Finanziato dal NIH (NS-18.205).

1. Preparazione di pesce per Recordings associati <ol><li> Prima di iniziare la procedura, preparare cinque soluzioni chiave, tra cui: Bagno Solution, Solution Bagno con Tricaine (MS222), Soluzione Bagno con Formammide, Neuron soluzione interna e la soluzione del muscolo interno. </li><li> Successivamente, raccogliere un pesce zebra singolo larvale di età compresa tra 72 a 96 ore e il luogo in Solution Bagno con Tricaine. Entro un minuto di esposizione al anestetico, il pesce non risponde al tatto. </li><li> Mettere il pesce su un piatto di plastica e, utilizzando uno stereomicroscopio, decapitare con un rasoio a doppia lama bordo. </li><li> Trasferire il pesce in una camera di registrazione di vetro rivestito con sylgard e riempito di soluzione del bagno. Fissare ad ogni estremità con l&#39;inserimento di perni di tungsteno elettroliticamente affinato attraverso la notocorda. </li><li> Creare un lembo cutaneo verso la fine rostrale del pesce con uno spillo che fornirà una presa adeguata per un paio di pinze fine. Togliere la pelle sovrastante afferrandolo vicino al pin rostrale con un paio di pinze fine. Sbucciare lentamente in direzione caudale per rimuovere la pelle. </li><li> Trattare il pesce con 3 ml di soluzione da bagno con Formammide per 3-5 minuti per bloccare le contrazioni muscolari che potrebbero interferire con le registrazioni. Lavare 5 volte con la soluzione di Bagno normale. Rimuovere parte dello strato superficiale del muscolo da 5 a 6 segmenti delicatamente rottamazione con il lato di uno spillo tungsteno. </li><li> Trasferire il pesce ad un microscopio verticale situato in una gabbia di Faraday per schermare il rumore elettrico. Il microscopio è dotato di un palco fisso, lunga obiettivo 40x distanza di 0,5 e zoom a 4x. Il microscopio è montato su un palcoscenico motorizzato traduzione XY in modo da spostare la preparazione tra nervo e muscolo in alta potenza. L&#39;uso di ottiche interferenza differenziale contrasto aiuta a visualizzare i neuroni. </li><li> Sotto zoom 0,5 x, utilizzare un ampio foro di 15 micron pipetta per rimuovere muscolare sovrastante ed esporre il midollo spinale. Pressione negativa viene applicata attraverso una siringa da 3 cc usa e getta e le cellule muscolari vengono rimosse una ad una. Questa procedura viene ripetuta per 5 o 6 segmenti dorsale del muscolo scheletrico. Lo stesso ampio foro pipetta viene utilizzato per rimuovere anche la parte superiore due strati di muscoli ventrali obiettivo di ottenere velocemente dei muscoli scheletrici. </li><li> Questo completa la preparazione per le registrazioni appaiati e lo zoom microscopio è aumentata a circa 1,6 volte. </li></ol> 2. Registrazioni accoppiato della PAC motore neurone e cellule muscolari target <ol><li> I segmenti pulite vengono analizzati per individuare i neuroni PAC. Ogni emi-segmento del midollo spinale contiene un grande, diametro 10 micron, Cap-neurone motore. Questa soma a forma di lacrima si trova superficialmente sotto la dura madre spinale verso la fine più caudale del confine segmentale. Un neurone CaP viene scelto per la registrazione e il muscolo bersaglio ventrale del segmento adiacente caudale viene analizzato per verificare l&#39;integrità. </li><li> Due elettrodi patch sono posizionati per la registrazione, una per il neurone e un secondo per il muscolo. L&#39;elettrodo superiore ha una conicità poco profonda per la penetrazione della dura spinale dura e un&#39;apertura di circa 2 micron. L&#39;elettrodo inferiore ha un cono più ripida per la registrazione a bassa resistenza morsetto di tensione del muscolo scheletrico </li><li> Posizionare l&#39;elettrodo neurone in un angolo di circa 30 gradi di penetrare la dura madre spinale, circa