proteina transmembrana con un dominio citoplasmico del C-terminale fuori la SV e un dominio luminale del N-terminale all'interno delle SV3. Durante l'esocitosi, il dominio luminale di Syt1 diventa esposte al mezzo esterno. A questo mezzo esterno, aggiungiamo gli anticorpi diretti contro il dominio citoplasmatico, che diventa interiorizzato durante endocitosi. Questi anticorpi possono essere che sia pre-coniugato con fluorofori o immunostained con anticorpi secondari4,5,6,7. L'intensità di fluorescenza del immunosignal risultante è proporzionale alla quantità di riciclaggio SV. Questo approccio può essere utilizzato per determinare sia costitutiva e indotto da depolarizzazione SV riciclaggio6,8.
Saggi di Syt1 l'assorbimento possono essere eseguite dopo virus-mediata del gene transfer a virtualmente tutte le cellule nel piatto o dopo trasfezione sparse di un piccolo numero di cellule. Il nostro metodo combina il dosaggio con doppia sparsa transfezione dei neuroni hippocampal primari con fosfato di calcio9. Usiamo una proteina ricombinante marcatore conosciuta ad accumularsi in presynapses, lo Synaptophysin fluorescente contrassegnato, per individuare i terminali presinaptici e overexpress nostra proteina d'interesse, Rogdi. Questo ci permette di testare o meno Rogdi obiettivi funzionali sinapsi e colpisce SV riciclaggio. Il gene che codifica per Rogdi originalmente è stato identificato in una schermata per mutanti Drosophila caratterizzate da indebolimento della memoria10. Nell'uomo, le mutazioni nel gene Rogdi provocare una rara e devastante malattia chiamata sindrome di Kohlschütter-Tönz. I pazienti soffrono di malformazioni dello smalto dentale, Sturm epilessia e ritardo psicomotorio; Tuttavia, la localizzazione subcellulare del prodotto del gene è rimasto inafferrabile11. Così, l'analisi di assorbimento Syt1 fornito la prova chiave per la localizzazione di GFP-etichettato Rogdi alle sinapsi funzionali9.
Questa tecnica di assorbimento ha diversi vantaggi. In primo luogo, SV riciclaggio possa essere osservato sia in tempo reale eseguendo dal vivo imaging7,12e dopo fissazione6,9 misurando l'intensità della fluorescenza dell'etichetta Syt1 fluorescenza. Inoltre, sono state sviluppate diverse varianti di anticorpo Syt1. Esistono varianti senza tag che possono essere etichettati con un anticorpo secondario seguendo un protocollo standard immunostaining dopo la fissazione e varianti pre-coniugati con un'etichetta di fluorescenza già fissata. Infine, anticorpo-basato fluorescenza è vantaggioso dovuto la vasta selezione di coloranti secondari o coniugati commercialmente disponibile che può essere utilizzato.
Quando fissaggio e immunostaining i neuroni, è anche possibile macchiare per altre proteine ed eseguire analisi di colocalizzazione. Questo può aiutare a determinare dove si trovano in relazione riciclaggio SVs. L'intensità dell'etichetta fluorescenza è la misura diretta della quantità di SV riciclaggio. Inoltre, gli anticorpi etichetta selettivamente strutture contenenti Syt1, con conseguente elevata specificità e piccolo sfondo fluorescenza4. Protocolli di stimolazione differenti possono essere utilizzati, ad esempio buffer di depolarizzazione o stimolazione elettrica protocolli9,12,13,14. Tuttavia, riciclaggio di SV basale può essere misurata senza stimolare le colture neuronali15.
Il nostro metodo affronta specificamente l'assorbimento dell'anticorpo Syt1 nei neuroni double-trasfettate con anticorpo secondario immunolabeling dopo la fissazione. Tuttavia, ci riferiamo a tutte le varianti ordinariamente usate del dosaggio nella nostra discussione di dare agli spettatori l'opportunità di adattare il protocollo alle esigenze specifiche.

<table>
	<tbody>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A7030-50g</td>
			<td></td>
		</tr>
		<tr>
			<td>CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C3306-100g</td>
			<td></td>
		</tr>
		<tr>
			<td>CoolSNAP HQ2</td>
			<td>Photometrics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>dH2O</td>
			<td>Invitrogen</td>
			<td>15230</td>
			<td></td>
		</tr>
		<tr>
			<td>DABCO</td>
			<td>Merck</td>
			<td>8.03456.0100</td>
			<td></td>
		</tr>
		<tr>
			<td>donkey anti mouse Alexa 647</td>
			<td>Jackson-Immunoresearch</td>
			<td>715605151</td>
			<td>antibody</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Invitrogen</td>
			<td>41966</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS</td>
			<td>Gibco</td>
			<td>14190</td>
			<td></td>
		</tr>
		<tr>
			<td>Eppendorf tubes</td>
			<td>Eppendorf</td>
			<td>30120094</td>
			<td></td>
		</tr>
		<tr>
			<td>multiwell 24 well</td>
			<td>Fisher Scientific</td>
			<td>087721H</td>
			<td></td>
		</tr>
		<tr>
			<td>tube (50 mL)</td>
			<td>Greiner Bio-One</td>
			<td>227261</td>
			<td></td>
		</tr>
		<tr>
			<td>FBS superior</td>
			<td>BiochromAG</td>
			<td>S0615</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td>1,083,421,000</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS</td>
			<td>Invitrogen</td>
			<td>14170</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Sigma</td>
			<td>H4034-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>Hera Cell 150 (Inkubator)</td>
			<td colspan="2">ThermoElectron Corporation</td>
			<td></td>
		</tr>
		<tr>
			<td>KCL</td>
			<td>Sigma-Aldrich</td>
			<td>P9541-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Glutamin</td>
			<td>Gibco</td>
			<td>25030</td>
			<td></td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Honeywell</td>
			<td>M0250-500g</td>
			<td></td>
		</tr>
		<tr>
			<td>microscope slides</td>
			<td>Fisher Scientific</td>
			<td>10144633CF</td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Mowiol4-88</td>
			<td>Calbiochem</td>
			<td>475904</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>BioFroxx</td>
			<td>1394KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4</td>
			<td>BioFroxx</td>
			<td>5155KG001</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Invitrogen</td>
			<td>21103049</td>
			<td></td>
		</tr>
		<tr>
			<td>OpenView Experiment Analysis Application</td>
			<td>Free software, see comments</td>
			<td></td>
			<td>written by Noam E. Ziv, Technion &#8211; Israel Institute of Technology, Haifa, Israel</td>
		</tr>
		<tr>
			<td>PBS (10x)</td>
			<td>Roche</td>
			<td>11666789001</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimem</td>
			<td>Invitrogen</td>
			<td>31985</td>
			<td></td>
		</tr>
		<tr>
			<td>Penstrep</td>
			<td>Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr>
			<td>PFA</td>
			<td>Sigma</td>
			<td>P6148-1kg</td>
			<td></td>
		</tr>
		<tr>
			<td>safety hood</td>
			<td>ThermoElectron</td>
			<td></td>
			<td>Serial No. 40649111</td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>neoFroxx</td>
			<td>1104kg001</td>
			<td></td>
		</tr>
		<tr>
			<td>Synaptotagmin1</td>
			<td>Synaptic Systems</td>
			<td>105311</td>
			<td>mouse monoclonal; clone 604.2</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Merck</td>
			<td>1,086,031,000</td>
			<td></td>
		</tr>
		<tr>
			<td>Vortex Genius 3&#160;</td>
			<td>IKA</td>
			<td>3340001</td>
			<td></td>
		</tr>
		<tr>
			<td>Water bath</td>
			<td>GFL</td>
			<td>1004</td>
			<td></td>
		</tr>
		<tr>
			<td>Zeiss Observer. Z1&#160;</td>
			<td>Zeiss</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

an optical assay for synaptic vesicle recycling in cultured neurons overexpressing presynaptic proteins

Alle terminazioni nervose presinaptiche attivo, vescicole sinaptiche sono sottoposti a cicli di exo - ed endocitosi. Durante il riciclaggio, i luminal domini delle proteine transmembrana SV diventano esposti alla superficie delle cellule. Una di queste proteine è Synaptotagmin-1 (Syt1). Un anticorpo diretto contro il dominio luminale di Syt1, una volta aggiunto al terreno di coltura, è ripreso durante il ciclo eso-endocitotico. Questo assorbimento è proporzionale alla quantità di SV riciclaggio e possa essere quantificato mediante immunofluorescenza. Qui, uniamo l'assorbimento di anticorpo Syt1 con doppia transfezione delle colture di neuroni ippocampali. Questo ci permette di (1) localizzare siti presinaptici basati sull'espressione del marcatore presinaptica ricombinante Synaptophysin, (2) determinare la loro funzionalità mediante l'assorbimento Syt1 e (3) caratterizzare il targeting e gli effetti di una proteina di interesse, GFP-Rogdi.

Un risultato atteso di questo approccio è l'individuazione dei circa 50 neuroni trasfettate con doppio al vetrino coprioggetti ad una densità di 50.000 neuroni per pozzetto. L'assone di ogni neurone dovrebbe mostrare più hotspot di etichetta fluorescente Synaptophysin accumulo, che indica i cluster ofSVs. A siti presinaptici funzionali, il segnale lo Synaptophysin ricombinante colocalizza con punctate Syt1 fluorescenza. Verrà co-espressi con ricombinante Synaptophysin mediante transfe...

Watch this Scientific Journal Video about An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins at JoVE.com

An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

Descriviamo un test ottico per vescicole sinaptiche (SV) riciclaggio in colture di neuroni. Combinando questo protocollo con doppia transfezione di esprimere un marcatore presinaptica e la proteina di interesse ci permette di individuare siti presinaptici, loro delle vescicole sinaptiche capacità di riciclaggio e di determinare il ruolo della proteina di interesse.

Un test ottico per riciclo delle vescicole sinaptiche in colture di neuroni che Overexpressing proteine presinaptiche

At active presynaptic nerve terminals, synaptic vesicles undergo cycles of exo- and endocytosis. During recycling, the luminal domains of SV transmembrane ...

an-optical-assay-for-synaptic-vesicle-recycling-cultured-neurons

Research

JoVE Journal

Neuroscience

7.4K Views.  University Medical Centre Göttingen. Descriviamo un test ottico per vescicole sinaptiche (SV) riciclaggio in colture di neuroni. Combinando questo protocollo con doppia transfezione di esprimere un marcatore presinaptica e la proteina di interesse ci permette di individuare siti presinaptici, loro delle vescicole sinaptiche capacità di riciclaggio e di determinare il ruolo della proteina di interesse.

Video: An Optical Assay for Synaptic Vesicle Recycling in Cultured Neurons Overexpressing Presynaptic Proteins

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

Fluorescence Recovery After Photobleaching (FRAP) of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached in the region of interest. The fluorescence of the region recovers when the unbleached GFP-tagged protein from outside of the region diffuses into the region of interest. The mobility of the protein is then analyzed by measuring the fluorescence recovery rate. This technique could be used to characterize protein mobility and turnover rate.
In this study, we express the (enhanced green fluorescent protein) EGFP vector in cultured hippocampal neurons. Using the Zeiss 710 confocal microscope, we photobleach the fluorescence signal of the GFP protein in a single spine, and then take time lapse images to record the fluorescence recovery after photobleaching. Finally, we estimate the percentage of mobile and immobile fractions of the GFP in spines, by analyzing the imaging data using ImageJ and Graphpad softwares.
This FRAP protocol shows how to perform a basic FRAP experiment as well as how to analyze the data.

Fluorescence Recovery After Photobleaching FRAP of Fluorescence Tagged Proteins in Dendritic Spines of Cultured Hippocampal Neurons

FRAP has been used to quantify the mobility of Green Fluorescence Protein (GFP)-tagged proteins in cultured cells. We examined the mobile/immobile fractions of the GFP by analyzing the fluorescence recovery percentage after photobleaching. In this study, FRAP was performed at spines of hippocampal neurons.

Recupero della fluorescenza dopo photobleaching (FRAP) di fluorescenza proteine ​​Tagged in spine dendritiche di colture di neuroni ippocampali

FRAP has been used to quantify the mobility of GFP-tagged proteins. Using a strong excitation laser, the fluorescence of a GFP-tagged protein is bleached ...

Vibrodissociation of Neurons from Rodent Brain Slices to Study Synaptic Transmission and Image Presynaptic Terminals

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of interest. This allows for examination of synaptic transmission under conditions where the extracellular and postsynaptic intracellular environments can be well controlled. A vibration-based technique without the use of proteases, known as vibrodissociation, is the most popular technique for mechanical isolation. A micropipette, with the tip fire-polished to the shape of a small ball, is placed into a brain slice made from a P1-P21 rodent. The micropipette is vibrated parallel to the slice surface and lowered through the slice thickness resulting in the liberation of isolated neurons. The isolated neurons are ready for study within a few minutes of vibrodissociation. This technique has advantages over the use of primary neuronal cultures, brain slices and enzymatically isolated neurons including: rapid production of viable, relatively mature neurons suitable for electrophysiological and imaging studies; superior control of the extracellular environment free from the influence of neighboring cells; suitability for well-controlled pharmacological experiments using rapid drug application and total cell superfusion; and improved space-clamp in whole-cell recordings relative to neurons in slice or cell culture preparations. This preparation can be used to examine synaptic physiology, pharmacology, modulation and plasticity. Real-time imaging of both pre- and postsynaptic elements in the living cells and boutons is also possible using vibrodissociated neurons. Characterization of the molecular constituents of pre- and postsynaptic elements can also be achieved with immunological and imaging-based approaches.

This report demonstrates a technique for mechanical isolation of individual viable neurons retaining attached presynaptic boutons. Vibrodissociated neurons have the advantages of rapid production, excellent pharmacological control and improved space-clamp without influence from neighboring cells. This method can be used for imaging of synaptic elements and patch-clamp recording.

Vibrodissociation di neuroni da fettine di cervello di roditore studio di trasmissione sinaptica e Immagine terminali presinaptici

Mechanical dissociation of neurons from the central nervous system has the advantage that presynaptic boutons remain attached to the isolated neuron of ...

Analysis of Dendritic Spine Morphology in Cultured CNS Neurons

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These structures are rich in actin and have been shown to be highly dynamic. In response to classical Hebbian plasticity 
as well as neuromodulatory signals, dendritic spines can change shape and number, which is thought to be critical for the refinement of neural 
circuits and the processing and storage of information within the brain. Within dendritic spines, a complex network of proteins link extracellular 
signals with the actin cyctoskeleton allowing for control of dendritic spine morphology and number. Neuropathological studies have demonstrated that 
a number of disease states, ranging from schizophrenia to autism spectrum disorders, display abnormal dendritic spine morphology or numbers. 
Moreover, recent genetic studies have identified mutations in numerous genes that encode synaptic proteins, leading to suggestions that these 
proteins may contribute to aberrant spine plasticity that, in part, underlie the pathophysiology of these disorders. In order to study the potential 
role of these proteins in controlling dendritic spine morphologies/number, the use of cultured cortical neurons offers several advantages. Firstly, 
this system allows for high-resolution imaging of dendritic spines in fixed cells as well as time-lapse imaging of live cells. Secondly, this in 
vitro system allows for easy manipulation of protein function by expression of mutant proteins, knockdown by shRNA constructs, or pharmacological 
treatments. These techniques allow researchers to begin to dissect the role of disease-associated proteins and to predict how mutations of these 
proteins may function in vivo.

Numerous recent studies have identified mutations in synaptic proteins associated with brain pathologies. Primary cultured cortical neurons offer great flexibility in examining the effects of these disease-associated proteins on dendritic spine morphology and motility.

Analisi di Morfologia spine dendritiche in colture di neuroni del SNC

Dendritic spines are the sites of the majority of excitatory connections within the brain, and form the post-synaptic 
compartment of synapses. These ...

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 ...

Inducing Dendritic Growth in Cultured Sympathetic Neurons

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs 4-6. Altered patterns of dendritic growth and plasticity are associated with impaired neurobehavioral function in experimental models 7, and are thought to contribute to clinical symptoms observed in both neurodevelopmental disorders 8-10 and neurodegenerative diseases 11-13. Such observations underscore the functional importance of precisely regulating dendritic morphology, and suggest that identifying mechanisms that control dendritic growth will not only advance understanding of how neuronal connectivity is regulated during normal development, but may also provide insight on novel therapeutic strategies for diverse neurological diseases.
Mechanistic studies of dendritic growth would be greatly facilitated by the availability of a model system that allows neurons to be experimentally switched from a state in which they do not extend dendrites to one in which they elaborate a dendritic arbor comparable to that of their in vivo counterparts. Primary cultures of sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rodents provide such a model. When cultured in defined medium in the absence of serum and ganglionic glial cells, sympathetic neurons extend a single process which is axonal, and this unipolar state persists for weeks to months in culture 14,15. However, the addition of either bone morphogenetic protein-7 (BMP-7) 16,17 or Matrigel 18 to the culture medium triggers these neurons to extend multiple processes that meet the morphologic, biochemical and functional criteria for dendrites. Sympathetic neurons dissociated from the SCG of perinatal rodents and grown under defined conditions are a homogenous population of neurons 19 that respond uniformly to the dendrite-promoting activity of Matrigel, BMP-7 and other BMPs of the decapentaplegic (dpp) and 60A subfamilies 17,18,20,21. Importantly, Matrigel- and BMP-induced dendrite formation occurs in the absence of changes in cell survival or axonal growth 17,18.
Here, we describe how to set up dissociated cultures of sympathetic neurons derived from the SCG of perinatal rats so that they are responsive to the selective dendrite-promoting activity of Matrigel or BMPs.

We describe a protocol for using bone morphogenetic protein-7 (BMP-7) or Matrigel to selectively induce dendritic growth in primary sympathetic neurons dissociated from the superior cervical ganglia (SCG) of perinatal rats.

Indurre crescita dendritica in colture di neuroni simpatici

The shape of the dendritic arbor determines the total synaptic input a neuron can receive 1-3, and influences the types and distribution of these inputs ...

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 ...

Examination of Synaptic Vesicle Recycling Using FM Dyes During Evoked, Spontaneous, and Miniature Synaptic Activities

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using styryl FM dyes, which reveal membrane turnover. Conventional protocols for the use of FM dyes were designed for analyzing neurons following stimulated (evoked) synaptic activity. Recently, protocols have become available for analyzing the FM signals that accompany weaker synaptic activities, such as spontaneous or miniature synaptic events. Analysis of these small changes in FM signals requires that the imaging system is sufficiently sensitive to detect small changes in intensity, yet that artifactual changes of large amplitude are suppressed. Here we describe a protocol that can be applied to evoked, spontaneous, and miniature synaptic activities, and use cultured hippocampal neurons as an example. This protocol also incorporates a means of assessing the rate of photobleaching of FM dyes, as this is a significant source of artifacts when imaging small changes in intensity.

We describe the use of styryl FM dyes to image synaptic vesicle recycling in functional nerve terminals. This protocol can be applied not only to evoked, but also spontaneous and miniature synaptic activities. The protocol expands the variety of synaptic events that can be effectively evaluated.

Esame delle vescicole sinaptiche Riciclaggio Uso FM coloranti Durante evocati, spontaneo, e miniatura Attività Synaptic

Synaptic vesicles in functional nerve terminals undergo exocytosis and endocytosis. This synaptic vesicle recycling can be effectively analyzed using ...

Quantification of Endosome and Lysosome Motilities in Cultured Neurons Using Fluorescent Probes

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, survival, and glial communications. To date, numerous studies have reported that defects in these systems cause various neuronal diseases. Thus, understanding the mechanisms underlying vesicle dynamics may provide influential clues that could aid in the treatment of several neuronal disorders. Here, we describe a method for quantifying vesicle motilities, such as motility distance and rate of movement, using a software plug-in for the ImageJ platform. To obtain images for quantification, we labeled neuronal endosome-lysosome structures with EGFP-tagged vesicle marker proteins and observed the movement of vesicles using a time-lapse microscopy. This method is highly useful and simplify measuring vesicle motility in neurites, such as axons and dendrites, as well as in the soma of both neurons and glial cells. Furthermore, this method can be applied to other cell lines, such as fibroblasts and endothelial cells. This approach could provide a valuable advancement of our understanding of membrane trafficking.

The investigation of membrane trafficking is crucial for understanding neuronal functions. Here, we introduce a method for quantifying vesicle motility in neurons. This is a convenient method that can be adapted to the quantification of membrane trafficking in the nervous system.

Quantificazione delle endosomi e dei motivi di lisosomi nei neuroni coltivati ​​utilizzando sonde fluorescenti

In the brain, membrane trafficking systems play important roles in regulating neuronal functions, such as neuronal morphology, synaptic plasticity, ...

Measuring Synaptic Vesicle Endocytosis in Cultured Hippocampal Neurons

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

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