Name: isolation of sensory neurons of aplysia californica for patch clamp recordings of glutamatergic currents
Uploaded: 2013-07-10T12:00:00
Description: Watch this Scientific Journal Video about Isolation of Sensory Neurons of Aplysia californica for Patch Clamp Recordings of Glutamatergic Currents at JoVE.com

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 d...

<ol>
	<li>Buttner, N., Siegelbaum, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antagonistic+modulation+of+a+hyperpolarization-activated+Cl(-)+current+in+Aplysia+sensory+neurons+by+SCP(B)+and+FMRFamide.">Antagonistic modulation of a hyperpolarization-activated Cl(-) current in Aplysia sensory neurons by SCP(B) and FMRFamide.</a> J. Neurophysiol. 90 (2), 586-598 (2003).</li><li>Byrne, J. H., Castellucci, V. F., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+individual+mechanoreceptor+sensory+neurons+to+defensive+gill-withdrawal+reflex+in+Aplysia.">Contribution of individual mechanoreceptor sensory neurons to defensive gill-withdrawal reflex in Aplysia.</a> J. Neurophysiol. 41 (2), 418-431 (1978).</li><li>Carlson, S. L., Fieber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physiological+evidence+that+D-Aspartate+activates+a+current+distinct+from+ionotropic+glutamate+receptor+currents+in+Aplysia+californica.">Physiological evidence that D-Aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica.</a> J. Neurophysiol. 106 (4), 1629-1636 (2011).</li><li>Carlson, S. L., Kempsell, A. T., Fieber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pharmacological+evidence+that+D-Aspartate+activates+a+current+distinct+from+ionotropic+glutamate+receptor+currents+in+Aplysia+californica.">Pharmacological evidence that D-Aspartate activates a current distinct from ionotropic glutamate receptor currents in Aplysia californica.</a> Br. Behav. 2 (4), 391-401 (2012).</li><li>Fieber, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+Na++and+Ca2++currents+in+bag+cells+of+sexually+immature+Aplysia+californica.">Characterization of Na+ and Ca2+ currents in bag cells of sexually immature Aplysia californica.</a> J. Exp. Biol. 201 (5), 745-754 (1998).</li><li>Fieber, L. .. A., Carlson, S. L., Capo, T. R., Schmale, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+D-Aspartate+ion+currents+in+the+Aplysia+nervous+system+with+aging.">Changes in D-Aspartate ion currents in the Aplysia nervous system with aging.</a> Br. Res. 1343, 28-36 (2010).</li><li>Glansman, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Habituation+in+Aplysia:+The+Cheshire+Cat+of+neurobiology.">Habituation in Aplysia: The Cheshire Cat of neurobiology.</a> Neurobiol. Learn. Mem. 92, 147-154 (2009).</li><li>Hawkins, R. D., Abrams, T. W., Carew, T. J., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Cellular+Mechanism+of+Classical+Conditioning+in+Aplysia:+Activity-Dependent+Amplification+of+Presynaptic+Facilitation.">A Cellular Mechanism of Classical Conditioning in Aplysia: Activity-Dependent Amplification of Presynaptic Facilitation.</a> Science, New. 219 (4583), 400-405 (1983).</li><li>Ichinose, M., Sawada, M., Maeno, T., McAdoo, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+acetylcholine+on+ventrocaudal+sensory+neurons+in+the+pleural+ganglia+of+Aplysia.">Effect of acetylcholine on ventrocaudal sensory neurons in the pleural ganglia of Aplysia.</a> Cell Mol. Neurobiol. 9, 233-245 (1989).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cellular basis of behavior. , (1976).</li><li>Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+of+memory+storage:+a+dialog+between+nerves+and+synapses.">The molecular biology of memory storage: a dialog between nerves and synapses.</a> Science. 294 (5544), 1030-1038 (2001).