la metà del segmento anteriore del neurone CaP selezionato. Anticipo l&#39;elettrodo con un manipolatore assiale, mentre applicando una leggera pressione positiva continua di circa 60 millimetri di mercurio. Come l&#39;elettrodo sfonda la dura, la PAC neurone può essere spostato dalla perfusione dall&#39;elettrodo, che richiedono un riaggiustamento della messa a fuoco. </li><li> Quando l&#39;elettrodo tocca il CaP neurone soma pressione positiva viene rilasciata e di solito provoca la formazione immediata di un sigillo gigaohm. Tuttavia, in alcuni casi in cui un sigillo non riesce a formarsi, diventa necessario applicare una leggera pressione negativa. Dopo la formazione di tenuta, il potenziale dell&#39;elettrodo viene regolata a 80mV e l&#39;applicazione di pressione negativa rottura della membrana cellulare. Il neurone CaP è caratterizzata da una resistenza di ingresso di 140-180 Mega-ohm e un potenziale d&#39;azione che il superamento 40 millivolt. Questo è testato iniettando aumento incrementale passi depolarizzanti sotto morsetto corrente fino a quando il potenziale di azione si manifesta. </li><li> Successivamente, il microscopio è trasferita a muscoli ventrali con il traduttore XY motorizzati con potenza elevata. Una cellula muscolare veloce è scelto dal campo di destinazione. </li><li> L&#39;elettrodo muscolo è avanzato verso la cellula muscolare bersaglio sotto pressione positiva, che quando viene rilasciato, forma un sigillo gigaohm. Il potenziale dell&#39;elettrodo viene regolata a -50 millivolt per inattivare i canali del sodio e sotto succhiare dolce la membrana cellulare si rompe. Un calo della resistenza a 100-200 Mega-ohm e l&#39;improvvisa apparizione del grande ingresso capacitivo transitori segnali. </li><li> Per testareper la registrazione appaiati, il motore-neurone è depolarizzata da iniezioni incrementale maggiore di corrente fino a quando un potenziale d&#39;azione si manifesta. A questo punto una corrente placca deve essere raggiunta in muscolo. Stimolazione continua del motorneuron a 1 risultati Hertz correnti placca motrice senza guasti. Come la frequenza di stimolo viene aumentata a 100 Hertz le correnti di placca variano in ampiezza e subire l&#39;esaurimento funzionale entro 10 secondi di stimolazione. </li></ol> 3. Rappresentante Risultati Tipicamente, il neurone di registrazione è stabile per un massimo di un&#39;ora, ma le cellule muscolari si deteriorano, come riflessi in un incremento della resistenza serie. Quando questo si verifica l&#39;esperimento può essere interrotto o un&#39;altra cellula muscolare è di patch bloccato. Tutte le registrazioni dovrebbero essere completati entro un&#39;ora. <img src="/files/ftp_upload/2351/2351fig1.jpg" alt="Figura 1" /> Figura 1. Registrazioni del potenziale d&#39;azione motorneuron (AP, in alto) e la placca muscolare associato corrente (EPC, in basso). Il potenziale d&#39;azione di un neurone PAC dovrebbe overshoot 40 mV e l&#39;EPC dovrebbe salire a 300 msec e di degrado lungo un corso a tempo esponenziale con una costante di tempo inferiore a 1 msec. <table border="1"><tbody><tr><td> Nome </td><td> Ricetta </td></tr><tr><td> Bagno Solution (in mm) </td><td> 134 NaCl, 2.9 KCl, 2.1 CaCl 2, 1.2 MgCl 2, 10 Glucosio, 10 Na-HEPES, pH 7,8 </td></tr><tr><td> Soluzione bagno con Tricaine </td><td> Bagno Soluzione contenente 0,02% tricaine </td></tr><tr><td> Soluzione bagno con Formammide </td><td> Bagno Soluzione contenente 2M formammide </td></tr><tr><td> Neuron interno Solution (in mm) </td><td> 115 K-gluconato, 15 KCl, 2 MgCl 2, 10 K-HEPES, 5 K-EGTA, 4 Mg-ATP, pH 7,2 </td></tr><tr><td> Soluzione interna del muscolo (in mm) </td><td> 120 KCl, 10 K-HEPES, 5 Bapta, pH 7,4 </td></tr></tbody></table> Tabella 1. Solutions.

