Name: high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses
Uploaded: 2016-02-18T14:00:00.000+00:00
Duration: 9 min 24 s
Description: Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

Questo lavoro è stato sostenuto dai programmi intramurale del National Institute of Neurological Disorder and Stroke (NINDS) e National Institute on Deafness altri disturbi della comunicazione e (NIDCD), del National Institutes of Health (NIH). Ringraziamo l&#39;impianto NINDS EM e il nucleo NIDCD Advanced Imaging (codice # ZIC DC 000.081-03) per l&#39;assistenza.

Cura e trattamento degli animali erano in conformità con NIH cura degli animali e linee guida del Comitato Usa. Postnatale giorno (P) 15-21 ratti Sprague-Dawley, iniettati con 1-1,2% CTB bilateralmente attraverso il collicolo superiore, sono stati mantenuti su un 12: luce 12-hr: ciclo scuro. 1. Fissazione tessuto retinico <ol><li> Montare i seguenti materiali e strumenti: un microscopio da dissezione, 2 pinze con punte molto fini, forbici, carta da filtro di cellulosa, pipetta di plastica e un vetrino da microscopio. </li><li> Anestetizzare ratto in una camera chiusa con 2,0 ml alotano (un anestetico inalante). Determinare un&#39;adeguata anestesia con questi metodi: la mancanza di ritiro della zampa posteriore dopo pizzico punta, o la mancanza di riflesso corneale. Poi decapitare immediatamente con una ghigliottina. Rimuovere gli occhi con un paio di forbici iris e mettere in un piatto di vetro contenente 4% paraformaldeide in tampone fosfato 0,1 M (PB) a pH 7,4. </li><li> Utilizzando il microscopio da dissezione, rimuovere il maisEA tagliando la parte anteriore del bulbo oculare. Rimuovere la lente e vitreale dalla superficie della retina interna con una pinza. </li><li> Sbucciare la sclera con le due pinze fino a quando la retina è isolato dal oculare. </li><li> Tagliare la retina immediatamente in 100 - 200 strisce micron di spessore con un rasoio, e soggetti a fissaggio variazione del pH. </li><li> Fix strisce retina in 4% paraformaldeide in 0.1 M PB a pH 6,0 per 20 - 30 min e poi in 4% paraformaldeide più 0,01% glutaraldeide a pH 10,5 per il 10 - 20 min a temperatura ambiente. </li><li> Dopo alcuni lavaggi in PB con 0.15 mM CaCl 2 (pH 7,4 a 4 ° C), cryoprotect le strisce retinici con glicerolo (60 min ogni a 10%, 20%, 30%, poi O / N a 30%) in 0,1 M PB prima di congelare la sostituzione. </li></ol> 2. Fermo-sostituzione NOTA: Questo metodo freeze-sostituzione viene modificato da un precedente protocollo pubblicato 19,20. Inoltre, è fondamentale che gli strumenti sono molto freddi (indossare guanti); otherwise, il tessuto può scongelare parzialmente quando viene toccato con gli strumenti. Tutti questi passaggi sono fatti all&#39;interno della camera di AFS e gli strumenti sono mai permesso di passare sopra il bordo della camera. Allo stesso modo, il corretto raffreddamento di tutte le sostanze chimiche utilizzate nella AFS è necessario. <ol><li> Tuffatevi-congelare le strisce della retina in propano liquido a -184 ° C in uno strumento EM crioconservazione (CPC). Utilizzando un pennello fine, posizionare i campioni su piccoli pezzi di nastro biadesivo attaccato ai mozziconi di metallo che vanno sulla fine delle aste a strapiombo (stoppino off tampone usando il pennello). </li><li> Plunge le aste in propano liquido e tenere lì per circa 5 secondi, e poi il trasferimento allo strumento freeze-sostituzione automatica (AFS) con una piccola camera di trasporto che viene riempito con azoto liquido ( &#39;LN 2 raffreddato contenitore di trasferimento crio&#39;). </li><li> Dopo aver posizionato il campione congelato e strumenti (pinze e bisturi) nel AFS, raffreddare gli strumenti nella camera per SEminuti veral prima di toccare il tessuto, o raffreddarli ponendo le punte degli strumenti per pochi secondi nella piccola camera di trasporto (riempito con azoto liquido ( &#39;LN 2 raffreddata contenitore di trasferimento crio&#39;)). </li><li> Una volta in AFS, togliere il campione e nastro dalla stub usando una multa bisturi. Inoltre, mantenere il controllo del flusso di gas di azoto, TF (TF è descritto come un &#39;controllo del regolatore per LN 2 vaporizzatore&#39;) aperto durante queste procedure. </li><li> Prima di posizionare il tessuto congelato nei supporti piatti incorporamento (cioè, prima di impostare la AFS), tagliare un cerchio sottile da un foglio di plastica trasparente e posizionarlo nella parte inferiore del supporto in modo da far corrispondere il fondo di ciascun pozzetto. Questo permette relativamente facile rimozione dei blocchi campione polimerizzati quando finito. NOTA: In precedenza, abbiamo utilizzato un metodo alternativo per i titolari di incorporamento piatte, e impiegando doppi Reichert + capsule di gelatina (vedi Petralia e Wenthold, 1999) &lt;sup&gt; 20. </li><li> Utilizzare la seguente sequenza automatica in AFS (usando la terminologia strumento): T1 = -90 ° C per 32 ore, S1 = aumento di temperatura di 4 ° C / ora (11,3 ore), T2 = -45 ° C per 50 ore, S2 = aumento di temperatura di 5 ° C / ora (9 ore), T3 = 0 ° C per 40 ore (totale = 142,3 ore). Si può prendere 2 - 4 ore di inserire campioni nel AFS (a -90 ° C) prima di iniziare la sequenza temporizzata. </li><li> Immergere le sezioni congelate di 1,5% di acetato di uranile in metanolo a -90 ° C per&gt; 32 ore in AFS. Mettere due pezzi di tessuti (in genere) in ciascuno dei sette pozzi nel supporto di alluminio piatto embedding (tre assemblata nel AFS). <ol><li> Posizionare il tessuto + nastro nella acetato di uranile / metanolo nei pozzetti e rimuovere il nastro dopo, appena prima dell&#39;inizio mezzo di inclusione (ad esempio Lowicryl HM20) infiltrazione, se è troppo difficile rimuovere il tessuto dal nastro. </li></ol></li><li> Poi aumentare la graduale della temperatura a -45 ° C (+ 4 ° C per ora; in sequenza automatica). </li><li> Lavare i campioni per tre volte in metanolo pre-raffreddata, utilizzando una pipetta di plastica a punta fine per rimuovere la vecchia soluzione da ciascun titolare piatta-embedding, e quindi utilizzando un&#39;altra pipetta di plastica standard per aggiungere il metanolo fresco preraffreddato. </li><li> Poi, usando lo stesso metodo, infiltrarsi campioni progressivamente con basse resine temperatura incorporamento come mezzo di inclusione (HM20 / metanolo 1: 1 e 2: 1, ciascuno per 2 ore, seguita da resina pura per 2 ore e quindi modificare le resine e mantenere O / N). </li><li> Cambiare la resina nuovamente il giorno successivo, regolando il livello della resina per raggiungere solo al bordo superiore dei pozzetti. </li><li> Polimerizzare i campioni (-45 ° C a 0 ° C in sequenza automatica; + 5 ° C per ora) con luce UV per 40 hr. </li><li> Quindi rimuovere i campioni dal AFS. In genere, i blocchi di esempio mostrano ancora un po &#39;di colore rosa a colori. Posizionare i campioni vicini alla luce fluorescente nella cappa, a RT O / N o più a lungo fino a quando non apporecchio del tutto chiaro. </li></ol> 3. Postembedding EM Immunogold Immunocitochimica NOTA: Postembedding immunocitochimica viene eseguita come descritto 23,24,25. <ol><li> Tagliare 1 micron sezioni con ultramicrotomo, sezioni macchia con 1% toluidina-blu, e li esaminerà per l&#39;orientamento sezione; orientare il blocco di tessuto per ottenere il piano trasversale ottimale di sezionamento. </li><li> Tagliare 70 nm sezioni ultrasottili di spessore con ultramicrotomo e alla loro raccolta di nichel di slot griglie Formvar-carbonio rivestite. </li><li> Lavare griglie una volta in H 2 O distillata O seguita da una soluzione salina tamponata con Tris tre volte (TBS, 0,05 M di tampone Tris, 0.7% NaCl, pH 7,6) lavaggio. </li><li> Incubare griglie 20 gocce microlitri di 5% BSA in TBS per 30 minuti, e poi in 20 gocce microlitri di una miscela contenente capra anti-CTB (1: 3.000) ed un anticorpo per un NMDAR subunità (anti-rabbit GluN2A 01:50 , GluN2B 1:30), o un anti-rabbit GluA2 / 3 (1:30)in TBS-Triton (TBST, 0,01% Triton X-100, pH 7,6) con 2% BSA e 0,02 M NaN 3 O / N a RT. </li><li> griglie Lavare su tre gocce separate (20 microlitri) di TBS (pH 7,6) per 10, 10, e 20 minuti, seguiti da TBS (pH 8,2) per 5 min. </li><li> Incubare griglie per 2 ore in gocce (20 mL) di una miscela contenente asino IgG anti-coniglio (1,20) accoppiata a 10 nm particelle d&#39;oro e IgG asino anti-capra (1:20) accoppiata a particelle d&#39;oro 18 nm in TBST (pH 8.2) con 2% BSA e 0,02 M NaN 3. </li><li> Lavare griglie in 20 gocce microlitri di TBS (pH 7,6) per 5, 5, e 10 min, poi in acqua ultrapura e poi asciugare. </li><li> Griglie di contrasto con il 5% di acetato di uranile e lo 0,3% di piombo citrato di H 2 O distillata per 8 e 5 minuti in condizioni di oscurità, rispettivamente. </li><li> Per gli esperimenti triple-etichettatura, incubare le griglie O / N a temperatura ambiente con 20 gocce microlitri di una miscela di anti-capra CTB (1: 3.000), anti-topo PSD-95 (1: 100), e anti-coniglio GluN2A (1 : 50). Poi incubare griglie per 2 ore su 20 gocce microlitri di una miscela di IgG accoppiata a 18, 10 e 5 particelle di oro nm in TBST (pH 8.2) con 2% BSA e 0,02 M NaN 3. Mantenere le stesse procedure doppia marcatura per il lavaggio e controcolorazione tra e dopo l&#39;incubazione degli anticorpi. </li><li> I controlli sono stati eseguiti in cui gli anticorpi secondari sono stati applicati solo. </li><li> Vedi griglie su un EM e digitalizzare immagini. Elaborare dati definitivi in Adobe Photoshop 6.0 ​​solo per la luminosità ed il contrasto se necessario 24. </li></ol> 4. Quantificazione <ol><li> Selezionare manualmente aree del livello plessiforme interno (IPL), senza buchi o crepe a 8,000X ingrandimento, quindi fotomontaggio a caso tutta la profondità di IPL a 25,000X ingrandimento. </li><li> Identificare dendriti RGC a cono diadi bipolari quando a) contengono il segnale CTB retrograda trasportato (grandi particelle d&#39;oro), b) mostrano ben definito membrane, crepacci, e densità post-sinaptici, c) contengono almeno due piccola parte in oroicles all&#39;interno del PSD, o più di una piccola particella d&#39;oro lungo la membrana plasmatica extrasinaptica. </li><li> Raccogliere 45 - 55 dendriti RGC, sulla base di criteri di cui sopra, per l&#39;analisi quantitativa. </li><li> Misurare le lunghezze del PSD e la membrana plasmatica extrasinaptica dei singoli dendriti RGC con il software NIH ImageJ. Conteggio particelle d&#39;oro entro 10 nm della membrana come associata alla membrana, basato su uno spessore medio di 7 - 9 nm per la membrana plasmatica 26. </li><li> Calcolare la densità di particelle come il numero di particelle d&#39;oro per micrometro lineare (oro / micron). Misurare la distanza tra il centro di ogni particella oro e la metà del PSD (per la posizione sinaptica) o il bordo del PSD (per la posizione extrasinaptica). </li><li> Effettuare analisi statistiche da parte del software. Utilizzare due code t-test per confrontare le medie. Concludere significato quando P &lt;0,05. Se non diversamente indicato, i valori di rapporto come media ± SEM 18. </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+metabotropic+glutamate+receptor+(mGluR1)+is+concentrated+at+perisynaptic+membrane+of+neuronal+subpopulations+as+detected+by+immunogold+reaction.">The metabotropic glutamate receptor (mGluR1) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.</a> Neuron. 11, 771-787 (1993).</li><li>Van Lookeren Campagne, M., Oestreicher, A. B., van der Krift, T. P., Gispen, W. H., Verkleij, A. 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Neurosci. 16, 4457-4467 (1996).</li><li>Petralia, R. 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=Organization+of+NMDA+receptors+at+extrasynaptic+locations.">Organization of NMDA receptors at extrasynaptic locations.</a> Neuroscience. 167, 68-87 (2010).</li><li>Merighi, A., Polak, J. M., Priestley, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Post-embedding+electron+microscopic+immunocytochemistry.">Post-embedding electron microscopic immunocytochemistry.</a> Electron Microscopic Immunocytochemistry: Principles and Practice. , 51-87 (1992).</li><li>Ottersen, O. P., Takumi, Y., Matsubara, A., Landsend, A. S., Laake, J. H., Usami, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+organization+of+a+type+of+peripheral+glutamate+synapse:+the+afferent+synapses+of+hair+cells+in+the+inner+ear.">Molecular organization of a type of peripheral glutamate synapse: the afferent synapses of hair cells in the inner ear.</a> Prog. Neurobiol. 54, 127-148 (1998).</li><li>Nusser, Z., Lujan, R., Laube, G., Roberts, J. D., Molnar, E., Somogyi, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+type+and+pathway+dependence+of+synaptic+AMPA+receptor+number+and+variability+in+the+hippocampus.">Cell type and pathway dependence of synaptic AMPA receptor number and variability in the hippocampus.</a> Neuron. 21, 545-559 (1998).</li><li>Takumi, Y., Ramirez-Leon, V., Laake, P., Rinvik, E., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+modes+of+expression+of+AMPA+and+NMDA+receptors+in+hippocampal+synapses.">Different modes of expression of AMPA and NMDA receptors in hippocampal synapses.</a> Nat. Neurosci. 2, 618-624 (1999).</li><li>Grimes, W. 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=Complex+inhibitory+microcircuitry+regulates+retinal+signaling+near+visual+threshold.">Complex inhibitory microcircuitry regulates retinal signaling near visual threshold.</a> J. Neurophysiol. 114, 341-353 (2015).</li><li>Sassoe-Pognetto, M., Ottersen, O. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+ionotropic+glutamate+receptors+at+dendrodendritic+synapses+in+the+rat+olfactory+bulb.">Organization of ionotropic glutamate receptors at dendrodendritic synapses in the rat olfactory bulb.</a> J. Neurosci. 20, 2192-2201 (2000).</li></ol>

The authors have no disclosures.

Abbiamo descritto quattro tecniche per il successo post-embedding immunogold EM quantitativa: 1) fissazione breve e debole, 2) freeze-sostituzione, 3) post-embedding colorazione immunogold, e 4) la quantificazione. EM immunogold permette il rilevamento di proteine ​​specifiche in sezioni di tessuto ultrasottili. Gli anticorpi marcati con particelle d&#39;oro possono essere visualizzati direttamente usando EM. Mentre potente nel rilevare la localizzazione subsinaptico di un recettore di m...

Il glutammato è il principale neurotrasmettitore eccitatorio nella retina 1. Cellule gangliari della retina (RGCs), che ricevono in ingresso sinaptica glutammatergica da cellule bipolari 2, sono i neuroni di uscita della retina che inviano le informazioni visive al cervello. Studi fisiologici hanno mostrato che l&#39;eccitazione sinaptica di RGCs è mediata da recettori NMDA postsinaptica (NMDARs) e recettori AMPA (AMPARs) 3,4,5. Anche se le correnti post-sinaptiche eccitatorie (EPSCs) in RGCs sono mediate da AMPARs e NMDARs 3,5,6,7,8, EPSCs miniatura spontanee (mEPSCs) su RGCs mostrano solo una componente AMPARs mediata 4,5,9. Tuttavia, la riduzione del glutammato captazione rivelato una componente NMDAR in EPSCs spontanee 5, suggerendo che NMDARs su dendriti RGC possono essere situati al di fuori delle sinapsi eccitatorie. Associata alla membrana guanilato chinasi (MAGUKs) come PSD-95 che i recettori grappolo neurotrasmettitori, tra cui i recettori del glutammato e canale ionicos nei siti sinaptici, mostrano anche diversi modelli di espressione subsinaptico 10,11,12,13,14. Negli ultimi decenni, immunoistochimica confocale e l&#39;incorporamento di pre-microscopio elettronico immunoistochimica (EM) sono stati impiegati per studiare l&#39;espressione del recettore di membrana. Anche se immunostaining confocale rivela ampi schemi di espressione dei recettori, la sua risoluzione più bassa rende impossibile l&#39;utilizzo di distinguere posizione subcellulare. -Embedding Pre studi EM in retina di mammifero indicano che subunità NMDAR sono presenti negli elementi post-sinaptici a cono bipolari sinapsi nastro cellule 15,16,17. Questo è in evidente contrasto con prove fisiologica. Tuttavia, la diffusione del prodotto di reazione è un artefatto noto nel metodo dell&#39;immunoperossidasi pre-embedding. Quindi, questo approccio di solito non dà dati statisticamente affidabili e può escludere distinzione tra la localizzazione di membrana sinaptica contro 18,19,20,21 membrana extrasinaptica. SopraD&#39;altra parte, i dati fisiologici e anatomici sono coerenti con una localizzazione sinaptica di AMPARs su RGCs 3,5,7,9,22. Così, i recettori del glutammato e MAGUKs a sinapsi nastro retinica sono localizzate non solo al postsinaptica ma anche ai compartimenti di membrana perisynaptic o extrasinaptica. Tuttavia, è ancora necessaria una ad alta risoluzione analisi quantitativa di queste proteine ​​di membrana in una sinapsi nastro della retina. Qui, abbiamo sviluppato una tecnica postembedding EM immunogold per esaminare la localizzazione subsinaptico di subunità NMDAR, subunità Ampar e PSD-95 seguita da stimare il numero, la densità e la variabilità di queste proteine ​​a livello delle sinapsi Onto RGCs ratto etichettati utilizzando tossina colerica subunità B (CTB) metodi di tracciamento retrogrado.

<table><tbody><tr><td>Paraformaldehyde</td><td>EMS</td><td>15710</td><td></td></tr><tr><td>Glutaraldehyde</td><td>EMS</td><td>16019</td><td></td></tr><tr><td>NaH2PO4</td><td>Sigma</td><td>S9638</td><td></td></tr><tr><td>Na2HPO4</td><td>Sigma</td><td>7782-85-6</td><td></td></tr><tr><td>CaCl2</td><td>Sigma</td><td>C-8106</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A-7030</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T-8787</td><td></td></tr><tr><td>NaOH</td><td>Sigma</td><td>221465</td><td></td></tr><tr><td>NaN3</td><td>JT Baker</td><td>V015-05</td><td></td></tr><tr><td>Glycerol</td><td>Gibco BRL</td><td>15514-011</td><td></td></tr><tr><td>Lowicryl HM 20</td><td>Polysciences</td><td>15924-1</td><td></td></tr><tr><td>Tris-Base</td><td>Fisher</td><td>BP151-500</td><td></td></tr><tr><td>Tris</td><td>Fisher</td><td>04997-100</td><td></td></tr><tr><td>Anti-GluN2A</td><td>Millipore</td><td>AB1555P</td><td>Dilution 1/50</td></tr><tr><td>Anti-GluN2B</td><td>Millipore</td><td>AB1557P</td><td>Dilution 1/30</td></tr><tr><td>Anti-GluA2/3</td><td>Millipore</td><td>AB1506</td><td>Dilution 1/30</td></tr><tr><td>Anti-PSD-95</td><td>Millipore</td><td>MA1&amp;ndash;046</td><td>Dilution 1/100</td></tr><tr><td>Donkey anti-rabbit IgG-10 nm gold particles</td><td>EMS</td><td>25704</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-10 nm gold particles</td><td>EMS</td><td>25814</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-mouse IgG-5 nm gold particles</td><td>EMS</td><td>25812</td><td>Dilution 1/20</td></tr><tr><td>Donkey anti-goat IgG-18 nm gold particles</td><td>Jackson ImmunoResearch</td><td>705-215-147</td><td>Dilution 1/20</td></tr><tr><td>Formvar-Carbon coated nickel-slot grids.</td><td>EMS</td><td>FCF2010-Ni</td><td></td></tr><tr><td>Uranyl acetate</td><td>EMS</td><td>22400-1</td><td></td></tr><tr><td>Methanol</td><td>EMS</td><td>67-56-1</td><td></td></tr><tr><td>Lead citrate</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM AFS</td><td>Leica</td><td></td><td></td></tr><tr><td>Leica EM CPC</td><td>Leica</td><td></td><td></td></tr><tr><td>Ultramicrotome</td><td>Leica</td><td></td><td></td></tr><tr><td>JEOL 1200 EM</td><td>JEOL</td><td></td><td></td></tr><tr><td>liquid nitrogen&amp;nbsp;</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>Propane</td><td>Roberts Oxygen</td><td></td><td></td></tr><tr><td>CTB</td><td>List Biological Laboratories</td><td>104</td><td>1 - 1.2%</td></tr><tr><td>Anti-CTB</td><td>List Biological Laboratories</td><td>703</td><td>Dilution 1/4,000</td></tr></tbody></table>

high-resolution quantitative immunogold analysis of membrane receptors at retinal ribbon synapses

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

I risultati qui presentati dimostrano sorprendentemente diversi modelli di localizzazione subsinaptico di GluA 2/3 e NMDARs sui dendriti RGC in retina di ratto, come descritto in precedenza 24,25. 77% di GluA 2/3 particelle immunogold in RGC profili dendritiche erano situati all&#39;interno del PSD (Figura 1A), simile alla maggior sinapsi centrali. Tuttavia, NMDARs erano situati sia sinapticamente o extrasynaptically. 83% di particelle immunogold GluN2A stati ...

Watch this Scientific Journal Video about High-Resolution Quantitative Immunogold Analysis of Membrane Receptors at Retinal Ribbon Synapses at JoVE.com

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

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

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

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

high-resolution-quantitative-immunogold-analysis-membrane-receptors

Research

JoVE Journal

Neuroscience

7.5K Views.  National Institutes of Health. The postembedding immunogold method is one of the most effective ways to provide high-resolution analyses of the subcellular localization of specific molecules. Here we describe a protocol to quantitatively analyze glutamate receptors at retinal ribbon synapses.

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

Quantifying Synapses: an Immunocytochemistry-based Assay to Quantify Synapse Number

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become imperative to develop objective approaches to quantify changes in synaptic connectivity. Starting from sample fixation, this protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers. The number of these colocalizations is quantified using a plug in Puncta Analyzer (written by Bary Wark, available upon request, c.eroglu@cellbio.duke.edu) under the ImageJ analysis software platform. The synapse assay described in this protocol can be applied to any neural tissue or culture preparation for which you have selective pre- and postsynaptic markers. This synapse assay is a valuable tool that can be widely utilized in the study of synaptic development.

This protocol details how to quantify synapse number both in dissociated neuronal culture and in brain sections using immunocytochemistry. Using compartment-specific antibodies, we label presynaptic terminals as well as sites of postsynaptic specialization. We define synapses as points of colocalization between the signals generated by these markers.

Sinapsi quantificare: un Immunocitochimica basato su analisi per quantificare Numero Synapse

One of the most important goals in neuroscience is to understand the molecular cues that instruct early stages of synapse formation. As such it has become ...

Ricerca

Neuroscienze

Imaging Exocytosis in Retinal Bipolar Cells with TIRF Microscopy

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective imaging of fluorescent molecules that are closest to a high refractive index substance such as glass1. In this article, we apply this technique to image exocytosis of synaptic vesicles in retinal bipolar cells isolated from the goldfish retina. These neurons are very suitable for this kind of study due to their large axon terminals. By simultaneously patch clamping the bipolar cells, it is possible to investigate the relationship between pre-synaptic voltage and synaptic release2,3. Synaptic vesicles inside the bipolar cell terminals are loaded with a fluorescent dye (FM 1-43&reg;) by co-puffing the dye and a ringer solution containing a high K+ concentration onto the synaptic terminals. This depolarizes the cells and stimulates endocytosis and consequent dye uptake into the glutamatergic vesicles. After washing the excess dye away for around 30 minutes, cells are ready for being patch clamped and imaged simultaneously with a 488 nm laser. The patch pipette solution contains a rhodamine-based peptide that binds selectively to the synaptic ribbon protein RIBEYE4, thereby labeling ribbons specifically when terminals are imaged with a 561 nm laser. This allows the precise localization of active zones and the separation of synaptic from extra-synaptic events.

In this video, we demonstrate how to label and visualize single synaptic vesicle exocytosis and trafficking in goldfish retinal bipolar cells using total internal reflectance fluorescence (TIRF) microscopy.

Esocitosi imaging in cellule bipolari della retina con microscopia TIRF

Total internal reflectance fluorescence (TIRF) microscopy is a technique that allows the study of events happening at the cell membrane, by selective ...

Biologia

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 ...

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for immunohistochemistry and, 2) calcium imaging for the study of voltage gated calcium channel (VGCC) mediated calcium signaling in retinal ganglion cells. The calcium imaging method we describe circumvents issues concerning non-specific loading of displaced amacrine cells in the ganglion cell layer.

Immunohistochemistry protocols are used to study the localization of a specific protein in the retina. Calcium imaging techniques are employed to study calcium dynamics in retinal ganglion cells and their axons.

Metodi di imaging immunoistochimici e di calcio in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

Combined Optogenetic and Freeze-fracture Replica Immunolabeling to Examine Input-specific Arrangement of Glutamate Receptors in the Mouse Amygdala

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to study the molecular composition of membranes produced a significant decline in its use. Recently, there has been a major revival in freeze-fracture electron microscopy thanks to the development of effective ways to reveal integral membrane proteins by immunogold labeling. One of these methods is known as detergent-solubilized Freeze-fracture Replica Immunolabeling (FRIL).
The combination of the FRIL technique with optogenetics allows a correlated analysis of the structural and functional properties of central synapses. Using this approach it is possible to identify and characterize both pre- and postsynaptic neurons by their respective expression of a tagged channelrhodopsin and specific molecular markers. The distinctive appearance of the postsynaptic membrane specialization of glutamatergic synapses further allows, upon labeling of ionotropic glutamate receptors, to quantify and analyze the intrasynaptic distribution of these receptors. Here, we give a step-by-step description of the procedures required to prepare paired replicas and how to immunolabel them. We will also discuss the caveats and limitations of the FRIL technique, in particular those associated with potential sampling biases. The high reproducibility and versatility of the FRIL technique, when combined with optogenetics, offers a very powerful approach for the characterization of different aspects of synaptic transmission at identified neuronal microcircuits in the brain.
Here, we provide an example how this approach was used to gain insights into structure-function relationships of excitatory synapses at neurons of the intercalated cell masses of the mouse amygdala. In particular, we have investigated the expression of ionotropic glutamate receptors at identified inputs originated from the thalamic posterior intralaminar and medial geniculate nuclei. These synapses were shown to relay sensory information relevant for fear learning and to undergo plastic changes upon fear conditioning.

This article illustrates how the expression of neurotransmitter receptors can be quantified and the pattern analyzed at synapses with identified pre and postsynaptic elements using a combination of viral transduction of optogenetic tools and the freeze-fracture replica immunolabeling technique.

Combinato optogenetic e Freeze-frattura Replica immunomarcatura per esaminare specifica disposizione-Input di glutammato recettori nell&#39;amigdala mouse

Freeze-fracture electron microscopy has been a major technique in ultrastructural research for over 40 years. However, the lack of effective means to ...

Imaging the Human Immunological Synapse

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) and an effector helper T lymphocyte (Th) cell, and to record the images corresponding to the first stages of the IS formation and the subsequent trafficking events (occurring both in the APC and in the Th cell). These events will eventually lead to polarized secretion at the IS. In this protocol, Jurkat cells challenged with Staphylococcus enterotoxin E (SEE)-pulsed Raji cells as a cell synapse model was used, because of the closeness of this experimental system to the biological reality (Th cell-APC synaptic conjugates). The approach presented here involves cell-to-cell conjugation, time-lapse acquisition, wide-field fluorescence microscopy (WFFM) followed by image processing (post-acquisition deconvolution). This improves the signal-to-noise ratio (SNR) of the images, enhances the temporal resolution, allows the synchronized acquisition of several fluorochromes in emerging synaptic conjugates and decreases fluorescence bleaching. In addition, the protocol is well matched with the end point cell fixation protocols (paraformaldehyde, acetone or methanol), which would allow further immunofluorescence staining and analyses. This protocol is also compatible with laser scanning confocal microscopy (LSCM) and other state-of-the-art microscopy techniques. As a main caveat, only those T cell-APC boundaries (called IS interfaces) that were at the right 90&#176; angle to the focus plane along the Z-axis could be properly imaged and analyzed. Other experimental models exist that simplify imaging in the Z dimension and the following image analyses, but these approaches do not emulate the complex, irregular surface of an APC, and may promote non-physiological interactions in the IS. Thus, the experimental approach used here is suitable to reproduce and to confront some biological complexities occurring at the IS.

This protocol images both the immunological synapse formation and the subsequent polarized secretory traffic towards the immunological synapse. Cellular conjugates were formed between a superantigen-pulsed Raji cell (acting as an antigen-presenting cell) and a Jurkat clone (acting as an effector helper T lymphocyte).

Immaginare la sinapsi immunologica umana

The purpose of the method is to generate an immunological synapse (IS), an example of cell-to-cell conjugation formed by an antigen-presenting cell (APC) ...

Immunologia e infezioni

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

<meta charset="utf8"></head><body>Protocollo ottimizzato per la preparazione di campioni di organoidi retinici per TEM

Preparing Retinal Organoid Samples for Transmission Electron Microscopy

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem cells (iPSCs) under specific conditions. Synapse development and maturation in ROs have been studied immunocytochemically and functionally. However, the direct evidence of the synaptic contact ultrastructure is limited, containing both special ribbon synapses and conventional chemical synapses. Transmission electron microscopy (TEM) is characterized by high resolution and a respectable history elucidating retinal development and synapse maturation in humans and various species. It is a powerful tool to explore synaptic structure in ROs and is widely used in the research field of ROs. Therefore, to better explore the structure of RO synaptic contacts at the nanoscale and obtain high-quality microscopic evidence, we developed a simple and repeatable method of RO TEM sample preparation. This paper describes the protocol, reagents used, and detailed steps, including RO fixation preparation, post fixation, embedding, and visualization.

<meta charset="utf8"></head><body>Autore in primo piano: Analisi dell'ultrastruttura sinaptica in organoidi retinici maturi utilizzando la TEM

This protocol provides an optimized and elaborate preparation procedure for retinal organoid samples for transmission electron microscopy. It is suitable for applications that involve the analysis of synapses in mature retinal organoids.

Preparazione di campioni di organoidi retinici per la microscopia elettronica a trasmissione

Retinal organoids (ROs) are a three-dimensional culture system mimicking human retinal features that have differentiated from induced pluripotent stem ...

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

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Capitoli in questo video

Sinapsi quantificare: un Immunocitochimica basato su analisi per quantificare Numero Synapse

Esocitosi imaging in cellule bipolari della retina con microscopia TIRF

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

Metodi di imaging immunoistochimici e di calcio in Wholemount Rat Retina

Combinato optogenetic e Freeze-frattura Replica immunomarcatura per esaminare specifica disposizione-Input di glutammato recettori nell'amigdala mouse

Immaginare la sinapsi immunologica umana

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Preparazione di campioni di organoidi retinici per la microscopia elettronica a trasmissione

Preparazione di campioni di organoidi retinici per la microscopia elettronica a trasmissione

Preparazione di campioni di organoidi retinici per la microscopia elettronica a trasmissione

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

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Capitoli in questo video

Sinapsi quantificare: un Immunocitochimica basato su analisi per quantificare Numero Synapse

Esocitosi imaging in cellule bipolari della retina con microscopia TIRF

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

Metodi di imaging immunoistochimici e di calcio in Wholemount Rat Retina

Combinato optogenetic e Freeze-frattura Replica immunomarcatura per esaminare specifica disposizione-Input di glutammato recettori nell'amigdala mouse

Immaginare la sinapsi immunologica umana

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Preparazione di campioni di organoidi retinici per la microscopia elettronica a trasmissione

Preparazione di campioni di organoidi retinici per la microscopia elettronica a trasmissione

Preparazione di campioni di organoidi retinici per la microscopia elettronica a trasmissione

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