</li><li>Kupfermann, I., Castellucci, V., Pinsker, H., Kandel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+correlates+of+habituation+and+habituation+of+the+gill-withdrawal+reflex+in+Aplysia.">Neuronal correlates of habituation and habituation of the gill-withdrawal reflex in Aplysia.</a> Science. 167 (3926), 1743-1745 (1970).</li><li>Magoski, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+an+Aplysia+bag-cell+neuron+cation+channel+by+closely+associated+protein+kinase+A+and+a+protein+phosphatase.">Regulation of an Aplysia bag-cell neuron cation channel by closely associated protein kinase A and a protein phosphatase.</a> J. Neurosci. 24 (30), 6833-6841 (2004).</li><li>R, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+transcriptome+of+Aplysia:+neuronal+compartments+and+circuitry.">Neuronal transcriptome of Aplysia: neuronal compartments and circuitry.</a> Cell. 127, 1453-1467 (2006).</li><li>Nick, T. A., Kaczmarek, L. K., Carew, T. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ionic+currents+underlying+developmental+regulation+of+repetitive+firing+in+Aplysia+bag+cell+neurons.">Ionic currents underlying developmental regulation of repetitive firing in Aplysia bag cell neurons.</a> J. Neurosci. 16 (23), 7583-7598 (1996).</li><li>Sakmann, B., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Single-channel recording. , (1995).</li><li>Tam, A. K., Geiger, J. E., Hung, A. Y., Groten, C. J., Magoski, N. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Persistent+Ca2++current+contributes+to+a+prolonged+depolarization+in+Aplysia+bag+cell+neurons.">Persistent Ca2+ current contributes to a prolonged depolarization in Aplysia bag cell neurons.</a> J. Neurophysiol. 102 (6), 3753-3765 (2009).</li><li>Schacher, S., Proshansky, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurite+regeneration+by+Aplysia+neurons+in+dissociated+cell+culture:+modulation+by+Aplysia+hemolymph+and+the+presence+of+the+initial+axonal+segment.">Neurite regeneration by Aplysia neurons in dissociated cell culture: modulation by Aplysia hemolymph and the presence of the initial axonal segment.</a> J. Neurosci. 3 (12), 2403-2413 (1983).</li><li>Walters, E. T., Byrne, J. H., Carew, T. J., Kandel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanoafferent+neurons+innervating+tail+of+Aplysia.+I.+Response+properties+and+synaptic+connections.">Mechanoafferent neurons innervating tail of Aplysia. I. Response properties and synaptic connections.</a> J. Neurophysiol. 50 (6), 1522-1542 (1983).</li><li>Wilson, G. F., Richardson, F. C., Fisher, T. E., Olivera, B. M., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+a+Ca(2+)-sensitive+nonspecific+cation+channel+underlying+prolonged+repetitive+firing+in+Aplysia+neurons.">Identification and characterization of a Ca(2+)-sensitive nonspecific cation channel underlying prolonged repetitive firing in Aplysia neurons.</a> J. Neurosci. 16 (11), 3661-3671 (1996).</li><li>White, B. H., Nick, T. A., Carew, T. J., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+kinase+C+regulates+a+vesicular+class+of+calcium+channels+in+the+bag+cell+neurons+of+Aplysia.">Protein kinase C regulates a vesicular class of calcium channels in the bag cell neurons of Aplysia.</a> J. Neurophysiol. 80 (5), 2514-2520 (1998).</li><li>Wilson, G. F., Magoski, N. S., Kaczmarek, L. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+a+calcium-sensitive+nonspecific+cation+channel+by+closely+associated+protein+kinase+and+phosphatase+activities.">Modulation of a calcium-sensitive nonspecific cation channel by closely associated protein kinase and phosphatase activities.</a> Proc. Natl. Acad. Sci. U.S.A. 95 (18), 10938-10943 (1998).</li><li>Zhao, Y., Wang, D. O., Martin, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+Aplysia+sensory-motor+neuronal+cell+cultures.">Preparation of Aplysia sensory-motor neuronal cell cultures.</a> J. Vis. Exp. (28), e1355 (2009).</li></ol>