<ol>
	<li>Fetcho, J. R., O'Malley, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Imaging+neuronal+networks+in+behaving+animals.">Imaging neuronal networks in behaving animals.</a> Current Opinion in Neurobiology. 7, 832-838 (1997).</li><li>Fetcho, J. R., S, H. i. g. a. s. h. i. j. i. 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=Optical+and+genetic+approaches+toward+understanding+neuronal+circuits+in+zebrafish.">Optical and genetic approaches toward understanding neuronal circuits in zebrafish.</a> Integr Comp Biol. 44, 57-70 (2004).</li><li>Iba&#241;ez-Tallon, I., Wen, H., Miwa, J., Xing, J., Aslantas-Tekinay, A., Ono, F., Brehm, P., Heintz, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tethering+naturally+occurring+peptide+toxins+for+cell+autonomous+modulation+of+ion+channels+and+receptors+in+vivo.">Tethering naturally occurring peptide toxins for cell autonomous modulation of ion channels and receptors in vivo.</a> Neuron. 43, 305-311 (2004).</li><li>Li, W., Ono, F., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+measurements+of+presynaptic+release+in+mutant+zebrafish+lacking+postsynaptic+receptors.">Optical measurements of presynaptic release in mutant zebrafish lacking postsynaptic receptors.</a> J. Neuroscience. , 23-10467 (2003).</li><li>Ono, F., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acetylcholine+receptors+direct+rapsyn+clusters+to+the+neuromuscular+synapse+in+zebrafish.">Acetylcholine receptors direct rapsyn clusters to the neuromuscular synapse in zebrafish.</a> J. Neuroscience. 24, 475-5481 (2004).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Fetcho, J., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paralytic+zebrafish+lacking+ACh+receptors+fail+to+localize+rapsyn+clusters+to+the+synapse.">Paralytic zebrafish lacking ACh receptors fail to localize rapsyn clusters to the synapse.</a> J. Neuroscience. 21, 5439-5448 (2001).</li><li>Ono, F., Shcherbatko, A., Higashijima, S., Mandel, G., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+motility+mutant+twitch+once+reveals+new+roles+for+rapsyn+in+synaptic+function.">The zebrafish motility mutant twitch once reveals new roles for rapsyn in synaptic function.</a> J. Neuroscience. 22, 6491-6498 (2002).</li><li>Wang, M., Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Function+of+neuromuscular+synapses+in+the+zebrafish+choline-acetyltransferase+mutant+bajan.">Function of neuromuscular synapses in the zebrafish choline-acetyltransferase mutant bajan.</a> J Neurophysiol. , 100-104 (2008).</li><li>Wen, H., Brehm, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paired+motor-neuron+muscle+recordings+in+zebrafish+test+the+receptor+blockade+model+for+shaping+synaptic+current.">Paired motor-neuron muscle recordings in zebrafish test the receptor blockade model for shaping synaptic current.</a> J. Neuroscience. 25, 8104-8111 (2005).</li></ol>

Nessun conflitto di interessi dichiarati.

Abbiamo utilizzato le registrazioni zebrafish accoppiati (Wen e Brehm, 2005) principalmente per studiare i processi di rilascio e riciclo delle vescicole durante la stimolazione ad alta frequenza. Questo processo viene interrotto in mutanti in cui la motilità normale trasmissione sinaptica è compromessa a causa di mutazioni puntiformi nelle principali componenti sinaptica. Per esempio, le mutazioni che interrompono aggregati recettore postsinaptico (Ono et al., 2001, 2002, 2004) e la sintesi del trasmettitore presinaptica (Wang et al., 2008) in conseguenza non coordinate nuoto. Registrazioni accoppiato fornire le informazioni che possono identificare l&#39;origine del difetto funzionale, collegando così il comportamento, la genetica e la fisiologia. Ora dovrebbe essere possibile sezionare ulteriormente i processi sottostanti la trasmissione sinaptica per mezzo di espressione transitoria della PAC motore-neuroni utilizzando promotori giudizioso. In questo modo i geni dominanti negativi o mutati possono essere specificamente indicato nella questi neuroni e sia le conseguenze comportamentali ed elettrofisiologiche determinato. L&#39;ulteriore vantaggio offerto dalla configurazione di cellula intera permette di dialisi del soma CaP con farmaci che possono potenzialmente alterare il riciclo delle vescicole, come le tossine clostridiali e buffer di calcio. A causa della breve distanza tra il soma e muscolare, la diffusione dovrebbe avvenire anche entro i tempi delle registrazioni. Il potenziale di questo preparato ha solo cominciato a essere sfruttato. La trasparenza dei pesci in questa posizione età e superficiale delle sinapsi facilita anche l&#39;uso di strumenti ottici (Fetcho e O&#39;Malley, 1997; Fetcho e Higashijima, 2004). Abbiamo avuto molto successo nella misurazione di endo ed esocitosi nei pesci che vivono con coloranti FM e gli indicatori genetici come Synaptophluorin (Li et al., 2003). Inoltre, fluorescente tossine coniugati possono essere usati per etichettare le proteine ​​sinaptiche per l&#39;imaging di pesci vivi (Ibanez-Tallon et al., 2004). Data la semplicità di questa preparazione è molto promettente per gli studi successivi.

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<td>Pneumatic transducer tester</td>
<td>&nbsp;</td>
<td>Fluke Biomedical Instruments</td>
<td>DPM1B</td>
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<td>0.002 x 3 inch tungsten rod</td>
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<td>A-M Systems Inc</td>
<td>715000</td>
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<td>Sylgard 184 Elastomer</td>
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<td>Dow Corning Corp.</td>
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<td>The thickness of application will affect the DIC optics</td>
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paired patch clamp recordings from motor-neuron and target skeletal muscle in zebrafish

Larvale zebrafish rappresentano il primo sistema modello vertebrato per consentire la registrazione simultanea patch clamp da un neurone spinale a motore e la destinazione dei muscoli. Questa è una conseguenza diretta della accessibilità per entrambi i tipi di cellule e la capacità di distinguere visivamente il tetto unico segmentale motore neurone sulla base della morfologia e la posizione. Questo video mostra i metodi microscopici utilizzato per identificare un CAP motore neuroni e cellule muscolari bersaglio così come le metodologie per la registrazione da ogni tipo di cellula. Identificazione del motore tipo-neurone CaP è confermato da uno riempimento tintura o dalle caratteristiche biofisiche, come forma d&#39;onda del potenziale d&#39;azione e resistenza d&#39;ingresso delle cellule. Motore neurone registrazioni di routine scorso per un&#39;ora consentendo a lungo termine le registrazioni da cellule bersaglio più diversi muscolare. Il controllo del motore neurone modello cottura consente misurazioni della frequenza-dipendenza della trasmissione sinaptica a livello della giunzione neuromuscolare. A causa di una grande dimensione quantica e basso rumore fornite dal morsetto intera tensione di cella, tutti gli eventi unitario può essere risolto nel muscolo. Questa funzione permette di studiare le proprietà fondamentali sinaptica come le proprietà di rilascio, il riciclo delle vescicole, così come la depressione sinaptica e facilitazione. I vantaggi offerti da questo in vivo preparazione eclissi precedenti sistemi neuromuscolari modello studiato in cui il motore neuroni sono di solito stimolati da elettrodi extracellulari ed i muscoli sono troppo grandi per delle patch clamp intera cellula. La preparazione zebrafish è riconducibile alla combinazione di analisi elettrofisiologiche con una vasta gamma di approcci tra cui linee transgeniche, knockdown morfolino, l&#39;intervento farmacologico e imaging in vivo. Questi approcci, insieme con il crescente numero di modelli di malattia neuromuscolare forniti da linee mutanti di zebrafish, aprire la porta per la nuova comprensione dei diritti umani malattie neuromuscolari.

Watch this Scientific Journal Video about Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish at JoVE.com

Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

Larvale zebrafish rappresentano il primo sistema modello vertebrato per consentire la registrazione simultanea patch clamp da un neurone spinale a motore e la destinazione del muscolo scheletrico. Questo video mostra i metodi microscopici utilizzato per identificare un CAP segmentale motore neuroni e cellule muscolari bersaglio così come le metodologie per la registrazione da ogni tipo di cellula.

Accoppiato a pinza registrazioni patch da Motor-neurone e muscolo scheletrico bersaglio in Zebrafish

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle.  ...

paired-patch-clamp-recordings-from-motor-neuron-target-skeletal

Research

JoVE Journal

Neuroscience

17.0K Views.  Oregon Health and Sciences University. Larvale zebrafish rappresentano il primo sistema modello vertebrato per consentire la registrazione simultanea patch clamp da un neurone spinale a motore e la destinazione del muscolo scheletrico. Questo video mostra i metodi microscopici utilizzato per identificare un CAP segmentale motore neuroni e cellule muscolari bersaglio così come le metodologie per la registrazione da ogni tipo di cellula.

Video: Paired Patch Clamp Recordings from Motor-neuron and Target Skeletal Muscle in Zebrafish

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

Our visual experience is initiated when the visual pigment in our retinal photoreceptors absorbs photons of light energy and initiates a cascade of intracellular events that lead to closure of cyclic-nucleotide-gated channels in the cell membrane. The resulting change in membrane potential leads in turn to reductions in the amount of neurotransmitter release from both rod and cone synaptic terminals. To measure how the light-evoked change in photoreceptor membrane potential leads to downstream activity in the retina, scientists have made electrophysiological recordings from retinal slice preparations for decades1,2. In the past these slices have been cut manually with a razor blade on retinal tissue that is attached to filter paper; in recent years another method of slicing has been developed whereby retinal tissue is embedded in low gelling temperature agar and sliced in cool solution with a vibrating microtome3,4. This preparation produces retinal slices with less surface damage and very robust light-evoked responses. Here we document how this procedure can be done under infrared light to avoid bleaching the visual pigment.

Here we describe a procedure for generating dark-adapted slices of the mouse retina for electrophysiological recordings.

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 ...

Ricerca

Neuroscienze

Patch Clamp Recordings in Inner Ear Hair Cells Isolated from 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 video represents the first application of those techniques to hair cells from zebrafish. Here we demonstrate a method to isolate healthy, intact hair cells from all of the inner ear end-organs: saccule, lagena, utricle and semicircular canals. Further, we demonstrate the diversity in hair cell size and morphology and give an example of the kinds of patch clamp recordings that can be obtained. The advantage of the use of this zebrafish model system over others stems from the availability of zebrafish mutants that affect both hearing and balance. In combination with the use of transgenic lines and other techniques that utilize genetic analysis and manipulation, the cell isolation and electrophysiological methods introduced here should facilitate greater insight into the roles hair cells play in mediating these sensory modalities.

The purpose of this video is to demonstrate procedures for obtaining healthy, intact hair cells from the inner ear organs of adult zebrafish and then using them for patch clamp studies aimed at characterizing the biophysical properties of their voltage-gated channels.

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

Mitochondria are involved in many important cellular functions including metabolism, survival1, development and, calcium signaling2. Two of the most important mitochondrial functions are related to the efficient production of ATP, the energy currency of the cell, by oxidative phosphorylation, and the mediation of signals for programmed cell death3.
The enzyme primarily responsible for the production of ATP is the F1FO-ATP synthase, also called ATP synthase4-5. In recent years, the role of mitochondria in apoptotic and necrotic cell death has received considerable attention. In apoptotic cell death, BCL-2 family proteins such as Bax enter the mitochondrial outer membrane, oligomerize and permeabilize the outer membrane, releasing pro-apoptotic factors into the cytosol6. In classic necrotic cell death, such as that produced by ischemia or excitotoxicity in neurons, a large, poorly regulated increase in matrix calcium contributes to the opening of an inner membrane pore, the mitochondrial permeability transition pore or mPTP. This depolarizes the inner membrane and causes osmotic shifts, contributing to outer membrane rupture, release of pro-apoptotic factors, and metabolic dysfunction. Many proteins including Bcl-xL7 interact with F1FO ATP synthase, modulating its function. Bcl-xL interacts directly with the beta subunit of F1FO ATP synthase, and this interaction decreases a leak conductance within the F1FOATPasecomplex, increasing the net transport of H+ by F1FO during F1FO ATPase activity8 and thereby increasing mitochondrial efficiency. To study the activity and modulation of the ATP synthase, we isolated from rodent brain submitochondrial vesicles (SMVs) containing F1FO ATPase. The SMVs retain the structural and functional integrity of the F1FO ATPase as shown in Alavian et al. Here, we describe a method that we have used successfully for the isolation of SMVs from rat brain and we delineate the patch clamp technique to analyze channel activity (ion leak conductance) of the SMVs.

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

A method to isolate submitochondrial vesicles enriched in F1FO ATP synthase complexes from rat brain is described. These vesicles allow the study of the activity of F1FO ATPase complex and its modulation using the technique of patch clamp recording.

<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 ...

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

The marine gastropod mollusk Aplysia californica has a venerable history as a model of nervous system function, with particular significance in studies of learning and memory. The typical preparations for such studies are ones in which the sensory and motoneurons are left intact in a minimally dissected animal, or a technically elaborate neuronal co-culture of individual sensory and motoneurons. Less common is the isolated neuronal preparation in which small clusters of nominally homogeneous neurons are dissociated into single cells in short term culture. Such isolated cells are useful for the biophysical characterization of ion currents using patch clamp techniques, and targeted modulation of these conductances. A protocol for preparing such cultures is described. The protocol takes advantage of the easily identifiable glutamatergic sensory neurons of the pleural and buccal ganglia, and describes their dissociation and minimal maintenance in culture for several days without serum.

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

We describe the dissection of the nervous system of the marine sea hare Aplysia after anesthesia, the isolation of neurons for short term-tissue culture, and recordings of single cell ion currents via the patch clamp technique.

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 ...

Patch Clamp Recordings from Embryonic Zebrafish Mauthner Cells

Mauthner cells (M-cells) are large reticulospinal neurons located in the hindbrain of teleost fish. They are key neurons involved in a characteristic behavior known as the C-start or escape response that occurs when the organism perceives a threat. The M-cell has been extensively studied in adult goldfish where it has been shown to receive a wide range of excitatory, inhibitory and neuromodulatory signals1. We have been examining M-cell activity in embryonic zebrafish in order to study aspects of synaptic development in a vertebrate preparation. In the late 1990s Ali and colleagues developed a preparation for patch clamp recording from M-cells in zebrafish embryos, in which the CNS was largely intact2,3,4. The objective at that time was to record synaptic activity from hindbrain neurons, spinal cord neurons and trunk skeletal muscle while maintaining functional synaptic connections within an intact brain-spinal cord preparation. This preparation is still used in our laboratory today. To examine the mechanisms underlying developmental synaptic plasticity, we record excitatory (AMPA and NMDA-mediated)5,6 and inhibitory (GABA and glycine) synaptic currents from developing M-cells. Importantly, this unique preparation allows us to return to the same cell (M-cell) from preparation to preparation to carefully examine synaptic plasticity and neuro-development in an embryonic organism. The benefits provided by this preparation include 1) intact, functional synaptic connections onto the M-cell, 2) relatively inexpensive preparations, 3) a large supply of readily available embryos 4) the ability to return to the same cell type (i.e. M-cell) in every preparation, so that synaptic development at the level of an individual cell can be examined from fish to fish, and 5) imaging of whole preparations due to the transparent nature of the embryos.

We have developed an intact brain-spinal cord preparation to record and monitor electrical activity via patch clamp recording from the Mauthner neurons and other reticulospinal cells in zebrafish embryos. Thus, we are able to record excitatory and inhibitory synaptic currents, voltage-gated channel activity and action potentials from key neurons in a developing embryo.

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 ...

Whole-cell Patch-clamp Recordings from Morphologically- and Neurochemically-identified Hippocampal Interneurons

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal population (10-20%), but show a high level of physiological, morphological, and neurochemical heterogeneity, reflecting their diverse functions. Therefore, investigation of interneurons provides important insights into the organization principles and function of neuronal circuits. This, however, requires an integrated physiological and neuroanatomical approach for the selection and identification of individual interneuron types. Whole-cell patch-clamp recording from acute brain slices of transgenic animals, expressing fluorescent proteins under the promoters of interneuron-specific markers, provides an efficient method to target and electrophysiologically characterize intrinsic and synaptic properties of specific interneuron types. Combined with intracellular dye labeling, this approach can be extended with post-hoc morphological and immunocytochemical analysis, enabling systematic identification of recorded neurons. These methods can be tailored to suit a broad range of scientific questions regarding functional properties of diverse types of cortical neurons.

Cortical networks are controlled by a small, but diverse set of inhibitory interneurons. Functional investigation of interneurons therefore requires targeted recording and rigorous identification. Described here is a combined approach involving whole-cell recordings from single or synaptically-coupled pairs of neurons with intracellular labeling, post-hoc morphological and immunocytochemical analysis.

Whole-cell patch-clamp registrazioni da Morphologically- e neurochimico-identificati interneuroni dell&#39;ippocampo

GABAergic inhibitory interneurons play a central role within neuronal circuits of the brain. Interneurons comprise a small subset of the neuronal ...

Paired Whole Cell Recordings in Organotypic Hippocampal Slices

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct electrophysiological characterization of the synaptic connections between individual neurons, but also pharmacological manipulation of either the presynaptic or the postsynaptic neuron. When carried out in organotypic hippocampal slice cultures, the probability that two neurons are synaptically connected is significantly increased. This preparation readily enables identification of cell types, and the neurons maintain their morphology and properties of synaptic function similar to that in native brain tissue. A major advantage of paired whole cell recordings is the highly precise information it can provide on the properties of synaptic transmission and plasticity that are not possible with other more crude techniques utilizing extracellular axonal stimulation. Paired whole cell recordings are often perceived as too challenging to perform. While there are challenging aspects to this technique, paired recordings can be performed by anyone trained in whole cell patch clamping provided specific hardware and methodological criteria are followed. The probability of attaining synaptically connected paired recordings significantly increases with healthy organotypic slices and stable micromanipulation allowing independent attainment of pre- and postsynaptic whole cell recordings. While CA3-CA3 pyramidal cell pairs are most widely used in the organotypic slice hippocampal preparation, this technique has also been successful in CA3-CA1 pairs and can be adapted to any neurons that are synaptically connected in the same slice preparation. In this manuscript we provide the detailed methodology and requirements for establishing this technique in any laboratory equipped for electrophysiology.

Pair recordings are simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling precise electrophysiological and pharmacological characterization of the synapses between individual neurons. Here we describe the detailed methodology and requirements for establishing this technique in organotypic hippocampal slice cultures in any laboratory equipped for electrophysiology.

Associati a cellule intere registrazioni in organotipiche fettine di ippocampo

Pair recordings involve simultaneous whole cell patch clamp recordings from two synaptically connected neurons, enabling not only direct ...

Electrophysiological and Morphological Characterization of Neuronal Microcircuits in Acute Brain Slices Using Paired Patch-Clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with simultaneous intracellular biocytin filling allows a correlated analysis of their structural and functional properties. With this method it is possible to identify and characterize both pre- and postsynaptic neurons by their morphology and electrophysiological response pattern. Paired recordings allow studying the connectivity patterns between these neurons as well as the properties of both chemical and electrical synaptic transmission. Here, we give a step-by-step description of the procedures required to obtain reliable paired recordings together with an optimal recovery of the neuron morphology. We will describe how pairs of neurons connected via chemical synapses or gap junctions are identified in brain slice preparations. We will outline how neurons are reconstructed to obtain their 3D morphology of the dendritic and axonal domain and how synaptic contacts are identified and localized. We will also discuss the caveats and limitations of the paired recording technique, in particular those associated with dendritic and axonal truncations during the preparation of brain slices because these strongly affect connectivity estimates. However, because of the versatility of the paired recording approach it will remain a valuable tool in characterizing different aspects of synaptic transmission at identified neuronal microcircuits in the brain.

Patch-clamp recordings and simultaneous intracellular biocytin filling of synaptically coupled neurons in acute brain slices allow a correlated analysis of their structural and functional properties. The aim of this protocol is to describe the essential technical steps of electrophysiological recording from neuronal microcircuits and their subsequent morphological analysis.

Elettrofisiologici e morfologica Caratterizzazione neuronali Microcircuiti in Acute Fette cervello Utilizzando accoppiati di patch-clamp Recordings

The combination of patch clamp recordings from two (or more) synaptically coupled neurons (paired recordings) in acute brain slice preparations with ...

Whole-cell Patch-clamp Recordings in Brain Slices

Whole-cell patch-clamp recording is an electrophysiological technique that allows the study of the&#160;electrical properties of a substantial part of the neuron. In this configuration, the micropipette is in tight contact with the cell membrane, which prevents current leakage and thereby provides more accurate ionic current measurements than the previously used intracellular sharp electrode recording method. Classically, whole-cell recording can be performed on neurons in various types of preparations, including cell culture models, dissociated neurons, neurons in brain slices, and in intact anesthetized or awake animals. In summary, this technique has immensely contributed to the understanding of passive and active biophysical properties of excitable cells. A major advantage of this technique is that it provides information on how specific manipulations (e.g., pharmacological, experimenter-induced plasticity) may alter specific neuronal functions or channels in real-time. Additionally, significant opening of the plasma membrane allows the internal pipette solution to freely diffuse into the cytoplasm, providing means for introducing drugs, e.g., agonists or antagonists of specific intracellular proteins, and manipulating these targets without altering their functions in neighboring cells. This article will focus on whole-cell recording performed on neurons in brain slices,&#160;a preparation that has the advantage of recording neurons in relatively well preserved brain circuits, i.e., in a physiologically relevant context. In particular,&#160;when combined with appropriate pharmacology, this technique is a powerful tool allowing identification of specific neuroadaptations that occurred following any type of experiences, such as learning, exposure to drugs of abuse, and stress. In summary, whole-cell patch-clamp recordings in brain slices provide means to measure in ex vivo preparation long-lasting changes in neuronal functions that have developed in intact awake animals.

This protocol describes basic procedural steps for performing whole-cell patch-clamp recordings. This technique allows the study of&#160;the electrical behavior of neurons, and when performed in brain slices, allows the assessment of various neuronal functions from neurons that are still integrated in relatively well preserved brain circuits.

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

Dendrites of dopaminergic neurons receive and convey synaptic input, support action potential back-propagation and neurotransmitter release. Understanding these fundamental functions will shed light on the information transfer in these neurons. Dendritic patch-clamp recordings provide the possibility to directly examine the electrical properties of dendrites and underlying voltage-gated ion channels. However, these fine structures are not easily accessible to patch pipettes because of their small diameter. This report describes a step-by-step procedure to collect stable and reliable recordings from the dendrites of dopaminergic neurons in acute slices. Electrophysiological measurements are combined with post hoc recovery of cell morphology. Successful experiments rely on improved preparation of slices, solutions and pipettes, adequate adjustment of the optics and stability of the pipette in contact with the recorded structure. Standard principles of somatic patch-clamp recording are applied to dendrites but with a gentler approach of the pipette. These versatile techniques can be implemented to address various questions concerning the excitable properties of dendrites.

Subcellular patch-clamp recordings offer the possibility to investigate the functional properties of dendrites. However these fine structures are not easily accessible due to their small diameter. The protocol described here aims to facilitate the collection of stable and reliable recordings from the dendrites of dopamine neurons in slices in vitro.

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 ...