Finanziato dal NIH P40 OD010952, la Fondazione Korein, una Università di Miami Fellowship a SLC e una borsa di studio per Maytag ATK. Gli autori ringraziano il personale delle Risorse Nazionale per l&#39;Aplysia, così come Lauren Simonitis e Hannah Peck, che ha fornito al microscopio per una figura.

1. Preparazione delle cellule <ol><li> Il giorno 1, pesare e anestetizzare animali. <ol><li> Pesare 30 g-1 kg di animale. Anestetizzare in 5-10 volumi di animali di acqua di mare: 01:01 MgCl isotonica. 6H 2 0 per 1 ora con aerazione ad esempio una pompa di aria dell&#39;acquario elettrica con airstone allegata. </li></ol></li><li> Preparare forniture di dissezione. <ol><li> Assemblare vassoio dissezione pulita con perni diritti inox quali spine tessuto. Assemblare diverse piastre di Petri contenenti acqua di mare artificiale (ASW; vedi tabelle 1 e 3) + penicillina / streptomicina (P / S). </li><li> Avere pronto un microscopio a bassa potenza. Avere gli strumenti di dissezione pulita pronta, come ad esempio il naso a coste e una pinza sottile chirurgici, e fine e grandi forbici chirurgiche. Spruzzare gli strumenti con il 70% di alcol isopropol prima dell&#39;uso e lasciare asciugare. </li></ol></li><li> Posizione animale sul vassoio dissezione. <ol><li> Posizione animale lato ventrale verso il basso nel vassoio dissezione. Animale Pinattraverso bordi della parete del corpo e confini parapodial, ma evitare la coda e la testa, per esporre la superficie dorsale e il solco genitale </li><li> Sciacquare la superficie dorsale in acqua fredda con esecuzione lenta. Applicare un leggero flusso di 70% di alcol isopropol da una bottiglia di compressione lungo la scanalatura genitale e sopra la parte superiore della testa. </li></ol></li><li> Fai l&#39;incisione iniziale. <ol><li> Utilizzando un microscopio a bassa potenza (vedere Tabella 2) e riflessa per osservare il punto in cui la scanalatura genitale origina dal margine anteriore guscio, teso tenere con una pinza Poly-naso una piega di tessuto a sinistra di questa origine, mentre snipping un superficiale Hole il percorso attraverso il liscio, zona pianeggiante della parete del corpo appena a destra della scanalatura genitale con sottili forbici angolate. </li><li> Emolinfa deve essere osservato per versare dal foro, talvolta portando con sé il ganglio addominale e stomaco. </li></ol></li><li> Espandere l&#39;incisione. <ol><li> Utilizzare forbici grandi per espandere °incisione e anteriormente ad un punto tra le rhinophores, mentre si tira verso l&#39;alto con la lama inferiore per evitare intaccare l&#39;intestino. Gli organi interni saranno osservate per fuoriuscire l&#39;incisione. </li></ol></li><li> Rimuovere i gangli. <ol><li> Riposizionare il vassoio e rimettere a fuoco il microscopio sulla regione della testa. </li><li> Utilizzando pinze pulite e forbici sottili, togliere i gangli testa da troncare a un certo punto i nervi che costituiscono i gangli testa in un anello intorno all&#39;esofago per rimuovere pleurico-pedale e cerebrale gangli come gruppo. </li><li> Rimuovere il ganglio buccale che aderisce al lato ventrale dell&#39;esofago. </li><li> Rimuovere il ganglio addominale tagliando le 4 grandi connettivi nervosi che emettono da questo ganglio. </li></ol></li><li> Isolare gangli di interesse. <ol><li> Tagliare testa gangli a parte, lasciando una lunghezza dei connettivi collegati a ciascun ganglio di interesse che è pari ad almeno il diametro del ganglio, questo ritarderà enzima sopra-digestionedelle cellule di interesse nella fase successiva. </li><li> Mettere ogni ganglio obiettivo attraverso 2 risciacqui di ASW + P / S, muovendosi tra risciacqui con pinzette pulite. </li><li> Se il targeting delle cellule del PVC, lasciare il giusto hemiganglion pleurico collegato al diritto hemiganglion pedale, e allo stesso modo lasciare il hemiganglion pleurico sinistro attaccato al pedale hemiganglion sinistra. </li><li> Scartare gangli indesiderati e il resto dell&#39;animale secondo la prassi di ricerca accettato. </li></ol></li><li> Enzimaticamente digerire i gangli. <ol><li> Per ciascun ganglio, preparare 1 ml di soluzione enzimatica costituito 3,75 mg dispasi, 1 mg ialuronidasi e 0,3 g collagenasi tipo XI per ml di ASW + P / S in un tubo da 15 ml polipropilene. </li><li> Luogo risciacquato gangli nella soluzione enzimatica e fissare il tappo. Posizionare il tubo su un lato su un agitatore rotante (vedi tabella 2) a bassa velocità per 13-5 ore durante la notte a circa 23 ° C (temperatura ambiente). </li></ol></li><li> Preparare ilpiatti della cultura. <ol><li> Prima di microdissezione (Day 2), preparare poli-D-lisina (PDL) rivestite piatti della cultura in una cappa di coltura di tessuti. Aggiungere 0,5 ml ~ PDL (0,2 mg / ml di acqua sterile) al centro di un piatto 35 millimetri a ~ 25 min. </li><li> Sciacquare PDL 3x con acqua sterile. Lasciare asciugare. UV-sterilizzare le piastre. </li></ol></li><li> Giorno 2 Isolare i neuroni. <ol><li> Riempire il centro di 35 piatti mm che erano PDL-rivestito UV e sterilizzato con circa 0,5 ml di ASW + P / S per creare un&#39;isola di supporto all&#39;interno del piatto altrimenti asciutto. Questo può essere fatto in panchina, fuori della cappa coltura tissutale. Un piatto deve essere preparato per ogni gruppo di cellule provenienti da una hemiganglion. </li><li> Avere pronto un diametro di 100 millimetri piatto dissezione fatta da Sylgard-rivestimento e guarire una mm superficie dissezione profonda 5, dotato di sottili perni dissezione di varie dimensioni da utilizzare come perni e sonde (cfr. tabella 3). Sciacquare questo piatto con il 70% di alcol isopropol, poi con ASW + P / S, e, infine, fill con ASW + P / S. </li></ol></li><li> Preparare microdissezione dei gangli digerito. <ol><li> Versare la soluzione enzimatica contenente il pleurico-pedale e buccale gangli digerito nel piatto dissezione. </li><li> Porre la capsula sul palco del microscopio contro una superficie nera sotto luce riflessa. </li><li> Utilizzando il tessuto connettivo allegata, definire con precisione ogni pleurico-pedale hemiganglion lato dorsale alto. I tessuti saranno morbidi e intollerante di stretching. </li></ol></li><li> Microdissect neuroni in PVC. <ol><li> Utilizzando due coppie di pinze sottili puliti, e in possesso di un perno di dissezione multa in un paio di pinze come sonda, isolare le cellule del PVC da un hemiganglion pleurico. Digestione enzimatica avrà rimosso o rotto a parte il tessuto connettivo che copre il cluster PVC, ma le cellule saranno aderire l&#39;una all&#39;altra. </li><li> Usare la sonda di staccare queste cellule dal connettivo pedale-pleurica 18, poi lasciare cadere il cluster cella al fondo della dissezionepiatto. </li></ol></li><li> Trasferire i neuroni al piatto cultura. <ol><li> Trasferire il gruppo di cellule di un mm piatto preparato 35 cultura delicatamente succhiare il cluster nella punta di un po &#39;incendio lucidato pipetta Pasteur di vetro usa e getta, quindi l&#39;erogazione lentamente nell&#39;isola dei media in una capsula di Petri, facendo attenzione ad evitare l&#39;introduzione di bolle d&#39;aria nella pipetta. Ripetere l&#39;estrazione del cluster PVC dell&#39;altro hemiganglion e posto in una capsula di Petri separato. </li></ol></li><li> Microdissect e trasferire i neuroni BSC. <ol><li> Piedinatura del ganglio buccale lato ventrale, con cura per risparmiare le cellule di interesse. Ripetere il passo 1.12 con le 2 BSC gruppi di cellule del ganglio buccale 10. </li><li> Isolamento di neuroni in PVC e BSC produrrà 4 capsule di Petri contenenti cluster di neuroni: 2 piatti contenenti cluster BSC e 2 piatti contenenti cluster PVC. Scartare il resto dei gangli e mettere da parte il piatto di 100 mm dissezione. </li></ol></li><li> Dissociate i gruppi di cellule. <ol><li> Posizionare un piatto di coltura contenente cellule sul palco del microscopio a bassa potenza e rimuovere il coperchio. </li><li> Dissociare il cluster PVC delicatamente sfogliando il fondo del piatto cultura adiacente al cluster cella con un perno dissezione multa tenutasi a pinza. Sfogliando è scavare la punta del perno nella plastica del fondo del piatto come se cercasse di scavare una macchia di plastica. Quando l&#39;attrito con la plastica rilascia il punto di perno, un&#39;onda percussione viene prodotta attraverso il mezzo che si rompe il vicino ammasso di cellule. </li><li> 5-10 colpi di frusta possono essere necessari fino a quasi dissociare completamente le cellule, ma è meglio lasciare piccoli gruppi di cellule, piuttosto che a sfogliare troppo timore le cellule distrutte dalla pura meccanica. </li><li> Rimontare il coperchio e ripetere con altri piatti della cultura. </li></ol></li><li> Conservare le colture cellulari. <ol><li> Posizionare le piastre di coltura contenenti isole dei media nei loro centri di cellule dissociate inun grande contenitore che permette la circolazione dell&#39;aria, come ad esempio una x 25 millimetri di plastica rotondo piatto 150 Petri, e collocare questo piatto in un incubatore a 17 ° C. </li><li> Installare nella camera di una vaschetta aperta di acqua come fonte di umidità. Lasciar riposare durante la notte. Le cellule aderiscono al rivestimento poli-D-lisina nel centro del piatto. </li></ol></li><li> Piatti della cultura diluvio. <ol><li> Il giorno 3, inondare delicatamente i piatti con 2,5 ml di ASW + P / S. Esaminare e contare le cellule utilizzando una cultura microscopio cellula invertito. Conservare nel 17 ° C incubatore. </li></ol></li></ol> 2. Elettrofisiologia Il metodo è di connessione standard di serraggio che è stato descritto in numerosi testi (es. Sakmann e Neher, 1995) 16. Questo protocollo funziona su cellule ≤ 100 pF capacitanza, o cellule &lt;60 micron di diametro nei giorni 3 e 4 del protocollo. Cellule senza processi sono ottimali per la registrazione. Il seguente considerat specialeapplicano gli ioni: <ol><li> Celle più grandi possono essere sistemati con l&#39;impostazione di configurazione dell&#39;amplificatore 200B Axopatch impostato a β = 0,1. Attivazione di grandissime correnti può indurre le cellule a sfuggire voltage clamp. Soluzioni ASW sostituita contenuto di sale (per esempio una frazione del NaCl sostituito con impermeabile cloruro N-metil-D-glucammina) possono essere utilizzate per ridurre intera ampiezza della corrente della cella. </li><li> Fibre piene di pipette di patch borosilicato tirato su un estrattore pipetta e riempita a metà con soluzione intracellulare (ICS), composto da 450 mM KCl e altri sali (vedi Tabella 1) sono adatti, e devono avere la resistenza di circa 0,5-1 MOhm. Se è desiderato isolamento di correnti ioniche specifici, per esempio Na + correnti in assenza di correnti di K, K + in questa soluzione può essere sostituita con altri ioni come Cs +. Cl - sostituzione di ICS con un anione impermeant è anche giustificato se si desidera un ICS più naturale9. </li><li> Le cellule sono robusti con&gt; 1 hr-lunghe registrazioni possibili a temperatura ambiente. La registrazione è ottimale nei giorni 3 e 4 del protocollo, che corrispondono a 24-48 ore in cultura. Dopo 48 hr vi è difficoltà a formare guarnizioni gigaohm, forse a causa di elaborazione di un glicocalice sulla superficie cellulare, e problemi di spazio clamp causati dal processo escrescenza. Le cellule sono utili, tuttavia, per gli studi di clamp non di patch, come la tossicologia, che possono essere completati in &lt;14 d. </li><li> ASW ed altre soluzioni di essere dispensato dal dispositivo soluzione alimentato a gravità, così come la soluzione intracellulare, devono essere filtrati con una siringa montata 0,2 micron filtro Acrodisk (Tabella 3) per eliminare le particelle fini che interferiscono con la formazione sigillo gigaohm. </li><li> Desensibilizzazione si verifica quando si utilizza agonisti come il D-aspartato (D-Asp), quindi ~ 1-2 minuti tra le applicazioni degli agonisti è raccomandato. Al contrario, il potenziamento è spesso osservato con una rapida applicazione ripetutazione di agonisti come L-glutammato (Glu-L). </li><li> Agonisti correnti attivati ​​come L-Glu possono essere studiate applicando agonisti con il picospritzer pipetta. Questa pipetta è tirato la stessa della pipetta di patch e contiene agonista in ASW. Quando la punta della pipetta questa è posizionato sotto il tubo di uscita del dispositivo soluzione alimentato a gravità, e con un angolo di 45 ° dal tubo di efflusso (Figura 2F), agonista verrà rapidamente lavato via dalla cella, e desensibilizzazione sarà minimizzata. La pipetta agonista dovrebbe creare un angolo di 90 ° o più rispetto alla pipetta di patch. </li></ol>

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Gli autori dichiarano di non avere interessi finanziari in competizione.

Le tecniche di dissociazione descritti qui resa culture di neuroni sensoriali contenenti 50-100 neuroni isolati intervallati da un piccolo numero di cellule gliali e di altre cellule non identificati. Le fasi più critiche del protocollo sono il momento i gangli rimangono in soluzione enzimatica, e sfogliando, la dissociazione dei gruppi di cellule digerite a spezzare il gruppo in singole cellule. Digestione enzimatica (passo 1.8) deve essere ottimizzato alla temperatura disponibile. A 23 ° C con agitazione lenta, 13 o...

Il mollusco opistobranch marino, Aplysia, è stato un modello neurobiologico utile per molti decenni. E &#39;meglio conosciuto come modello di assuefazione e di condizionamento classico 7, 8. Gli studi sull&#39;apprendimento e la memoria in questo modello ha vinto il premio Nobel per la fisiologia o la medicina nel 2000 per Eric R. Kandel, in un premio che divideva con Arvid Carlsson e Paul Greengard 10. Gli studi che coinvolgono registrazioni elettriche da preparativi ridotti, in cui gli elementi del sistema nervoso di questo invertebrato sono sezionati dall&#39;animale con nervi e muscoli che si trovino, hanno contribuito a chiarire i ruoli dei singoli neuroni in Aplysia. Identificazione dei meccanismi molecolari precisi che costituiscono l&#39;apprendimento in Aplysia però, spesso utilizzato un&#39;altra tecnica, a lungo termine co-colture di un neurone sensoriale e di un motoneurone, ottenuto uno per uno dai singoli animali donatori e ha permesso di formare una sinapsi nel piatto cultura 21 . Noi e altri 1, 3, 6, 14, 15, 16, abbiamo sfruttato la facilità con cui identificare i neuroni possono essere mirati in questo modello, così come la loro resistenza in esperimenti a lungo termine per rendere le culture a breve termine dissociate di gruppi di neuroni nominalmente omogenee in cui studiarne correnti ioniche sotto voltage clamp nella configurazione di patch clamp. Molti neuroni Aplysia resistono a ripetuti cicli di patch di bloccaggio per consentire il tempo per le manipolazioni sperimentali di lunga durata. La tecnica è utile per i neuroni, come le cellule del sacchetto neurosecretory del ganglio addominale, ei neuroni sensoriali dei gangli pleurico e buccali cui dissociazione descriviamo qui, ma non per molto grandi neuroni&gt; 60 micron di diametro, come L7 o R2 di il ganglio addominale. Noi non utilizziamo Aplysia siero nelle nostre culture, a differenza dei sensori-motoneurone co-culture descritte altrove. La maggior parte dei neuroni ottenuti con questa procedura sono senza processi per tegli prima 48 ore di cultura, facilitando intera registrazione tensione di cella, ma poi germogliare e elaborare assoni e di altri processi per circa 14 giorni prima di morire per mancanza di nutrienti e / o fattori di crescita. Questa tecnica produce colture primarie di neuroni per 50-100 piatto da regioni fisiologicamente documentate dei gangli buccale e della pleura. Questo protocollo è utile per i ricercatori che studiano gli aspetti della singola fisiologia cellulare in esperimenti che richiedono numerosi sperimentale repliche per animale. Si produce una coppia di culture dovute alla separazione anatomica delle cellule bersaglio in hemiganglia destra e sinistra, permettendo studi che beneficio dal trattamento accoppiati e colture di controllo. Il protocollo mira buccali S a grappolo (BSC) neuroni del ganglio buccale e pleuriche ventrocaudal (PVC) neuroni del ganglio pleurico. Queste cellule sono in una dimensione appropriata per intere registrazioni di tensione delle cellule e ROBUS visualizzazionet risposte glutamatergiche. La metodologia discusso è appropriato per la maggior gangli del sistema nervoso Aplysia.

<table><tbody><tr><td>Artificial seawater ASW</td><td>Sigma-Aldrich</td><td>assorted</td><td>(mM): 417 NaCl, 10 KCl, 10 CaCl&lt;sub&gt;2&lt;/sub&gt; (2 H&lt;sub&gt;2&lt;/sub&gt;O), 5MgCl&lt;sub&gt;2&lt;/sub&gt; (6H&lt;sub&gt;2&lt;/sub&gt;O), 15 HEPES-NaOH, pH 7.6</td></tr><tr><td>Intracellular solution</td><td>Sigma</td><td>assorted</td><td>(mM): 450 KCl, 2.9 CaCl&lt;sub&gt;2&lt;/sub&gt; (2 H&lt;sub&gt;2&lt;/sub&gt;O), 2.5 MgCl&lt;sub&gt;2&lt;/sub&gt; (6 H&lt;sub&gt;2&lt;/sub&gt;O), 5 Na&lt;sub&gt;2&lt;/sub&gt;ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4</td></tr><tr><td>Poly-D-lysine</td><td>Sigma</td><td>P6407</td><td>&amp;nbsp;</td></tr><tr><td>penicillin/streptomycin added to ASW at 1:100</td><td>Lonzo Walkersville, Inc.</td><td>17-603E</td><td>5,000 Units/ml penicillin plus 5,000 mg/ml streptomycin</td></tr><tr><td>Neutral dispase II</td><td>Roche Diagnostics</td><td>10165859001</td><td>&amp;nbsp;</td></tr><tr><td>hyaluronidase</td><td>Sigma-Aldrich</td><td>H4272</td><td>&amp;nbsp;</td></tr><tr><td>collagenase type XI</td><td>Sigma-Aldrich</td><td>C9407</td><td>&amp;nbsp;</td></tr><tr><td>L-Glutamate (L-Glu)</td><td>Sigma-Aldrich</td><td>49601-100G</td><td>&amp;nbsp;</td></tr><tr><td>D-Aspartate (D-Asp)</td><td>Sigma-Aldrich</td><td>11200-10G</td><td>&amp;nbsp;</td></tr><tr><td>N-methyl-D-aspartate (NMDA)</td><td>Biomol</td><td>100002-268</td><td>&amp;nbsp;</td></tr><tr><td>L-Asp</td><td>Sigma</td><td>A6683-25G</td><td>&amp;nbsp;</td></tr><tr><td>alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionic acid (AMPA)</td><td>Sigma</td><td>A6816-5MG</td><td>&amp;nbsp;</td></tr><tr><td>L-Glu R antagonists</td><td>various</td><td>various</td><td>&amp;nbsp;</td></tr><tr><td>agar</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>kynurenate</td><td>Sigma-Aldrich</td><td>61250</td><td>&amp;nbsp;</td></tr><tr><td>APV</td><td>Sigma-Aldrich</td><td>A5282</td><td>&amp;nbsp;</td></tr><tr><td>DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist)</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>2-propanol</td><td>VWRSP</td><td>BDH1133</td><td>&amp;nbsp;</td></tr><tr><td>Chloriding solution</td><td>Sigma</td><td>assorted</td><td>25 g FeCl&lt;sub&gt;3&lt;/sub&gt; + 25 ml concentrated HCl + 50 ml H&lt;sub&gt;2&lt;/sub&gt;O</td></tr><tr><td>Sylgard silicone 2-part polymer</td><td>World Precision Instruments (WPI)</td><td>SYL184</td><td>Provides pin-out surface for small dissection dishes</td></tr><tr><td>0-40x zoom magnification microscope for dissections</td><td>Wild</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Techniquip 150 Watts Fiber Optic Illuminator</td><td>Microoptics of Florida</td><td>TQ FOI-150</td><td>&amp;nbsp;</td></tr><tr><td>RotoMix 50800 orbital mixer</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) &amp;amp; 40x objectives</td><td>SR Research Ltd.</td><td>Eyelink II</td><td>&amp;nbsp;</td></tr><tr><td>Tektronix digital oscilloscope</td><td>SR Research Ltd.</td><td></td><td>&amp;nbsp;</td></tr><tr><td>pClamp 10 data acquisition and analysis software</td><td>Molecular Devices</td><td></td><td>&amp;nbsp;</td></tr><tr><td>PC with Windows XP or higher operating system</td><td>PC Solutions</td><td></td><td>Thinkserver with solid state hard drives (80GB) and low noise monitors</td></tr><tr><td>Flaming/Brown P87 micropipette puller</td><td>Sutter Instruments, Novato, CA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Axon Instruments Axopatch 200B clamp amplifier with a capacitance compensation range of 1-1000 pF; preamplifier</td><td>Molecular Devices, Sunnyvale, CA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Axon instruments electrode holder assembly for Axopatch 200B preamplifier</td><td>Molecular Devices, Sunnyvale, CA</td><td>CV203BU</td><td>&amp;nbsp;</td></tr><tr><td>Digidata 1200 A/D converter</td><td>Molecular Devices, Sunnyvale, CA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Picospritzer, powered by N2 adjustable for pressure and duration</td><td>Parker Hannifin, Cleveland, OH</td><td></td><td>&amp;nbsp;</td></tr><tr><td>TMC Micro-G Vibration isolation table</td><td>Ametek</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Faraday cage</td><td></td><td></td><td>custom manufacture</td></tr><tr><td>Burleigh Piezoelectric Clamshell Micromanipulators</td><td>Burleigh Instruments; Thorlabs</td><td></td><td>presently PCS-5000; -6000 series + mounts</td></tr><tr><td>Narishige M-152 manual manipulators (for perfusion system and picospritzer)</td><td>Narishige USA</td><td></td><td>&amp;nbsp;</td></tr><tr><td>Filament pipette glass,1.5 mm OD, 0.84 mm ID -</td><td>WPI</td><td>1B150-3</td><td>&amp;nbsp;</td></tr><tr><td>3 inch length</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>Ag/AgCl half cell</td><td>WPI</td><td>EP4</td><td>&amp;nbsp;</td></tr><tr><td>15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes</td><td>VWRSP</td><td>21008-918</td><td>&amp;nbsp;</td></tr><tr><td>Angled Scissors</td><td>Fine Science Tools</td><td>15006-09</td><td>&amp;nbsp;</td></tr><tr><td>Dumostar Fine forceps</td><td>Fine Science Tools</td><td>11295-00</td><td>&amp;nbsp;</td></tr><tr><td>35 mm falcon tissue culture dishes</td><td>VWRSP</td><td>25382-064</td><td>&amp;nbsp;</td></tr><tr><td>falcon 150 x 25 mm tissue culture dishes; 1013</td><td>VWRSP</td><td>1013</td><td>also can be made into small dissection dishes with sylgard</td></tr><tr><td>sylgard</td><td>WPI</td><td>SYL184</td><td>&amp;nbsp;</td></tr><tr><td>animal dissection tray</td><td>various</td><td></td><td>&amp;nbsp;</td></tr><tr><td>15 ml centrifuge tubes, 35-2097 BD Falcon</td><td>VWRSP</td><td>21008-918</td><td>For 6-bore gravity-fed perfusion system</td></tr><tr><td>Aluminum clips with screw hole ends</td><td>hardware store</td><td></td><td>For perfusion system</td></tr><tr><td>23 gauge needles (manually file off points)</td><td>VWRSP</td><td></td><td>For perfusion system</td></tr><tr><td>Polyethylene tubing 0.022&quot;ID x 0.042&quot;OD; 427411</td><td>Becton-Dickinson</td><td></td><td>For perfusion system</td></tr><tr><td>H-7 pipette stand/holder for microcap perfusion array</td><td>Narishige USA</td><td></td><td>For perfusion system</td></tr><tr><td>one-way valves</td><td></td><td></td><td>For perfusion system</td></tr><tr><td>Drummond Microcaps 1 &amp;mu;l</td><td>VWRSP</td><td></td><td>For perfusion system</td></tr><tr><td>18 gauge needles for suction (filed off points)</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td>Polyethylene tubing</td><td>Cole Parmer</td><td>4.27436E+11</td><td>&amp;nbsp;</td></tr><tr><td>fine dissection pins</td><td>Fine Science Tools</td><td>26002-20</td><td>&amp;nbsp;</td></tr><tr><td>capillary tubes</td><td>Kimble 71900-100</td><td></td><td>fire-polished and U-shaped in a Bunsen burner flame and filled with 3% agar in ECS</td></tr><tr><td>modeling clay</td><td>craft store</td><td></td><td>&amp;nbsp;</td></tr><tr><td>dish holder for microscope stage with isolated ground bath</td><td></td><td></td><td>Custom manufacture</td></tr><tr><td>pasteur pipettes</td><td>VWRSP</td><td>14672-412</td><td>&amp;nbsp;</td></tr><tr><td>pipette bulbs</td><td>VWRSP</td><td>53283-911</td><td>&amp;nbsp;</td></tr><tr><td>acrodisk syringe filters</td><td>VWRSP</td><td>28144-040</td><td>&amp;nbsp;</td></tr><tr><td>thick-walled 1.5 mm diameter borosilicate filament glass</td><td>WPI</td><td>1B150F-3</td><td>&amp;nbsp;</td></tr><tr><td>High purity nitrogen cylinder and bifurcating regulator</td><td></td><td></td><td>&amp;nbsp;</td></tr><tr><td colspan="4">&lt;p class=&quot;jove_content&quot;&gt;&lt;strong&gt;Tables 1-3. Lists of Reagents, Materials, and Equipment.&lt;/strong&gt;&lt;/p&gt;</td></tr></tbody></table>

isolation of sensory neurons of aplysia californica for patch clamp recordings of glutamatergic currents

La marina Aplysia californica mollusco gasteropode ha una storia venerabile come un modello di funzione del sistema nervoso, con particolare rilievo negli studi di apprendimento e memoria. Le preparazioni tipiche per questo tipo di studi sono quelli in cui la sensoriali e motoneuroni sono lasciati intatti in un animale minimamente sezionato, o di un elaborato tecnico neuronale co-coltura di sensoriale individuale e motoneuroni. Meno comune è l&#39;isolato preparazione neuronale in cui piccoli gruppi di neuroni nominalmente omogenee sono dissociate in singole cellule in coltura a breve termine. Tali cellule isolate sono utili per la caratterizzazione biofisica di correnti ioniche con tecniche di patch clamp, e modulazione mirato di questi conduttanze. Un protocollo per la preparazione di tali colture è descritto. Il protocollo sfrutta le facilmente identificabili neuroni sensoriali glutammatergiche dei gangli pleurico e buccale, e descrive la loro dissociazione e la manutenzione minima in coltura per diversi giorni senza siero.

Le posizioni dei neuroni sensoriali all&#39;interno dei gangli che sono indirizzate in questo protocollo, i neuroni BSC e PVC sono mostrati in Figura 1. I neuroni BSC si trovano in 2 simmetriche grappoli ovale sul lato ventrale del ganglio buccale, la superficie che si affaccia dalla massa buccale nel ganglio intatto (Figura 1A). I neuroni formano PVC, cluster a forma di V bilaterali che avvolgono la superficie dorsale del ganglio pleurico verso l&#39;asse centrale (Figura 1B).<...

Watch this Scientific Journal Video about Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents at JoVE.com

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Descriviamo la dissezione del sistema nervoso della marina mare lepre<em&gt; Aplysia</em&gt; Dopo l&#39;anestesia, l&#39;isolamento dei neuroni per termine cultura dei tessuti di breve, e registrazioni di correnti di singolo ione delle cellule attraverso la tecnica del patch clamp.

Isolamento dei neuroni sensoriali<em&gt; Aplysia californica</em&gt; Per Bloccare Registrazioni patch di correnti glutamatergico

The marine gastropod mollusk Aplysia californica has a venerable  history as a model of nervous system function, with particular significance in  studies ...

isolation-sensory-neurons-aplysia-californica-for-patch-clamp

Research

JoVE Journal

Neuroscience

Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

12.2K Views.  University of Miami. Descriviamo la dissezione del sistema nervoso della marina mare lepre Aplysia Dopo l'anestesia, l'isolamento dei neuroni per termine cultura dei tessuti di breve, e registrazioni di correnti di singolo ione delle cellule attraverso la tecnica del patch clamp.

Video: Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal. Simultaneously recording from multiple nerves and muscles gives us precise information about the timing of neural activity. Previous recording methods have worked for two electrodes, but the study of additional nerves or muscles required combining and averaging the recordings of multiple animals, which made it difficult to determine fine details of timing and phasing, because of variability from response to response, and from animal to animal. Implanting four individual electrodes has a very low success rate due to the formation of adhesions that prevent animals from performing normal feeding movements. We developed a new method of electrode fabrication that reduces the bulk of the electrodes inside the animal allowing for normal feeding movements. Using a combination of glues to attach the electrodes results in a more reliable insulation of the electrode which lasts longer, making it possible to record for periods as long as a week. The fabrication technique that we describe could be extended to incorporate several additional electrodes, and would be applicable to vertebrate animals.

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals

A technique is described for implanting four in vivo electrodes to monitor the neuromuscular control of feeding behavior in Aplysia californica.

Fabbricazione e impianto di elettrodi nel Aplysia californica Per registrazioni multi-canale neurale e muscolare in intatto, Animali liberamente Comportarsi

Recording from key nerves and muscles of Aplysia during feeding behavior allows us to study the patterns of neural control in an intact animal.  ...

Ricerca

Neuroscienze

Neuronal Cell Cultures from Aplysia for High-Resolution Imaging of Growth Cones

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information into directional movement towards the appropriate target cell. To fully understand how guidance information is transmitted from the cell surface to the underlying dynamic cytoskeletal networks, one needs a model system suitable for live cell imaging of protein dynamics at high temporal and spatial resolution. Typical vertebrate growth cones are too small to quantitatively analyze F-actin and microtubule dynamics. Neurons from the sea hare Aplysia californica are 5-10 times larger than vertebrate neurons, can easily be kept at room temperature and are very robust cells for micromanipulation and biophysical measurements. Their growth cones have very defined cytoplasmic regions and a well-described cytoskeletal system. The neuronal cell bodies can be microinjected with a variety of probes for studying growth cone motility and guidance. In the present protocol we demonstrate a procedure for dissection of the abdominal ganglion, culture of bag cell neurons and setting up an imaging chamber for live cell imaging of growth cones.

Aplysia californica neurons develop large growth cones in culture that are excellent for high-resolution imaging of growth cone motility and guidance. Here, we present a protocol for dissection and plating of Aplysia bag cell neurons as well as for setting up a chamber for live cell imaging.

Colture cellulari neuronali da Aplysia per imaging ad alta risoluzione di coni di crescita

Neuronal growth cones are the highly motile structures at the tip of axons that can detect guidance cues in the environment and transduce this information ...

Biologia

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific molecular mechanisms are identified, new therapeutic strategies can be developed to treat abnormalities in specific behaviors caused by degenerative diseases or aging of the nervous system. The marine snail Aplysia californica is well suited for the investigations of cellular and molecular basis of behavior because neural circuitry underlying a specific behavior could be easily determined and the individual components of the circuitry could be easily manipulated. These advantages of Aplysia have led to several fundamental discoveries of neurobiology of learning and memory. Here we describe a preparation of the Aplysia nervous system for the electrophysiological and molecular analyses of individual neurons. Briefly, ganglion dissected from the nervous system is exposed to protease to remove the ganglion sheath such that neurons are exposed but retain neuronal activity as in the intact animal. This preparation is used to carry out electrophysiological measurements of single or multiple neurons. Importantly, following the recording using a simple methodology, the neurons could be isolated directly from the ganglia for gene expression analysis. These protocols were used to carry out simultaneous electrophysiological recordings from L7 and R15 neurons, study their response to acetylcholine and quantitating expression of CREB1 gene in isolated single L7, L11, R15, and R2 neurons of Aplysia.

Aplysia Ganglia Preparation for Electrophysiological and Molecular Analyses of Single Neurons

Marine snail Aplysia californica has been widely used as a neurobiology model for the studies on cellular and molecular basis of behavior. Here a methodology is described for exploring the nervous system of Aplysia for the electrophysiological and molecular analyses of single neurons of identified neural circuitry.

Alisia Preparazione ganglia per analisi elettrofisiotiche e molecolari di singoli neuroni

A major challenge in neurobiology is to understand the molecular underpinnings of neural circuitry that govern a specific behavior. Once the specific ...

Bioingegneria

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., homosynaptic facilitation) 1. These hypotheses can be tested by simultaneously monitoring changes in intracellular calcium and alterations in synaptic efficacy. We demonstrate how this can be accomplished by combining calcium imaging with intracellular recording techniques. Our experiments are conducted in a buccal ganglion of the mollusc Aplysia californica. This preparation has a number of experimentally advantageous features: Ganglia can be easily removed from Aplysia and experiments use adult neurons that make normal synaptic connections and have a normal ion channel distribution. Due to the low metabolic rate of the animal and the relatively low temperatures (14-16 &deg;C) that are natural for Aplysia, preparations are stable for long periods of time. 
To detect changes in intracellular free calcium we will use the cell impermeant version of Calcium Orange 2 which is easily 'loaded' into a neuron via iontophoresis. When this long wavelength fluorescent dye binds to calcium, fluorescence intensity increases. Calcium Orange has fast kinetic properties 3 and, unlike ratiometric dyes (e.g., Fura 2), requires no filter wheel for imaging. It is fairly photo stable and less phototoxic than other dyes (e.g., fluo-3) 2,4. Like all non-ratiometric dyes, Calcium Orange indicates relative changes in calcium concentration. But, because it is not possible to account for changes in dye concentration due to loading and diffusion, it can not be calibrated to provide absolute calcium concentrations. 
An upright, fixed stage, compound microscope was used to image neurons with a CCD camera capable of recording around 30 frames per second. In Aplysia this temporal resolution is more than adequate to detect even a single spike induced alteration in the intracellular calcium concentration. Sharp electrodes are simultaneously used to induce and record synaptic transmission in identified pre- and postsynaptic neurons. At the conclusion of each trial, a custom script combines electrophysiology and imaging data. To ensure proper synchronization we use a light pulse from a LED mounted in the camera port of the microscope. Manipulation of presynaptic calcium levels (e.g. via intracellular EGTA injection) allows us to test specific hypotheses, concerning the role of intracellular calcium in mediating various forms of plasticity.

Monitoring Changes in the Intracellular Calcium Concentration and Synaptic Efficacy in the Mollusc Aplysia

We demonstrate how changes in the intracellular free calcium concentration and synaptic efficacy can be simultaneously monitored in a ganglion preparation of Aplysia. We image intracellular calcium using a fluorescent dye, Calcium Orange, and induce and monitor synaptic transmission with sharp (intracellular) electrodes.

I cambiamenti di monitoraggio della concentrazione di calcio intracellulare ed efficacia Synaptic nel Mollusco<em&gt; Aplysia</em

It has been suggested that changes in intracellular calcium mediate the induction of a number of important forms of synaptic plasticity (e.g., ...

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

Multifunctionality, the ability of one peripheral structure to generate multiple, distinct behaviors1, allows animals to rapidly adapt their behaviors to changing environments. The marine mollusk Aplysia californica provides a tractable system for the study of multifunctionality. During feeding, Aplysia generates several distinct types of behaviors using the same feeding apparatus, the buccal mass. The ganglia that control these behaviors contain a number of large, identified neurons that are accessible to electrophysiological study. The activity of these neurons has been described in motor programs that can be divided into two types, ingestive and egestive programs, based on the timing of neural activity that closes the food grasper relative to the neural activity that protracts or retracts the grasper2. However, in isolated ganglia, the muscle movements that would produce these behaviors are absent, making it harder to be certain whether the motor programs observed are correlates of real behaviors. In vivo, nerve and muscle recordings have been obtained corresponding to feeding programs2,3,4, but it is very difficult to directly record from individual neurons5. Additionally, in vivo, ingestive programs can be further divided into bites and swallows1,2, a distinction that is difficult to make in most previously described in vitro preparations.
The suspended buccal mass preparation (Figure 1) bridges the gap between isolated ganglia and intact animals. In this preparation, ingestive behaviors - including both biting and swallowing - and egestive behaviors (rejection) can be elicited, at the same time as individual neurons can be recorded from and stimulated using extracellular electrodes6. The feeding movements associated with these different behaviors can be recorded, quantified, and related directly to the motor programs. The motor programs in the suspended buccal mass preparation appear to be more similar to those observed in vivo than are motor programs elicited in isolated ganglia. Thus, the motor programs in this preparation can be more directly related to in vivo behavior; at the same time, individual neurons are more accessible to recording and stimulation than in intact animals. Additionally, as an intermediate step between isolated ganglia and intact animals, findings from the suspended buccal mass can aid in interpretation of data obtained in both more reduced and more intact settings. The suspended buccal mass preparation is a useful tool for characterizing the neural control of multifunctionality in Aplysia.

An In Vitro Preparation for Eliciting and Recording Feeding Motor Programs with Physiological Movements in Aplysia californica

We describe a technique to extracellularly record and stimulate from nerves, muscles, and individual identified neurons in vitro while eliciting and observing different types of feeding behaviors in the feeding apparatus of Aplysia.

Un<em&gt; In Vitro</emPreparazione&gt; per stimolare e registrare i programmi motori Nutrire con i movimenti fisiologici in<em&gt; Aplysia californica</em

Multifunctionality, the ability of one peripheral  structure to generate multiple, distinct behaviors1, allows animals  to rapidly adapt their behaviors ...

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons. The neurons are large (up to 1 mm in diameter) and identifiable, with distinct sizes, shapes, positions and pigmentations, and the cell bodies are externally exposed in five paired ganglia distributed throughout the body of the animal. These properties have allowed investigators to delineate the circuitry underlying specific behaviors in the animal1. The monosynaptic connection between sensory and motor neurons is a central component of the gill-withdrawal reflex in the animal, a simple defensive reflex in which the animal withdraws its gill in response to tactile stimulation of the siphon. This reflex undergoes forms of non-associative and associative learning, including sensitization, habituation and classical conditioning. Of particular benefit to the study of synaptic plasticity, the sensory-motor synapse can be reconstituted in culture, where well-characterized stimuli elicit forms of plasticity that have direct correlates in the behavior of the animal2,3. Specifically, application of serotonin produces a synaptic strengthening that, depending on the application protocol, lasts for minutes (short-term facilitation), hours (intermediate-term facilitation) or days (long-term facilitation). In contrast, application of the peptide transmitter FMRFamide produces a synaptic weakening or depression that, depending on the application protocol, can last from minutes to days (long-term depression). The large size of the neurons allows for repeated sharp electrode recording of synaptic strength over periods of days together with microinjection of expression vectors, siRNAs and other compounds to target specific signaling cascades and molecules and thereby identify the molecular and cell biological steps that underlie the changes in synaptic efficacy.
An additional advantage of the Aplysia culture system comes from the fact that the neurons demonstrate synapse-specificity in culture4,5. Thus, sensory neurons do not form synapses with themselves (autapses) or with other sensory neurons, nor do they form synapses with non-target identified motor neurons in culture. The varicosities, sites of synaptic contact between sensory and motor neurons, are large enough (2-7 microns in diameter) to allow synapse formation (as well as changes in synaptic morphology) with target motor neurons to be studied at the light microscopic level.
In this video, we demonstrate each step of preparing sensory-motor neuron cultures, including anesthetizing adult and juvenile Aplysia, dissecting their ganglia, protease digestion of the ganglia, removal of the connective tissue by microdissection, identification of both sensory and motor neurons and removal of each cell type by microdissection, plating of the motor neuron, addition of the sensory neuron and manipulation of the sensory neurite to form contact with the cultured motor neuron.

Preparation of Aplysia Sensory-motor Neuronal Cell Cultures

Primary cultures of Aplysia sensory-motor neurons provide a model preparation for studying synapse formation and synaptic plasticity in vitro. This video demonstrates the identification and microdissection of sensory and motor neurons from Aplysia ganglia as well as the methods for establishing and maintaining sensory-motor neurons in culture.

Preparazione del Aplysia Senso-motorio colture cellulari neuronali

The nervous system of the marine mollusk Aplysia californica is relatively simple, consisting of approximately 20,000 neurons.  The neurons are large (up ...

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.
This extracellular technique has advantages. First, extracellular electrodes can stimulate and record neurons through the sheath5, so it does not need to be removed. Thus, neurons will be healthier in extracellular experiments than in intracellular ones. Second, if ganglia are rotated by appropriate pinning of the sheath, extracellular electrodes can access neurons on both sides of the ganglion, which makes it easier and more efficient to identify multiple neurons in the same preparation. Third, extracellular electrodes do not need to penetrate cells, and thus can be easily moved back and forth among neurons, causing less damage to them. This is especially useful when one tries to record multiple neurons during repeating motor patterns that may only persist for minutes. Fourth, extracellular electrodes are more flexible than intracellular ones during muscle movements. Intracellular electrodes may pull out and damage neurons during muscle contractions. In contrast, since extracellular electrodes are gently pressed onto the sheath above neurons, they usually stay above the same neuron during muscle contractions, and thus can be used in more intact preparations.
To uniquely identify motor neurons for a motor pool (in particular, the I1/I3 muscle in Aplysia) using extracellular electrodes, one can use features that do not require intracellular measurements as criteria: soma size and location, axonal projection, and muscle innervation4,6,7. For the particular motor pool used to illustrate the technique, we recorded from buccal nerves 2 and 3 to measure axonal projections, and measured the contraction forces of the I1/I3 muscle to determine the pattern of muscle innervation for the individual motor neurons.
We demonstrate the complete process of first identifying motor neurons using muscle innervation, then characterizing their timing during motor patterns, creating a simplified diagnostic method for rapid identification. The simplified and more rapid diagnostic method is superior for more intact preparations, e.g. in the suspended buccal mass preparation8 or in vivo9. This process can also be applied in other motor pools10,11,12 in Aplysia or in other animal systems2,3,13,14.

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica

In animals with large identified neurons (e.g. mollusks), analysis of motor pools is done using intracellular techniques1,2,3,4. Recently, we developed a technique to extracellularly stimulate and record individual neurons in Aplysia californica5. We now describe a protocol for using this technique to uniquely identify and characterize motor neurons within a motor pool.

Extracellulare Identificazione neuroni motori per un pool di motore muscolare in<em&gt; Aplysia californica</em

In animals with large  identified neurons (e.g. mollusks), analysis of motor pools is done using  intracellular techniques1,2,3,4. Recently,  we developed ...

Acute Dissociation of Lamprey Reticulospinal Axons to Enable Recording from the Release Face Membrane of Individual Functional Presynaptic Terminals

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated calcium channels (VGCCs) located in the release face membrane, is the trigger for vesicle fusion and neurotransmitter release. Crucial to the rapidity of synaptic transmission is the spatial and temporal synchrony between the arrival of the action potential, VGCCs and the neurotransmitter release machinery. The ability to directly record Ca2+&#160;currents from the release face membrane of individual presynaptic terminals is imperative for a precise understanding of the relationship between presynaptic Ca2+&#160;and neurotransmitter release. Access to the presynaptic release face membrane for electrophysiological recording is not available in most preparations and presynaptic Ca2+&#160;entry has been characterized using imaging techniques and macroscopic current measurements &#8211; techniques that do not have sufficient temporal resolution to visualize Ca2+ entry. The characterization of VGCCs directly at single presynaptic terminals has not been possible in central synapses and has thus far been successfully achieved only in the calyx-type synapse of the chick ciliary ganglion and in rat calyces. We have successfully addressed this problem in the giant reticulospinal synapse of the lamprey spinal cord by developing an acutely dissociated preparation of the spinal cord that yields isolated reticulospinal axons with functional presynaptic terminals devoid of postsynaptic structures. We can fluorescently label and identify individual presynaptic terminals and target them for recording. Using this preparation, we have characterized VGCCs directly at the release face of individual presynaptic terminals using immunohistochemistry and electrophysiology approaches. Ca2+ currents have been recorded directly at the release face membrane of individual presynaptic terminals, the first such recording to be carried out at central synapses.

Recording Ca2+ currents at the presynaptic release face membrane is key to a precise understanding of Ca2+ entry and neurotransmitter release. We present an acute dissociation of the lamprey spinal cord that yields functional isolated reticulospinal axons, permitting recording directly from the release face membrane of individual presynaptic terminals.

Acuta dissociazione di Lamprey reticulospinal assoni per attivare la registrazione dal rilascio Volto membrana di singoli funzionali terminali presinaptici

Synaptic transmission is an extremely rapid process. Action potential driven influx of Ca2+&#160;into the presynaptic terminal, through voltage-gated ...

Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter receptors that contribute to cell diversity across this population. This protocol outlines the steps necessary for successful dissecting, dissociating, and short-term culturing of the somata of inner ear bipolar neurons for the purpose of patch-clamp recordings. Detailed instructions for preparing vestibular ganglion neurons are provided with the necessary modifications needed for plating spiral ganglion neurons. The protocol includes instructions for performing whole-cell patch-clamp recordings in the perforated-patch configuration. Example results characterizing the voltage-clamp recordings of hyperpolarization-activated cyclic nucleotide-gated (HCN)-mediated currents highlight the stability of perforated-patch recording configuration in comparison to the more standard ruptured-patch configuration. The combination of these methods, isolated somata plus perforated-patch-clamp recordings, can be used to study cellular processes that require long, stable recordings and the preservation of intracellular milieu, such as signaling through G-protein coupled receptors.

Presented here are methods providing detailed instructions for dissecting, dissociating, culturing, and patch-clamp recording from vestibular ganglion and spiral ganglion neurons of the inner ear.

Isolare e coltivare i somata gangliari vestibolari e spirali da roditori neonatali per registrazioni con patch-clamp

The compact morphology of isolated and cultured inner ear ganglion neurons allows for detailed characterizations of the ion channels and neurotransmitter ...

Perforated Patch-Clamp Recordings of Inner Ear Sensory Neuron Cell Bodies

Source: Iyer, M. R., et al. <a href="https://app.jove.com/v/64908">Isolating and Culturing Vestibular and Spiral Ganglion Somata from Neonatal Rodents for Patch-Clamp Recordings.</a> J. Vis. Exp. (2023).
This video demonstrates the patch-clamp technique for measuring ionic currents in inner ear sensory neuron cell bodies. It details the preparation and use of a polished patch-clamp pipette filled with an internal solution containing amphotericin B, which perforates the membrane to allow ion movement while preserving intracellular conditions. Voltage pulses are applied to trigger and record ion channel activity, providing insights into their properties and behavior.

Registrazioni perforate Patch-Clamp dei corpi cellulari dei neuroni sensoriali dell'orecchio interno

1. Recording
NOTE: In this procedure, patch-clamp recordings from the isolated ganglion are typically performed 12-24 h after the plating. Other labs have ...

Isolamento dei neuroni sensoriali

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