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 border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Artificial seawater ASW</td>
 <td>Sigma-Aldrich</td>
 <td>assorted</td>
 <td>(mM): 417 NaCl, 10 KCl, 10 CaCl2 (2 H2O), 5MgCl2 (6H2O), 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 CaCl2 (2 H2O), 2.5 MgCl2 (6 H2O), 5 Na2ATP, 10 EGTA, and 40 HEPES-KOH, pH 7.4</td>
 </tr>
 <tr>
 <td>Poly-D-lysine</td>
 <td>Sigma</td>
 <td>P6407</td>
 <td>&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>&nbsp;</td>
 </tr>
 <tr>
 <td>hyaluronidase</td>
 <td>Sigma-Aldrich</td>
 <td>H4272</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>collagenase type XI</td>
 <td>Sigma-Aldrich</td>
 <td>C9407</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glutamate (L-Glu)</td>
 <td>Sigma-Aldrich</td>
 <td>49601-100G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>D-Aspartate (D-Asp)</td>
 <td>Sigma-Aldrich</td>
 <td>11200-10G</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>N-methyl-D-aspartate (NMDA)</td>
 <td>Biomol</td>
 <td>100002-268</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Asp</td>
 <td>Sigma</td>
 <td>A6683-25G</td>
 <td>&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>&nbsp;</td>
 </tr>
 <tr>
 <td>L-Glu R antagonists</td>
 <td>various</td>
 <td>various</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>agar</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>kynurenate</td>
 <td>Sigma-Aldrich</td>
 <td>61250</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>APV</td>
 <td>Sigma-Aldrich</td>
 <td>A5282</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>DL-2-Amino-5-phosphonopentanoic acid (NMDAR antagonist)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2-propanol</td>
 <td>VWRSP</td>
 <td>BDH1133</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Chloriding solution</td>
 <td>Sigma</td>
 <td>assorted</td>
 <td>25 g FeCl3 + 25 ml concentrated HCl + 50 ml H2O</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>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Techniquip 150 Watts Fiber Optic Illuminator</td>
 <td>Microoptics of Florida</td>
 <td>TQ FOI-150</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>RotoMix 50800 orbital mixer</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Nikon Diaphot inverted phase-contrast microscope with 4x, 20x (optional) &amp; 40x objectives</td>
 <td>SR Research Ltd.</td>
 <td>Eyelink II</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Tektronix digital oscilloscope</td>
 <td>SR Research Ltd.</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pClamp 10 data acquisition and analysis software</td>
 <td>Molecular Devices</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>PC with Windows XP or higher operating system</td>
 <td>PC Solutions</td>
 <td>&nbsp;</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>&nbsp;</td>
 <td>&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>&nbsp;</td>
 <td>&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>&nbsp;</td>
 </tr>
 <tr>
 <td>Digidata 1200 A/D converter</td>
 <td>Molecular Devices, Sunnyvale, CA</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Picospritzer, powered by N2 adjustable for pressure and duration</td>
 <td>Parker Hannifin, Cleveland, OH</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>TMC Micro-G Vibration isolation table</td>
 <td>Ametek</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Faraday cage</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>custom manufacture</td>
 </tr>
 <tr>
 <td>Burleigh Piezoelectric Clamshell Micromanipulators</td>
 <td>Burleigh Instruments; Thorlabs</td>
 <td>&nbsp;</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>&nbsp;</td>
 <td>&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>&nbsp;</td>
 </tr>
 <tr>
 <td>3 inch length</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Ag/AgCl half cell</td>
 <td>WPI</td>
 <td>EP4</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>15 ml centrifuge tubes, 35-2097 BD Falcon* Centrifuge Tubes</td>
 <td>VWRSP</td>
 <td>21008-918</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Angled Scissors</td>
 <td>Fine Science Tools</td>
 <td>15006-09</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Dumostar Fine forceps</td>
 <td>Fine Science Tools</td>
 <td>11295-00</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>35 mm falcon tissue culture dishes</td>
 <td>VWRSP</td>
 <td>25382-064</td>
 <td>&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>&nbsp;</td>
 </tr>
 <tr>
 <td>animal dissection tray</td>
 <td>various</td>
 <td>&nbsp;</td>
 <td>&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>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>23 gauge needles (manually file off points)</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Polyethylene tubing 0.022"ID x 0.042"OD; 427411</td>
 <td>Becton-Dickinson</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>H-7 pipette stand/holder for microcap perfusion array</td>
 <td>Narishige USA</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>one-way valves</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>Drummond Microcaps 1 &mu;l</td>
 <td>VWRSP</td>
 <td>&nbsp;</td>
 <td>For perfusion system</td>
 </tr>
 <tr>
 <td>18 gauge needles for suction (filed off points)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Polyethylene tubing</td>
 <td>Cole Parmer</td>
 <td>4.27436E+11</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>fine dissection pins</td>
 <td>Fine Science Tools</td>
 <td>26002-20</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>capillary tubes</td>
 <td>Kimble 71900-100</td>
 <td>&nbsp;</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>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>dish holder for microscope stage with isolated ground bath</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Custom manufacture</td>
 </tr>
 <tr>
 <td>pasteur pipettes</td>
 <td>VWRSP</td>
 <td>14672-412</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>pipette bulbs</td>
 <td>VWRSP</td>
 <td>53283-911</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>acrodisk syringe filters</td>
 <td>VWRSP</td>
 <td>28144-040</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>thick-walled 1.5 mm diameter borosilicate filament glass</td>
 <td>WPI</td>
 <td>1B150F-3</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>High purity nitrogen cylinder and bifurcating regulator</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>
Tables 1-3. Lists of Reagents, Materials, and Equipment.

