Gli autori vorrebbero riconoscere NanoVentures Australia per sostegno finanziario di viaggio e il Prof. John Haycock per aver ospitato in parte questa ricerca presso l&#39;Università di Sheffield e la signora Jaimee Mayne per il suo aiuto durante le riprese.

1. Au NR Preparazione Nota: Au NR può essere sintetizzato da un numero di ricette 16, o acquistati da fornitori commerciali. <ol><li> Misurare la densità ottica iniziale (OD) della soluzione Au NR tramite spettroscopia UV-Vis, registrando i valori di assorbimento da 300 nm a 1000 nm con una risoluzione di 0,5-2 nm. Variare il volume della soluzione da usare con la cuvetta disponibili. </li><li> Valutare la concentrazione iniziale NP molare con una tecnica 17 Ideale (ad esempio la spettroscopia UV-Vis, singola particella ICP-MS, microscopia elettronica a trasmissione) o utilizzare i valori di concentrazione forniti dal venditore. </li><li> Preparare una soluzione madre 5 ml diluendo il campione iniziale Au NR raggiungere un OD = 1. Per una migliore ripetibilità, mantenere costante OD per tutti i campioni testati. Seguire protocollo del fornitore commerciale per la composizione del solvente di diluizione. Se non sei sicuro, Usare acqua deionizzata. </li><li> Centrifugare 1 ml della soluzione Au NR due volte per 15 minuti a 7800 xg per rimuovere qualsiasi eccesso chimico dalla soluzione. Cicli di centrifugazione può variare in vigore e nel tempo (ad esempio, 20 min a 5.600 × g) 18. </li><li> Rimuovere il surnatante e risospendere le NRs in acqua deionizzata. Come particelle risospeso potrebbero formare aggregati in soluzione, prepararli giorno per avere i migliori risultati. In alternativa, conservare in frigorifero per non più di 1 settimana. Non congelare. </li><li> Prima di utilizzare in condizioni di coltura cellulare, sonicare la soluzione Au NR per 5 minuti e poi sterilizzare con la luce UV per 30 minuti (intensità di radiazione UV non meno di 400 mW ∙ m -2 a 254 nm). </li></ol> 2. NG108-15 cellule neuronali Linea Cultura e differenziazione <ol><li> Per mezzo di coltura cellulare, preparare 500 ml di mezzo sterile modificato di Dulbecco Eagle (DMEM) contenente 10% (w / v) di siero fetale bovino (FCS), 1% (w / v) L-glutammina, 1%(W / v) di penicillina / streptomicina e 0,5% (w / v) anfotericina B. Nota: Gli integratori possono essere aliquotati, conservati a -20 ° C e aggiunti ai media il giorno desiderato. Mezzo di coltura cellulare può essere refrigerato in condizioni sterili per un massimo di 1 mese. </li><li> Per il mezzo differenziazione cellulare, preparare 50 ml di DMEM sterile contenente 1% (w / v) L-glutammina, 1% (w / v) di penicillina / streptomicina e 0,5% (w / v) anfotericina B. </li><li> Grow NG108-15 cellule neuronali in 10 ml di mezzo di coltura cellulare in fiasche T75 in polistirolo in un incubatore con un&#39;atmosfera umidificata (5% CO 2 a 37 ° C). Normalmente, sementi 1,5-2 x 10 5 cellule in ogni contenitore per essere pronto in 3-4 giorni. Cambiare mezzo di coltura cellulare ogni due giorni. Nota: per evitare derive genetiche o di variazione, non usare le cellule di età superiore a 21 passaggio per gli esperimenti. </li><li> Quando 70-80% confluenti nella cultura, cambia il mezzo con mezzo di caldo coltura cellulare fresca. Meccanicamente staccare le cellule delicatamente Knocre il fondo del pallone confluenti. Non usare tripsina. </li><li> Centrifugare la sospensione cellulare per 5 minuti a 600 xg e risospendere il pellet cellulare in 2 ml di mezzo caldo differenziazione cellulare. </li><li> Seed 2 × 10 4 cellule / cm 2 in una piastra di coltura tissutale polistirene ben 96 con 200 ml di terreno di differenziamento cellulare. Incubare l&#39;esperimento per 1 giorno al 5% di CO 2/37 ° C. </li><li> Aggiungi tra 3,2 × 10 9 -4.2 × 1 0 10 particelle / ml di soluzione Au NR il giorno 2 ed incubare per ulteriori 24 ore. Non aggiungere le particelle per gli esperimenti di controllo. Nota: Come controllo alternativo, Au NP con un picco d&#39;onda di assorbimento ben differenziati possono essere utilizzati ai fini del confronto 14. Per un comportamento cellulare più coerente, mantenere la densità costante di cella e non modificare la superficie ben prima della semina delle cellule. </li></ol> 3. crescita dei neuriti Enhancement <ol><li> Coppiail laser con una fibra ottica monomodale (apertura numerica = 0.13) e terminare con un connettore in fibra (connettori FC sono convenienti e comunemente disponibili). Misurare la potenza del laser di uscita con un misuratore di potenza standard. Per ottenere risultati ottimali, abbinare il picco di lunghezza d&#39;onda del laser al picco di risonanza plasmonica di NR. </li><li> Il giorno 3 dopo incubazione NR, fissare il connettore FC al pozzo. Irradiare campioni ed i controlli a temperatura ambiente per 1 minuto in onda continua, per le diverse potenze laser. Lasciare che la cultura di procedere per 3 giorni aggiuntivi in 5% CO 2/37 ° C. Ripetere la radiazione laser per un minimo di 3 misurazioni indipendenti. Nota: differenti tempi di irradiazione e possono essere selezionati frequenze impulsi, a seconda dell&#39;applicazione. </li><li> Caratterizzano il laser in termini di diametro del raggio e di irraggiamento laser (W ∙ cm -2). <ol><li> Per una singola fibra modalità standard, utilizzare NA = n ∙ sin θ,dove n è l&#39;indice di rifrazione del mezzo in uso e θ è la metà-angolo del cono di luce uscenti dalla fibra (vedere Figura 1A). Da trigonometria, r = tan θ ∙ d, dove r è il raggio del fascio e d è la distanza tra la fibra e il campione. Il connettore FC corrisponde al diametro del pozzo, quindi illuminando il campione ad una distanza d = 2,70 ± 0,20 mm. Luce esce la fibra in acqua (n = 1.33), dando r = tan (sin-1 (NA / n)) ∙ d. </li><li> Utilizzando quest&#39;ultima equazione, calcolare il raggio del fascio laser, l&#39;area corrispondente trave e l&#39;irradianza media del laser (potenza del laser divisa per l&#39;area del fascio) sul bersaglio (si veda il grafico esempio nella Figura 1B). Questi valori rappresentano l&#39;irraggiamento medio sopra l&#39;area illuminata. </li><li> Valutare gli errori per le misure con la teoria generale di propagazione degli errori. Nota: La distanza bra il connettore FC e il campione possono essere misurati con fotografie del dispositivo sperimentale e post-elaborazione delle immagini utilizzando il software appropriato (per esempio ImageJ). </li></ol></li><li> Al giorno 5, rimuovere terreno di differenziamento cellulare dagli esperimenti e fissare i campioni con 3,7% (v / v) di formaldeide per 10 minuti, poi permeabilize le cellule con 0,1% (v / v) Triton X-100 per 20 min. </li><li> Aggiungere 3% (w / v) di albumina di siero bovino (BSA) per i campioni per 60 min per bloccare siti di legame della proteina non reagito. Etichettare i campioni per il pernottamento anti-βIII-tubulina (5 mg / ml in PBS integrato con 1% di BSA) a 4 ° C. </li><li> Incubare le cellule per 90 minuti al buio con un anticorpo secondario appropriato (topo anti-anticorpo es TRITC-coniugato IgG) utilizzando una concentrazione di 0,4-2 mg / ml in 1% BSA in PBS. Etichettare i nuclei delle cellule con DAPI (0,1 mg / ml in acqua deionizzata) per 10 minuti. Nota: coniugazione di anticorpi e concentrazione migHT variano secondo il protocollo aziendale. Lavare i campioni con PBS due volte per 5 minuti dopo ogni fase di colorazione. <img alt="Figura 1" src="/files/ftp_upload/52566/52566fig1.jpg" /></li><li> Campioni Immagine di epifluorescenza o microscopia confocale utilizzando almeno un 20 × obiettivo. Scegli il microscopio filtri di conseguenza per l&#39;anticorpo secondario. Selezionare un filtro DAPI (λ = 358 nm EX; λ = EM 488 nm) per visualizzare i nuclei delle cellule. </li><li> Analizzare le immagini valutando: i) la lunghezza massima dei neuriti (registrare la lunghezza dalla punta del neurite all&#39;inizio del corpo cellulare), ii) il numero di neuriti per cellula neuronale (riassumere tutti i neuriti per cella) e iii) la percentuale di cellule con neuriti (dividere il numero totale di cellule che esprimono βIII-tubulina per il numero totale di cellule con un nucleo tinto positivamente) 19. </li></ol> 4. laser-indotta intracellulare Ca 2+ Imaging <ol&gt; <li> Preparare un 20% (w / v) di soluzione stock di Pluronic F-127 sciogliendo 2 g di soluto in 10 ml di dimetilsolfossido anidro (DMSO). Riscaldare la soluzione di Pluronic F-127 a 40 ° C per circa 20 minuti per aumentare la solubilità. Preparare in anticipo se conservato a temperatura ambiente. </li><li> Il giorno 3 dopo NR incubazione, preparare una soluzione salina bilanciata (BSS; 135 mM NaCl, 4,5 mM KCl, CaCl 1,5 mm 2, MgCl 0,5 mm 2, 5.6 mM glucosio, e HEPES 10 mM, pH 7.4) e integrato con 5 pM di Fluo-4 sono nella DMSO e 0,1% (w / v) di soluzione Pluronic F-127. Nota: soluzione BSS può essere preparata in anticipo e refrigerato per un massimo di 1 mese. Aggiungere i supplementi (Fluo-4 AM ed Pluronic F-127), il giorno dell&#39;esperimento. </li><li> Rimuovere terreno di differenziamento cellulare e sostituirlo con la soluzione BSS integrata. Per ottenere i migliori risultati con un microscopio confocale invertito, incubare le cellule in un micro-pozzetto. </li><li> Carica NG108-15 cellule con Fluo-04:00per 30 minuti al buio a 5% di CO 2/37 ° C. Dopo il tempo di incubazione, lavare campioni due volte con BSS per rimuovere eventuali fluoroforo scarico extracellulare. </li><li> Coppia il laser con una fibra ottica monomodale e fende la punta utilizzando tecniche standard. Osservare la punta risultante sotto un microscopio ottico, per assicurare una superficie di alta qualità (cioè la punta deve essere perpendicolare all&#39;asse della fibra e piatto su controllo microscopia). <ol><li> Misurare la potenza del laser di uscita con un misuratore di potenza standard. Inserire la fibra di consegna luce in un supporto in fibra ottica e applicarla ad un micropositioner. Fare riferimento a Brown et al. 20 per maggiori dettagli sulla preparazione fibra ottica e il posizionamento. Attenzione: Seguire le regole generali per la sicurezza laser durante la misurazione della potenza del laser (per esempio non guardare direttamente il raggio laser, indossare occhiali di sicurezza laser durante la manipolazione, evitare l&#39;esposizione alla luce diffusa ad altri utenti del laboratorio) &lt;./ Li&gt; </ol></li><li> Collegare un oscilloscopio per il laser portatile per monitorare la modulazione ottica. Utilizzare un segnale binario con frequenze variabili (0,5-2 Hz) e lunghezze di impulso (20-100 msec). Collegare il laser utilizzando il segnale di modulazione come logica transistor-transistor (TTL) ingresso per microscopio, in seguito alla configurazione mostrata in Paviolo et al. 13. <ol><li> Collocare le cellule con un microscopio confocale invertito e posizionare la fibra consegna luce 250 ± 50 micron dalla cellula bersaglio in modalità di trasmissione di illuminazione. A questa distanza, utilizzare le equazioni presentati in 3.3.1 per calcolare il raggio del fascio sul bersaglio. </li></ol></li><li> Utilizzare un laser ad argon-ion (488 nm) per eccitare l&#39;interiorizzato Fluo-04:00 dye (λ = 494 nm EX; λ PN = 516 nm) e la LD sincronizzato per l&#39;eccitazione delle NRs endocitosi. Eseguire la immagini a RT di campioni e controlli utilizzando almeno un obiettivo 40 × raccogliendo scansioni time-seriescon 256 × 256 pixel / risoluzione fotogramma in modalità di andata e ritorno con una frequenza di almeno 4 Hz. Eseguire una registrazione senza alcuna eccitazione LD per identificare qualsiasi interferenza laser argon-ion (rumore di fondo). </li><li> Rimuovere lo sfondo dei dati utilizzando adattivo ponderati penalizzati minimi quadrati algoritmi 21. Tracciare le indotte Ca 2+ variazioni in funzione del tempo. Analizzare i picchi transitori di intensità di fluorescenza utilizzando immagine software di post-elaborazione per soglia ad un livello tre volte la deviazione standard del rumore di fondo (3σ). </li></ol>

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	<li>Richter, C. P., Matic, A. I., Wells, J. D., Jansen, E. D., Walsh, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stimulation+with+optical+radiation.">Neural stimulation with optical radiation.</a> Laser. Photonics Rev. 5 (1), 68-80 (2011).</li><li>Dittami, G. M., Rajguru, S. M., Lasher, R. A., Hitchcock, R. W., Rabbitt, R. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+calcium+transients+evoked+by+pulsed+infrared+radiation+in+neonatal+cardiomyocytes.">Intracellular calcium transients evoked by pulsed infrared radiation in neonatal cardiomyocytes.</a> J. Physiol. 589 (6), 1295-1306 (2011).</li><li>Thompson, A. C., Wade, S. A., Brown, W. G. A., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+light+absorption+in+tissue+during+infrared+neural+stimulation.">Modeling of light absorption in tissue during infrared neural stimulation.</a> J. Biomed. Opt. 17 (7), 075002-075002 (2012).</li><li>Thompson, A. C., Wade, S. A., Cadusch, P. J., Brown, W. G., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+of+the+temporal+effects+of+heating+during+infrared+neural+stimulation.">Modeling of the temporal effects of heating during infrared neural stimulation.</a> J. Biomed. Opt. 18 (3), 035004 (2013).</li><li>Huang, H., Delikanli, S., Zeng, H., Ferkey, D. M., Pralle, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+control+of+ion+channels+and+neurons+through+magnetic-field+heating+of+nanoparticles.">Remote control of ion channels and neurons through magnetic-field heating of nanoparticles.</a> Nat. Nanotechnol. 5 (8), 602-606 (2010).</li><li>Farah, 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=Holographically+patterned+activation+using+photo-absorber+induced+neural-thermal+stimulation.">Holographically patterned activation using photo-absorber induced neural-thermal stimulation.</a> J. Neural. Eng. 10 (5), (2013).</li><li>Ciofani, 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=Enhancement+of+neurite+outgrowth+in+neuronal-like+cells+following+boron+nitride+nanotube-mediated+stimulation.">Enhancement of neurite outgrowth in neuronal-like cells following boron nitride nanotube-mediated stimulation.</a> ACS Nano. 4 (10), 6267-6277 (2010).</li><li>Kim, J. 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=Enhancement+of+neurite+outgrowth+in+PC12+cells+by+iron+oxide+nanoparticles.">Enhancement of neurite outgrowth in PC12 cells by iron oxide nanoparticles.</a> Biomaterials. 32 (11), 2871-2877 (2011).</li><li>Myroshnychenko, V., 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=Modelling+the+optical+response+of+gold+nanoparticles.">Modelling the optical response of gold nanoparticles.</a> Chem. Soc. Rev. 37 (9), 1792-1805 (2008).</li><li>Choi, W. I., Sahu, A., Kim, Y. H., Tae, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photothermal+cancer+therapy+and+imaging+based+on+gold+nanorods.">Photothermal cancer therapy and imaging based on gold nanorods.</a> Ann. Biomed. Eng. 40 (2), 534-546 (2011).</li><li>Zhan, Q., Qian, J., Li, X., He, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+study+of+mesoporous+silica-encapsulated+gold+nanorods+as+enhanced+light+scattering+probes+for+cancer+cell+imaging.">A study of mesoporous silica-encapsulated gold nanorods as enhanced light scattering probes for cancer cell imaging.</a> Nanotechnology. 21 (5), 055704 (2010).</li><li>Paviolo, 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=Laser+exposure+of+gold+nanorods+can+increase+neuronal+cell+outgrowth.">Laser exposure of gold nanorods can increase neuronal cell outgrowth.</a> Biotechnol. Bioeng. 110 (8), 2277-2291 (2013).</li><li>Paviolo, C., Haycock, J. W., Cadusch, P. J., McArthur, S. L., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laser+exposure+of+gold+nanorods+can+induce+intracellular+calcium+transients.">Laser exposure of gold nanorods can induce intracellular calcium transients.</a> J. Biophotonics. 7 (10), 761-765 (2014).</li><li>Yong, 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=Gold-nanorod-assisted+near-infrared+stimulation+of+primary+auditory+neurons.">Gold-nanorod-assisted near-infrared stimulation of primary auditory neurons.</a> Adv. Healthcare Mater. , (2014).</li><li>Eom, 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=Enhanced+infrared+neural+stimulation+using+localized+surface+plasmon+resonance+of+gold+nanorods.">Enhanced infrared neural stimulation using localized surface plasmon resonance of gold nanorods.</a> Small. , (2014).</li><li>Juste, J., Pastoriza-Santos, I., Liz-Marz&#225;n, L. M., Mulvaney, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanorods:+Synthesis,+characterization+and+applications.">Gold nanorods: Synthesis, characterization and applications.</a> Coordination Chemistry Reviews. (17-18), 1870-1901 (2005).</li><li>Shang, J., Gao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoparticle+counting:+towards+accurate+determination+of+the+molar+concentration.">Nanoparticle counting: towards accurate determination of the molar concentration.</a> Chem. Soc. Rev. 43 (21), 7267-7278 (2014).</li><li>Sharma, V., Park, K., Srinivasarao, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shape+separation+of+gold+nanorods+using+centrifugation.">Shape separation of gold nanorods using centrifugation.</a> Proc. Natl. Acad. Sci. 106 (13), 4981-4985 (2009).</li><li>Kaewkhaw, R., Scutt, A. M., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anatomical+site+influences+the+differentiation+of+adipose-derived+stem+cells+for+schwann-cell+phenotype+and+function.">Anatomical site influences the differentiation of adipose-derived stem cells for schwann-cell phenotype and function.</a> Glia. 59 (5), 734-749 (2011).</li><li>Brown, W. G. A., Needham, K., Nayagam, B. A., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+cell+patch+clamp+for+investigating+the+mechanisms+of+infrared+neural+stimulation.">Whole cell patch clamp for investigating the mechanisms of infrared neural stimulation.</a> JoVE. (77), (2013).</li><li>Cadusch, P. J., Hlaing, M. M., Wade, S. A., McArthur, S. L., Stoddart, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+methods+for+fluorescence+background+subtraction+from+Raman+spectra.">Improved methods for fluorescence background subtraction from Raman spectra.</a> J. Raman Spectrosc. 44 (11), 1587-1595 (2013).</li><li>Daud, M. F. B., Pawar, K. C., Claeyssens, F., Ryan, A. J., Haycock, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+aligned+3D+neuronal-glial+co-culture+model+for+peripheral+nerve+studies.">An aligned 3D neuronal-glial co-culture model for peripheral nerve studies.</a> Biomaterials. 33 (25), 5901-5913 (2012).</li><li>Jung, 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=Intracellular+gold+nanoparticles+increase+neuronal+excitability+and+aggravate+seizure+activity+in+the+mouse+brain.">Intracellular gold nanoparticles increase neuronal excitability and aggravate seizure activity in the mouse brain.</a> PLoS ONE. 9 (3), e91360 (2014).</li><li>Salinas, K., Kereselidze, Z., DeLuna, F., Peralta, X., Santamaria, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transient+extracellular+application+of+gold+nanostars+increases+hippocampal+neuronal+activity.">Transient extracellular application of gold nanostars increases hippocampal neuronal activity.</a> J. Nanobiotechnology. 12 (1), 31 (2014).</li><li>Ebbesen, C. L., Bruus, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+laser-induced+heating+in+optical+neuronal+guidance.">Analysis of laser-induced heating in optical neuronal guidance.</a> J. Neurosci. Meth. 209 (1), 168-177 (2012).</li><li>Iwanaga, 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=Location-dependent+photogeneration+of+calcium+waves+in+HeLa+cells.">Location-dependent photogeneration of calcium waves in HeLa cells.</a> Cell Biochem. Biophys. 45 (2), 167-176 (2006).</li><li>Huang, X., Jain, P. K., El-Sayed, I. H., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+the+minimum+temperature+required+for+selective+photothermal+destruction+of+cancer+cells+with+the+use+of+immunotargeted+gold+nanoparticles.">Determination of the minimum temperature required for selective photothermal destruction of cancer cells with the use of immunotargeted gold nanoparticles.</a> Photochem. Photobiol. 82 (2), 412-417 (2006).</li><li>Connor, E. E., Mwamuka, J., Gole, A., Murphy, C. J., Wyatt, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanoparticles+are+taken+up+by+human+cells+but+do+not+cause+acute+cytotoxicity.">Gold nanoparticles are taken up by human cells but do not cause acute cytotoxicity.</a> Small. 1 (3), 325-327 (2005).</li><li>Isomaa, B., Reuter, J., Djupsund, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+subacute+and+chronic+toxicity+of+cetyltrimethylammonium+bromide+(CTAB),+a+cationic+surfactant,+in+the+rat.">The subacute and chronic toxicity of cetyltrimethylammonium bromide (CTAB), a cationic surfactant, in the rat.</a> Arch. Toxicol. 35 (2), 91-96 (1976).</li><li>Juste, J., Pastoriza-Santos, I., Liz-Marz&#225;n, L., Mulvaney, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gold+nanorods:+synthesis,+characterization+and+applications.">Gold nanorods: synthesis, characterization and applications.</a> Coord. Chem. Rev. 249 (17-18), 1870-1901 (2005).</li><li>Dreaden, E. C., Alkilany, A. M., Huang, X., Murphy, C. J., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+golden+age:+gold+nanoparticles+for+biomedicine.">The golden age: gold nanoparticles for biomedicine.</a> Chem. Soc. Rev. 41 (7), 2740-2779 (2012).</li><li>Huang, X., Jain, P. K., El-Sayed, I. H., El-Sayed, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasmonic+photothermal+therapy+(PPTT)+using+gold+nanoparticles.">Plasmonic photothermal therapy (PPTT) using gold nanoparticles.</a> Lasers Med. Sci. 23 (3), 217-228 (2008).</li><li>Albert, E. 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=TRPV4+channels+mediate+the+infrared+laser-evoked+response+in+sensory+neurons.">TRPV4 channels mediate the infrared laser-evoked response in sensory neurons.</a> J. Neurophysiol. 107 (12), 3227-3234 (2012).</li><li>Garcia-Elias, 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=Phosphatidylinositol-4,5-biphosphate-dependent+rearrangement+of+TRPV4+cytosolic+tails+enables+channel+activation+by+physiological+stimuli.">Phosphatidylinositol-4,5-biphosphate-dependent rearrangement of TRPV4 cytosolic tails enables channel activation by physiological stimuli.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (23), 9553-9558 (2013).</li><li>Shapiro, M. G., Homma, K., Villarreal, S., Richter, C. -. P., Bezanilla, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Infrared+light+excites+cells+by+changing+their+electrical+capacitance.">Infrared light excites cells by changing their electrical capacitance.</a> Nat. Commun. 3 (736), (2012).</li><li>Roggan, A., Friebel, M., D&#246;rschel, K., Hahn, A., M&#252;ller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+properties+of+circulating+human+blood+in+the+wavelength+range+400-2500+nm.">Optical properties of circulating human blood in the wavelength range 400-2500 nm.</a> J. Biomed. Opt. 4 (1), 36-46 (1999).</li><li>Byrnes, K. R., 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=Light+promotes+regeneration+and+functional+recovery+and+alters+the+immune+response+after+spinal+cord+injury.">Light promotes regeneration and functional recovery and alters the immune response after spinal cord injury.</a> Lasers Surg. Med. 36 (3), 171-185 (2005).</li><li>Wu, X., 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=810+nm+wavelength+light:+an+effective+therapy+for+transected+or+contused+rat+spinal+cord.">810 nm wavelength light: an effective therapy for transected or contused rat spinal cord.</a> Lasers Surg. Med. 41 (1), 36-41 (2009).</li><li>Grossman, N., Schneid, N., Reuveni, H., Halevy, S., Lubart, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=780+nm+low+power+diode+laser+irradiation+stimulates+proliferation+of+keratinocyte+cultures:+involvement+of+reactive+oxygen+species.">780 nm low power diode laser irradiation stimulates proliferation of keratinocyte cultures: involvement of reactive oxygen species.</a> Lasers Surg. Med. 22 (4), 212-218 (1998).</li><li>Wong-Riley, M. T. 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=Photobiomodulation+directly+benefits+primary+neurons+functionally+inactivated+by+toxins+-+Role+of+cytochrome+c+oxidase.">Photobiomodulation directly benefits primary neurons functionally inactivated by toxins - Role of cytochrome c oxidase.</a> J. Biol. Chem. 280 (6), 4761-4771 (2005).</li><li>Beauvoit, B., Kitai, T., Chance, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+mitochondrial+compartment+to+the+optical+properties+pf+the+rat+liver:+a+theoretical+and+practical+approach.">Contribution of the mitochondrial compartment to the optical properties pf the rat liver: a theoretical and practical approach.</a> Biophys. J. 67 (6), 2501-2510 (1994).</li></ol>

The authors have no competing interests to disclose.

I protocolli descritti in questa presentazione descrivono come la cultura, differenziare e otticamente stimolare le cellule neuronali utilizzando assorbitori estrinseci. Le caratteristiche NR (ad esempio le dimensioni, la forma, risonanza plasmonica di lunghezza d&#39;onda e chimica di superficie) ei parametri di stimolazione laser (come la lunghezza d&#39;onda, la lunghezza di impulso, frequenza di ripetizione, ecc) possono essere variate per soddisfare diverse esigenze sperimentali. Gli effetti sul c...

Recenti studi hanno dimostrato che il riscaldamento transitoria associata con l&#39;assorbimento di luce infrarossa da acqua (lunghezza d&#39;onda&gt; 1.400 nm) può essere utilizzato per indurre potenziali di azione nel tessuto nervoso 1 e transienti di calcio intracellulare in cardiomiociti 2. L&#39;uso di luce infrarossa ha suscitato notevole interesse per applicazioni in protesi neurali, a causa della potenziale risoluzione spaziale più fine, di un contatto diretto con il tessuto, la minimizzazione degli artefatti di stimolazione, e la rimozione della necessità di modificare geneticamente le cellule prima della stimolazione ( come richiesto in optogenetics) 1. Nonostante tutti questi vantaggi, modelli termici recentemente sviluppati suggerito che i destinatari dei tessuti / cellule possono essere influenzati da effetti di riscaldamento cumulativi, quando più siti di stimolo e / o alti tassi di ripetizione sono utilizzati 3,4. In risposta a queste sfide, i ricercatori hanno riconosciuto il potenziale di utilizzare absor estrinsecabri per la stimolazione del nervo a produrre effetti di riscaldamento più localizzati nel tessuto. Huang et al. Ha dimostrato questo principio usando nanoparticelle di ferrite superparamagnetiche per attivare da remoto i canali TRPV1 sensibili alla temperatura in HEK 293 cellule con una radio-frequenza di campo magnetico 5. Sebbene questa tecnica può consentire una penetrazione più profonda (campi magnetici relativamente interagiscono debolmente con tessuto), le risposte sono state registrate solo su periodi di secondi, piuttosto che le durate di millisecondi richiesti dispositivi bionici 5. Allo stesso modo, Farah et al. Stimolazione elettrica dimostrato di neuroni corticali di ratto con neri micro particelle in vitro. Hanno mostrato di precisione a livello di cella nella stimolazione con durate di impulso dell&#39;ordine di centinaia di ms ed energie nella gamma di μJ, potenzialmente permette tassi di ripetizione più veloce 6. L&#39;uso di assorbitori estrinseci è stata applicata anche per indurrecambiamenti morfologici in vitro. Ciofani et al. Hanno evidenziato un aumento del ~ 40% nella crescita neuronale utilizzando piezoelettrici boro nitruro nanotubi eccitato da ultrasuoni 7. Analogamente, ferro endocitosi nanoparticelle di ossido in cellule PC12 sono stati riportati per migliorare la differenziazione dei neuriti in modo dose-dipendente, causa l&#39;attivazione di molecole di adesione delle cellule con l&#39;ossido di ferro 8. Recentemente, l&#39;interesse assorbitori estrinseci per assistere stimolazione neurale è anche concentrato sull&#39;uso di nanoparticelle di oro (Au NP). Au NP hanno la capacità di assorbire efficacemente luce laser al picco plasmonica e dissiparlo nell&#39;ambiente circostante nella forma di calore 9. Tra tutte le forme di particelle disponibili, l&#39;assorbimento ottico di nanotubi d&#39;oro (Au NR) corrisponde comodamente la finestra terapeutica di tessuti biologici (vicino infrarosso - NIR, lunghezza d&#39;onda tra 750-1,400 nm) 10. Inoltre, nel context di stimolazione neurale, l&#39;uso di Au NR offre biocompatibilità relativamente favorevole e una vasta gamma di opzioni di funzionalizzazione superficiale 11. Recenti studi hanno dimostrato che un effetto stimolante sulla differenziazione può essere indotta dopo esposizione laser continue di Au NR in NG108-15 cellule neuronali 12. Allo stesso modo, transienti intracellulari di calcio sono stati registrati in cellule neuronali coltivate con Au NRs dopo irradiazione laser modulato con frequenze variabili e lunghezze di impulso 13. Membrana cellulare depolarizzazione si è registrato anche dopo l&#39;illuminazione laser NIR di Au NRs in colture primarie di neuroni gangliari spirale 14. La prima applicazione in vivo con irradiato Au NR è stata dimostrata solo di recente. EOM e collaboratori esposti Au NRs al loro picco plasmonica e hanno registrato un aumento di sei volte in ampiezza dei potenziali d&#39;azione del nervo composto (CNaPS) e una diminuzione di tre volte nella soglia di stimolazione in ratti nervi sciatico. L&#39;enrisposta ampliate è stato attribuito a effetti di riscaldamento locali derivanti dalla eccitazione del NR picco plasmonica 15. Nel presente lavoro, vengono specificati protocolli per studiare gli effetti della stimolazione laser a NG108-15 cellule neuronali coltivate con Au NR. Questi metodi forniscono una semplice, ma potente, modo di irradiare popolazioni cellulari in vitro usando tecniche biologiche standard e materiali. Il protocollo si basa su un diodo laser accoppiato in fibra (LD) che permette un funzionamento sicuro e ripetibile allineamento. La preparazione del campione e laser metodi di irraggiamento Au NR possono essere ulteriormente estesi a diverse forme di particelle e colture cellulari neuronali, a condizione che si conoscono, rispettivamente, i protocolli di sintesi e di cultura specifica.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Au NR</td>
			<td>Sigma Aldrich</td>
			<td>716812</td>
			<td></td>
		</tr>
		<tr>
			<td>NG108-15</td>
			<td>Sigma Aldrich</td>
			<td>8811230</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Sigma Aldrich</td>
			<td>D6546</td>
			<td></td>
		</tr>
		<tr>
			<td>FCS</td>
			<td>Life Technologies</td>
			<td>10100147</td>
			<td></td>
		</tr>
		<tr>
			<td>L-glutamine</td>
			<td>Sigma Aldrich</td>
			<td>G7513</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Life Technologies</td>
			<td>15140122</td>
			<td></td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Life Technologies</td>
			<td>15290018</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma Aldrich</td>
			<td>F8775</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>BDH</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma Aldrich</td>
			<td>A2058</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#946;III-tubulin</td>
			<td>Promega</td>
			<td>G7121</td>
			<td></td>
		</tr>
		<tr>
			<td>TRITC-conjugated anti-mouse IgG antibody</td>
			<td>Sigma Aldrich</td>
			<td>T5393</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Invitrogen</td>
			<td>D1306</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluo-4 AM</td>
			<td>Invitrogen</td>
			<td>F14201</td>
			<td></td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Sigma Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Pluronic F-127</td>
			<td>Invitrogen</td>
			<td>P6867</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment name</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td></td>
		</tr>
		<tr>
			<td>UV-Vis spectrometer</td>
			<td>Varian Medical Systems Inc.</td>
			<td>Cary 50 Bio</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini centrifuge</td>
			<td>Eppendorf</td>
			<td>Mini Spin</td>
			<td></td>
		</tr>
		<tr>
			<td>Sonic bath</td>
			<td>Unisonics Australia</td>
			<td>FPX 10D</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture incubator</td>
			<td>Kendro</td>
			<td>Hera Cell 150</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell culture centrifuge</td>
			<td>Hettich</td>
			<td>Rotofix 32A</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser diode</td>
			<td>Optotech</td>
			<td>780 nm single mode fibre - coupled LD</td>
			<td></td>
		</tr>
		<tr>
			<td>Optical fiber</td>
			<td>Thorlabs</td>
			<td>780 HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Power meter</td>
			<td>Coherent</td>
			<td>Laser Check</td>
			<td></td>
		</tr>
		<tr>
			<td>ImageJ</td>
			<td>http://rsb.info.nih.gov/ij/index.html</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluorescent microscope</td>
			<td>Axon Instruments</td>
			<td>ImageX-press 5000A</td>
			<td></td>
		</tr>
		<tr>
			<td>&#956;-slide well</td>
			<td>Ibidi</td>
			<td>80826</td>
			<td></td>
		</tr>
		<tr>
			<td>Inverted confocal microscope</td>
			<td>Carl Zeiss Microscopy Ltd.</td>
			<td>LSM 510 meta-confocal microscope</td>
			<td></td>
		</tr>
		<tr>
			<td>Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS210</td>
			<td></td>
		</tr>
	</tbody>
</table>

gold nanorod-assisted optical stimulation of neuronal cells

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared light by water in neuronal tissue. This technique holds great potential for replacing or complementing standard stimulation techniques, due to the potential for increased localization of the stimulus and minimization of mechanical contact with the tissue. However, optical approaches are limited by the inability of visible light to penetrate deep into tissues. Moreover, thermal modelling suggests that cumulative heating effects might be potentially hazardous when multiple stimulus sites or high laser repetition rates are used. The protocol outlined below describes an enhanced approach to the infrared stimulation of neuronal cells. The underlying mechanism is based on the transient heating associated with the optical absorption of gold nanorods, which can cause triggering of neuronal cell differentiation and increased levels of intracellular calcium activity. These results demonstrate that nanoparticle absorbers can enhance and/or replace the process of infrared neural stimulation based on water absorption, with potential for future applications in neural prostheses and cell therapies.

Utilizzando protocolli 1, 2, e 3 qui descritte, un effetto stimolante sulla differenziazione è stata osservata in NG108-15 cellule neuronali coltivate con NP Au (Au NR, poli (styrenesulfonate) Rivestiti Au NRs e silice rivestite Au NR) dopo laser esposizioni tra 1,25 e 7,5 W · cm -2. Immagini confocale di rhodamineB marcato Au NRs dimostrato che le particelle sono stati internalizzati dal giorno 1 di incubazione 12. La localizzazione è stata osservata prevalentemente nel citoplasma cellulare, in...

Watch this Scientific Journal Video about Gold Nanorod-assisted Optical Stimulation of Neuronal Cells at JoVE.com

Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Oro nanorod-assistito stimolazione ottica di cellule neuronali

Recent studies have demonstrated that nerves can be stimulated in a variety of ways by the transient heating associated with the absorption of infrared ...

gold-nanorod-assisted-optical-stimulation-of-neuronal-cells

Research

JoVE Journal

Neuroscience

8.9K Views.  Swinburne University of Technology. This protocol outlines how to use the transient heating associated with the optical absorption of gold nanorods to stimulate differentiation and intracellular calcium activity in neuronal cells. These results potentially open up new applications in neural prostheses and fundamental studies in neuroscience.

Video: Gold Nanorod-assisted Optical Stimulation of Neuronal Cells

Combining Transcranial Magnetic Stimulation and fMRI to Examine the Default Mode Network

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful rest."1,2,3 It is thought the default mode network corresponds to self-referential or "internal mentation".2,3
It has been hypothesized that, in humans, activity within the default mode network is correlated with certain pathologies (for instance, hyper-activation has been linked to schizophrenia 4,5,6 and autism spectrum disorders 7 whilst hypo-activation of the network has been linked to Alzheimer's and other neurodegenerative diseases 8). As such, noninvasive modulation of this network may represent a potential therapeutic intervention for a number of neurological and psychiatric pathologies linked to abnormal network activation. One possible tool to effect this modulation is Transcranial Magnetic Stimulation: a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability (either increasing or decreasing it) via the application of localized magnetic field pulses.9
In order to explore the default mode network's propensity towards and tolerance of modulation, we will be combining TMS (to the left inferior parietal lobe) with functional magnetic resonance imaging (fMRI). Through this article, we will examine the protocol and considerations necessary to successfully combine these two neuroscientific tools.

In this article, we examine the methodology and considerations relevant to the combination of TMS and fMRI to examine the effects of brain stimulation on the default network.

La combinazione di stimolazione magnetica transcranica e fMRI per esaminare il modo predefinito della rete

The default mode network is a group of brain regions that are active when an individual is not focused on the outside world and the brain is at "wakeful ...

Ricerca

Neuroscienze

TMS: Using the Theta-Burst Protocol to Explore Mechanism of Plasticity in Individuals with Fragile X Syndrome and Autism

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS display abnormalities in the expression of FMR1 - a protein required for typical, healthy neural development.3 Recent data has suggested that the loss of this protein can cause the cortex to be hyperexcitable thereby affecting overall patterns of neural plasticity.4,5
In addition, Fragile X shows a strong comorbidity with autism: in fact, 30% of children with FXS are diagnosed with autism, and 2 - 5% of autistic children suffer from FXS.6
Transcranial Magnetic Stimulation (a non-invasive neurostimulatory and neuromodulatory technique that can transiently or lastingly modulate cortical excitability via the application of localized magnetic field pulses 7,8) represents a unique method of exploring plasticity and the manifestations of FXS within affected individuals. More specifically, Theta-Burst Stimulation (TBS), a specific stimulatory protocol shown to modulate cortical plasticity for a duration up to 30 minutes after stimulation cessation in healthy populations, has already proven an efficacious tool in the exploration of abnormal plasticity.9,10 
Recent studies have shown the effects of TBS last considerably longer in individuals on the autistic spectrum - up to 90 minutes.11 This extended effect-duration suggests an underlying abnormality in the brain's natural plasticity state in autistic individuals - similar to the hyperexcitability induced by Fragile X Syndrome.
In this experiment, utilizing single-pulse motor-evoked potentials (MEPs) as our benchmark, we will explore the effects of both intermittent and continuous TBS on cortical plasticity in individuals suffering from FXS and individuals on the Autistic Spectrum.

In this article, we examine the effects of Theta-Burst TMS stimulation on cortical plasticity in individuals suffering from Fragile X syndrome and individuals on the autistic spectrum.

TMS: Utilizzo del Theta Burst protocollo Esplora Mechasnism di plasticità in individui con Sindrome X Fragile e autismo

Fragile X Syndrome (FXS), also known as Martin-Bell Syndrome, is a genetic abnormality found on the X chromosome.1,2 Individuals suffering from FXS ...

The NeuroStar TMS Device: Conducting the FDA Approved Protocol for Treatment of Depression

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic fields. These rapidly alternating fields induce electrical currents within localized, targeted regions of the cortex which are associated with various physiological and functional brain changes.1,2,3
In 2007, O'Reardon et al., utilizing the NeuroStar device, published the results of an industry-sponsored, multisite, randomized, sham-stimulation controlled clinical trial in which 301 patients with major depression, who had previously failed to respond to at least one adequate antidepressant treatment trial, underwent either active or sham TMS over the left dorsolateral prefrontal cortex (DLPFC). The patients, who were medication-free at the time of the study, received TMS five times per week over 4-6 weeks.4
The results demonstrated that a sub-population of patients (those who were relatively less resistant to medication, having failed not more than two good pharmacologic trials) showed a statistically significant improvement on the Montgomery-Asberg Depression Scale (MADRS), the Hamilton Depression Rating Scale (HAMD), and various other outcome measures. In October 2008, supported by these and other similar results5,6,7, Neuronetics obtained the first and only Food and Drug Administration (FDA) approval for the clinical treatment of a specific form of medication-refractory depression using a TMS Therapy device (FDA approval K061053).
In this paper, we will explore the specified FDA approved NeuroStar depression treatment protocol (to be administered only under prescription and by a licensed medical profession in either an in- or outpatient setting).

In this article, we examine the methodology and considerations relevant to the FDA approved depression treatment protocol using the Neuronetics NeuroStar TMS device.

Il dispositivo TMS NeuroStar: Condurre la FDA ha approvato protocollo per il trattamento della depressione

The Neuronetics NeuroStar Transcranial Magnetic Stimulation (TMS) System is a class II medical device that produces brief duration, pulsed magnetic ...

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding fundamental biological mechanisms. Striking progress has been particularly evident in Drosophila, whose extensive toolkit of mutants and transgenic lines provides a convenient model to study evolutionarily-conserved developmental and cell biological mechanisms. We are interested in understanding the mechanisms that control cell fate specification in the adult peripheral nervous system (PNS) in Drosophila. Bristles that cover the head, thorax, abdomen, legs and wings of the adult fly are individual mechanosensory organs, and have been studied as a model system for understanding mechanisms of Notch-dependent cell fate decisions. Sensory organ precursor (SOP) cells of the microchaetes (or small bristles), are distributed throughout the epithelium of the pupal thorax, and are specified during the first 12 hours after the onset of pupariation. After specification, the SOP cells begin to divide, segregating the cell fate determinant Numb to one daughter cell during mitosis. Numb functions as a cell-autonomous inhibitor of the Notch signaling pathway.
Here, we show a method to follow protein dynamics in SOP cell and its progeny within the intact pupal thorax using a combination of tissue-specific Gal4 drivers and GFP-tagged fusion proteins 1,2.This technique has the advantage over fixed tissue or cultured explants because it allows us to follow the entire development of an organ from specification of the neural precursor to growth and terminal differentiation of the organ. We can therefore directly correlate changes in cell behavior to changes in terminal differentiation. Moreover, we can combine the live imaging technique with mosaic analysis with a repressible cell marker (MARCM) system to assess the dynamics of tagged proteins in mitotic SOPs under mutant or wildtype conditions. Using this technique, we and others have revealed novel insights into regulation of asymmetric cell division and the control of Notch signaling activation in SOP cells (examples include references 1-6,7 ,8).

Live-cell Imaging of Sensory Organ Precursor Cells in Intact Drosophila Pupae

In this video, we describe a method for live cell imaging of asymmetrically dividing sensory organ progenitor cells and epidermal cells in intact Drosophila pupae

Live-cellula Imaging di cellule sensoriali Precursore in Organo intatto Drosophila Pupae

Since the discovery of Green Fluorescent Protein (GFP), there has been a revolutionary change in the use of live-cell imaging as a tool for understanding ...

Optical Control of a Neuronal Protein Using a Genetically Encoded Unnatural Amino Acid in Neurons

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to photo-regulate endogenous proteins expressed in their native environment. Here, we present an approach to optically control the function of a neuronal protein directly in neurons using a genetically encoded unnatural amino acid (Uaa). By using an orthogonal tRNA/aminoacyl-tRNA synthetase pair to suppress the amber codon, a photo-reactive Uaa 4,5-dimethoxy-2-nitrobenzyl-cysteine (Cmn) is site-specifically incorporated in the pore of a neuronal protein Kir2.1, an inwardly rectifying potassium channel. The bulky Cmn physically blocks the channel pore, rendering Kir2.1 non-conducting. Light illumination instantaneously converts Cmn into a smaller natural amino acid Cys, activating Kir2.1 channel function. We express these photo-inducible inwardly rectifying potassium (PIRK) channels in rat hippocampal primary neurons, and demonstrate that light-activation of PIRK ceases the neuronal firing due to the outflux of K+ current through the activated Kir2.1 channels. Using in utero electroporation, we also express PIRK in the embryonic mouse neocortex in vivo, showing the light-activation of PIRK in neocortical neurons. Genetically encoding Uaa imposes no restrictions on target protein type or cellular location, and a family of photoreactive Uaas is available for modulating different natural amino acid residues. This technique thus has the potential to be generally applied to many neuronal proteins to achieve optical regulation of different processes in brains. The current protocol presents an accessible procedure for intricate Uaa incorporation in neurons in vitro and in vivo to achieve photo control of neuronal protein activity on the molecular level.

Here, a procedure to selectively activate a neuronal protein with a short pulse of light by genetically encoding a photo-reactive unnatural amino acid into a target neuronal protein expressed in neurons in culture or in vivo is presented.

Controllo ottico di una proteina neuronale Utilizzando un acido Innaturale Amino geneticamente codificata in neuroni

Photostimulation is a noninvasive way to control biological events with excellent spatial and temporal resolution. New methods are desired to ...

Time-dependent Increase in the Network Response to the Stimulation of Neuronal Cell Cultures on Micro-electrode Arrays

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network dynamics in neuronal cultures. In contrast with more traditional methods, such as patch-clamping, which can only record activity from a single cell, MEAs can record simultaneously from multiple sites in a network, without requiring the arduous task of placing each electrode individually. Moreover, numerous control and stimulation configurations can be easily applied within the same experimental setup, allowing for a broad range of dynamics to be explored. One of the key dynamics of interest in these in vitro studies has been the extent to which cultured networks display properties indicative of learning. Mouse neuronal cells cultured on MEAs display an increase in response following training induced by electrical stimulation. This protocol demonstrates how to culture neuronal cells on MEAs; successfully record from over 95% of the plated dishes; establish a protocol to train the networks to respond to patterns of stimulation; and sort, plot, and interpret the results from such experiments. The use of a proprietary system for stimulating and recording neuronal cultures is demonstrated. Software packages are also used to sort neuronal units. A custom-designed graphical user interface is used to visualize post-stimulus time histograms, inter-burst intervals, and burst duration, as well as to compare the cellular response to stimulation before and after a training protocol. Finally, representative results and future directions of this research effort are discussed.

Mouse neuronal cells cultured on multi-electrode arrays display an increase in response following electrical stimulation. This protocol demonstrates how to culture neurons, how to record activity, and how to establish a protocol to train the networks to respond to patterns of stimulation.

Aumento del tempo della risposta alla rete alla stimolazione delle colture cellulari neuronali sulle matrici di microelettrodi

Micro-electrode arrays (MEAs) can be used to investigate drug toxicity, design paradigms for next-generation personalized medicine, and study network ...

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in vivo&#8212;rapidly, repetitively, and at a high resolution. This protocol describes OCT imaging in the mouse retina as a powerful tool to study optic neuropathies (OPN). The OCT system is an interferometry-based, non-invasive alternative to common post mortem histological assays. It provides a fast and accurate assessment of retinal thickness, allowing the possibility to track changes, such as retinal thinning or thickening. We present the imaging process and analysis with the example of the Opa1delTTAG mouse line. Three types of scans are proposed, with two quantification methods: standard and homemade calipers. The latter is best for use on the peripapillary retina during radial scans; being more precise, is preferable for analyzing thinner structures. All approaches described here are designed for retinal ganglion cells (RGC) but are easily adaptable to other cell populations. In conclusion, OCT is efficient in mouse model phenotyping and has the potential to be used for the reliable evaluation of therapeutic interventions.

Optical Coherence Tomography: Imaging Mouse Retinal Ganglion Cells In Vivo

This manuscript describes a protocol for the in vivo imaging of the mouse retina with high-resolution spectral domain optical coherence tomography (SD-OCT). It focuses on retinal ganglion cells (RGC) in the peripapillary region, with several scanning and quantifying approaches described.

Tomografia a coerenza ottica: Imaging Mouse ganglionari cellule In Vivo

Structural changes in the retina are common manifestations of ophthalmic diseases. Optical coherence tomography (OCT) enables their identification in ...

Chronic Transcranial Electrical Stimulation and Intracortical Recording in Rats

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a closed-loop event-triggered manner. Although many studies are focusing on the possible benefits and side-effects of TES in healthy and pathologic brains, there are still many fundamental open questions regarding the mechanism of action of the stimulation. Therefore, there is a clear need for a robust and reproducible method to test the acute and the chronic effects of TES in rodents. TES can be combined with regular behavioral, electrophysiological, and imaging techniques to investigate neuronal networks in vivo. The implantation of transcranial stimulation electrodes does not impose extra constraints on the experimental design while it offers a versatile, flexible tool to manipulate brain activity. Here we provide a detailed, step-by-step protocol to fabricate and implant transcranial stimulation electrodes to influence brain activity in a temporally constrained manner for months.

This detailed protocol describes transcranial stimulation electrode placement on the temporal bone in order to investigate the short- and long-term effects of transcranial electrical stimulation in freely moving rats.

Stimolazione elettrica transcranica cronica e registrazione intracorticale in ratti

Transcranial electrical stimulation (TES) is a powerful and relatively simple approach to diffusely influence brain activity either randomly or in a ...

Neuronal Differentiation from Mouse Embryonic Stem Cells In vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and potentially aid in regenerative medicine. Here, we established an efficient and low cost method for neuronal differentiation from mESCs in vitro, using the strategy of combinatorial screening. Under the conditions defined here, the 2-day embryoid body formation + 6-day retinoic acid induction protocol permits fast and efficient differentiation from mESCs into neural precursor cells (NPCs), as seen by the formation of well-stacked and neurite-like A2lox and 129 derivatives that are Nestin positive. The healthy state of embryoid bodies and the timepoint at which retinoic acid (RA) is applied, as well as the RA concentrations, are critical in the process. In the subsequent differentiation from NPCs into neurons, N2B27 medium II (supplemented by Neurobasal medium) could better support the long term maintenance and maturation of neuronal cells. The presented method is highly efficiency, low cost and easy to operate, and can be a powerful tool for neurobiology and developmental biology research.

Here, we established a low cost and easy to operate method that directs fast and efficient differentiation from embryonic stem cells into neurons. This method is suitable for popularization among laboratories and can be a useful tool for neurological research.

Differenziazione neuronale dalle cellule staminali embrionali del topo in vitro

The neural differentiation of mouse embryonic stem cells (mESCs) is a potential tool for elucidating the key mechanisms involved in neurogenesis and ...

GM-Free Generation of Blood-Derived Neuronal Cells

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other therapeutic strategies, there is currently no cure available for the injured or diseased brain. Cell replacement appears as a promising therapeutic strategy for neurodegenerative conditions. To this day, neural stem cells (NSCs) have been successfully generated from fetal tissues, human embryonic cells (ES) or induced pluripotent stem cells (iPSC). A process of dedifferentiation was initiated by activation of the novel human GPI-linked glycoprotein, which leads to generation of pluripotent stem cells. These blood-derived pluripotent stem cells (BD-PSCs) differentiate in vitro into cells with a neural phenotype as shown by brightfield and immunofluorescence microscopy. Ultrastructural analysis of these cells by means of electron microscopy confirms their primitive structure as well as neuronal-like morphology and subcellular characteristics.

We present a genetically modified-free (GM-free) method to obtain cells with a neuronal phenotype from reprogrammed peripheral blood cells. Activation of a signaling pathway linked to novel human GPI-linked protein reveals an efficient GM-free method for obtaining human pluripotent stem cells.

Generazione priva di OGM di cellule neuronali derivate dal sangue

Many human&#160;neurological disorders are caused by degeneration of neurons and glial cells in the brain. Due to limitations in pharmacological and other ...