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 (Video) | JoVE 

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 ...

Research

JoVE Journal

Neuroscience

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

Electrode Fabrication and Implantation in Aplysia californica for Multi-channel Neural and Muscular Recordings in Intact, Freely Behaving Animals (Video) | JoVE 

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

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation

Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation (Video) | JoVE 

Bloccare Registrazioni patch da neuroni della retina del mouse in un Dark-adattato Preparazione Slice

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of ...

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons (Video) | JoVE 

Live-imaging di traslocazione della PKC in SF9 cellule e nei neuroni sensoriali Aplysia

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and ...

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from Zebrafish (Video) | JoVE 

Registrazioni patch clamp in cellule interne dell&#39;orecchio Capelli Isolato da Zebrafish

Patch  clamp analyses of the voltage-gated channels in sensory hair cells isolated  from a variety of species have been described previously1-4 but this  ...

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes

F1FO ATPase Vesicle Preparation and Technique for Performing Patch Clamp Recordings of Submitochondrial Vesicle Membranes (Video) | JoVE 

<sub&gt; 1</sub&gt; F<sub&gt; O</sub&gt; ATPasi Vesicle Preparazione e tecnica per permettere Bloccare Registrazioni patch di membrane delle vescicole submitocondriali

Mitochondria are involved in many important cellular  functions including metabolism, survival1, development and, calcium signaling2. Two of the most ...

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

Extracellularly Identifying Motor Neurons for a Muscle Motor Pool in Aplysia californica (Video) | JoVE 

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 ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells (Video) | JoVE 

Bloccare Registrazioni patch da cellule embrionali Zebrafish Mauthner

Mauthner cells (M-cells) are large  reticulospinal neurons located in the hindbrain of teleost fish. They are key  neurons involved in a characteristic ...

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor

Perforated Patch-clamp Recording of Mouse Olfactory Sensory Neurons in Intact Neuroepithelium: Functional Analysis of Neurons Expressing an Identified Odorant Receptor (Video) | JoVE 

Perforato Patch-clamp registrazione di topo neuroni sensoriali olfattivi In neuroepithelium intatto: Analisi funzionale di neuroni Esprimendo un Identificato odorizzante Receptor

Analyzing the physiological responses of olfactory sensory neurons (OSN) when stimulated with specific ligands is critical to understand the basis of ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell Patch-clamp Recordings in Brain Slices (Video) | JoVE 

Registrazioni Whole-cell patch-clamp nel cervello fette

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the ...

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons

Subcellular Patch-clamp Recordings from the Somatodendritic Domain of Nigral Dopamine Neurons (Video) | JoVE 

Subcellulari patch-clamp dal dominio Somatodendritic di nigral dopamina neuroni

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding ...