Questo lavoro è stato sostenuto da NIH di Grant EY17606 (APS).

1. Elettrodi preparazione <ol><li> Preparare gli elettrodi di registrazione e di riferimento per immersione entrambi in 1M NaCl e guida 15V tra di loro utilizzando un 1 sinusoide Hz per ~ 20 s. </li><li> Rendere gli elettrodi di registrazione con un estrattore micropipetta (Sutter Instruments P-97). Per le patch-pipette utilizzare un vetro borosilicato con un diametro esterno di 1,2 mm e un diametro interno di 0,69 mm (Sutter Instruments, Cat # B120-69-10). Misurare la resistenza serie attraverso la punta dell&#39;elettrodo. In base alle dimensioni delle cellule bersaglio scegliere un diametro puntale; per corpi cellulari ~ 20 micron di diametro utilizzare ~ 5 MΩ per corpi cellulari ~ 5 micron di diametro utilizzare ~ 12 MW. </li><li> Preparare terra / elettrodi di riferimento. Utilizzando una siringa riempire un piccolo tubo di polietilene sezione (Intramedic, PE205) con 1 mM di NaCl, che include 3,5% agar (Sigma; Cat # A7002) in NaCl 1M. Posto questo ponte agar negli Ag / AgCl elettrodo di riferimento. </li></ol> 2. Soluzioni per la preparazione <ol><li> Bagno di soluzione esterna, Ames &#39;supporti contenenti bicarbonato (1 L): 1 bottiglia di Ames&#39; Media (Sigma; Cat # A1420), 1,9 g di NaHCO 3, 7 ml di penicillina-streptomicina (Sigma; Cat # P0781) per evitare che batteri crescita. Regolare osmolarità a ~ 280 mOsm. </li><li> Affettare soluzione, Ames &#39;supporti contenenti il ​​HEPES pH (1 L): 1 bottiglia di Ames&#39; media, 0,887 g di NaCl, 2,38 g di HEPES, 7 ml di penicillina-streptomicina. Portare il pH a ~ 7,4 con NaOH 1M e / o HCl 1M. Regolare osmolarità a ~ 280 mOsm. </li><li> Potassio-aspartato (K-Asp) soluzione di pipetta interne (in mm): 125 K-aspartato, 10 KCl, 10 HEPES, 5 NMG-HEDTA, 0,5 CaCl 2, 1 ATP-Mg, 0,2 GTP-Mg, pH viene regolato ~ 7.3 con NMG-OH, e osmolarità è regolato ~ 280 mOsm. </li><li> Backstop agar per la stabilizzazione a bassa temperatura di gelificazione agarosio contenente la retina (5% in peso), 7,5 g di agar (Sigma; Cat # A7002) sciolto in 150 ml di dH 2 O. </li></ol> 3. Giorno di Esperimento <ol><li> Scongelare ~ 1,5 mL di K-Asp soluzione interna e il fluoroforo di vostra scelta dal congelatore. Diluire il fluoroforo alla concentrazione adeguata, che dovrebbe essere empiricamente determinato. </li><li> Versare Ames &#39;supporti contenenti NaHCO 3 (~ 200 mL) nella luce contenitore a tenuta stagna di stoccaggio e poi posto nella vasca d&#39;acqua (30-32 ° C) ed equilibrare con il 5% di CO 2 / 95% O 2 gas </li><li> Riempire bottiglie con ~ 110 mL di media Ames &#39;contenente HEPES, posto su ghiaccio ed equilibrare con il 100% O 2. </li><li> Mettere il restante Ames &#39;supporti contenenti NaHCO 3 in un serbatoio riscaldato in cima alla gabbia di Faraday, ed equilibrare con il 5% di CO 2 / 95% O 2 gas. Parafilm può essere utilizzato per intrappolare il gas nello spazio sovrastante la soluzione. </li><li> Preparare la bassa temperatura di gelificazione agarosio (3%) con l&#39;aggiunta di 0,75 g (Sigma; Cat # A0701) a 25 mL di media Ames &#39;con HEPES e riscaldare la soluzione a ~ 115 ° C per sciogliere l&#39;agarosio. Mescolare fino a quando la soluzione limpida senza bolle, e conservare in un bagno d&#39;acqua (~ 42 ° C). </li><li> Riempire una pipetta di registrazione con il K-Asp soluzione interna e controllare la resistenza punta. </li></ol> 4. Iniziare Esperimento <ol><li> Euthanize il mouse e rimuovere rapidamente gli occhi con le forbici curve. Occhiali a raggi infrarossi deve essere utilizzato per tutte le procedure per prevenire lo sbiancamento del pigmento visivo. </li><li> Mettere gli occhi su un pezzo di carta da filtro per impedire loro di rotolamento, e utilizzando una sottile lama a doppio lato, praticare un&#39;incisione nella cornea mentre si tiene l&#39;occhio con le pinze. </li><li> Non appena il bulbo oculare è forato, trasferimento che gli occhi in un piatto pieno di mezzi di Ames &#39;. </li><li> Utilizzando la fessura nella cornea come punto di ingresso, forbicine sono usati per tagliare via le parti rimanenti della cornea, dopo di che l&#39;obiettivo e il corpo vitreo può essere rimossa. Fare attenzione che la retina rimane attaccato al oculare, e conservare l&#39;oculare nel buio a 32 ° C in media equilibrata con 5% di CO 2 / 95% O 2 Ames &#39;. </li></ol> 5. Affettare <ol><li> Hemisect uno degli oculari e dissociare la retina da dell&#39;epitelio pigmentato retinico. Rimuovere i bordi della retina per permettere al tessuto di mentire piatto. </li><li> Utilizzare un piccolo bicchiere pipetta Pasteur con un bulbo in lattice per riempire un piatto 35 millimetri Petri con agar fuso, e trascinare il forcipe che attraversi l&#39;agar per testare la sua consistenza. </li><li> Quando si può cominciare a vedere l&#39;agar solidificare: <ol class="lalpha"><li> Trasferire la retina alla parte superiore della agar. </li><li> Usare un pezzo ritorto di Kimwipe per assorbire l&#39;eccesso di soluzione attorno alla retina. </li><li> Luogo 2-3 gocce di Agar direttamente sulla retina e rapidamente posto un cilindro di plastica sopra la retina per formare un muro intorno. Poi a goccia ulteriori agar fuso ai lati del cilindro di plastica mentre centrare la retina all&#39;interno del agar. </li><li> Trasferire la piastra di Petri to un bagno di acqua ghiacciata per raffreddare. </li></ol></li><li> Dopo ~ 45 secondi rimuovere il tessuto dal bagno acqua ghiacciata e utilizzare un pistone per estrudere l&#39;agar blocco fuori dal cilindro di plastica, spingendo dal lato più lontano dalla retina. </li><li> Utilizzare il unilaterale lama di rasoio per tagliare un piccolo blocco di agar contenente la retina e supercolla il blocco in posizione contro l&#39;arresto agar sul disco campione. </li><li> Trasferire il disco di preparato al vassoio microtomo vibrante e versare il ghiaccio freddo HEPES Ames nel cassetto che permette il blocco con la retina di essere completamente sommerso. </li><li> Fette della retina con uno spessore di circa 200 micron può essere tagliato al tasso massimo lama vibrante e una velocità di lama in avanti impostato in modo che ci vogliono 4-5 secondi per tagliare la retina su ogni fetta. </li><li> Utilizzare una lama di bisturi n ° 11 per rimuovere ognuna utilizzabile fetta alla volta </li><li> Fette di buon contenenti retina sono inseriti in alloggiamenti di registrazione e sono ponderati con le ancore fetta con fili di nylon che li attraversano. I fili di nylon tenere premuto l&#39;agar circostanti della retina, lasciando la retina senza ostacoli per le registrazioni. </li><li> Trasferire la camera di registrazione alla fase del microscopio in posizione verticale, chiudere le tende e accendere la sorgente di luce a infrarossi per visualizzare la fetta su una telecamera a raggi infrarossi sensibile. </li></ol> 6. Registrazione <ol><li> Una volta che una buona fetta della retina (fotorecettori con intatta e l&#39;angolo di taglio del caso) viene identificato iniziare perfusione la retina ad un tasso superiore a 5 ml / min con i media riscaldata Ames &#39;che viene riscaldato a 35 37 ° C ed equilibrata con 5% CO 2 / 95% O 2. </li><li> Alcuni danni superficie può essere evidente a causa del processo di taglio. Questo strato di solito sottile di cellule possono essere rimosse con una pipetta con una punta rotta, e delicatamente succhiare via i corpi delle cellule morte. Questo genere rivelare cellule vive sotto. Identificare un corpo sano cellula la cui posizione si trova negli strati di interesse. </li><li> Riempire una micropipetta con la soluzione KAsp interno e applicare positivo (verso l&#39;esterno) della pressione attraverso la punta della pipetta, e abbassare la punta della pipetta nella camera di registrazione e fino al livello della cellula con un micromanipolatore. </li><li> Spostare la punta della pipetta tale da fossette la membrana cellulare, e applicare dolce negativa (verso l&#39;interno), la pressione per fare un sigillo ad alta resistenza. A cellula intera modalità può essere ottenuto mediante l&#39;applicazione di pressione negativa per l&#39;elettrodo di entrare in cella. </li><li> Stimolare il tessuto retinico tramite LED possono evocare reazioni alla luce. </li><li> Fotografare la cella da cui è stata effettuata la registrazione evocando fluorescenza dal fluoroforo pipetta nella soluzione interna. </li></ol> 7. Rappresentante Risultati <img src="/files/ftp_upload/2107/2107fig1.jpg" alt="Figura 1" /> Figura 1. Qui, la luce potenziali evocati di un adattato all&#39;oscurità dei neuroni della retina di lampi di luce di forza maggiore sono mostrati. <img src="/files/ftp_upload/2107/2107fig2.jpg" alt="Figura 2" /> Figura 2. Un&#39;immagine fluorescenza da una fetta della retina in cui le cellule sono stati riempiti con il fluoroforo, Lucifero Giallo. Fluorescenza evocato mostra la morfologia delle cellule da cui sono state fatte le registrazioni di patch.

<ol>
	<li>Weblin, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transmission+along+and+between+rods+in+the+tiger+salamander+retina.">Transmission along and between rods in the tiger salamander retina.</a> J Physiol. 280, 449-470 (1978).</li><li>Wu, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptic+connections+among+retina+neurons+in+living+slices.">Synaptic connections among retina neurons in living slices.</a> J Neurosci Methods. 20, 139-149 (1987).</li><li>Rieke, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temporal+contrast+adaptation+in+salamander+bipolar+cell.">Temporal contrast adaptation in salamander bipolar cell.</a> J Neurosci. 21, 9445-9454 (2001).</li><li>Armstrong-Gold, C., &amp;amp, R. i. e. k. e., F, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bandpass+filtering+at+the+rod+to+second-order+cell+synapse+in+salamander+(Ambystoma+tigrinum)+retina.">Bandpass filtering at the rod to second-order cell synapse in salamander (Ambystoma tigrinum) retina.</a> J Neurosci. 23, 3796-3806 (2003).</li><li>Masland, R. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fundamental+plan+of+the+retina.">The fundamental plan of the retina.</a> Nat Neurosci. 4, 877-886 (2001).</li><li>Wassle, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Parallel+processing+in+the+mammalian+retina.">Parallel processing in the mammalian retina.</a> Nat Rev Neurosci. 5, 747-757 (2004).</li></ol>

Nessun conflitto di interessi dichiarati.

Registrazione fetta di retina dei vertebrati sono state fatte per decenni 1,2, e sono stati molto efficaci nel fornire una comprensione più profonda di come i circuiti della retina codifica luce incidente. I vantaggi del taglio con un microtomo vibrante, piuttosto che direttamente con una lama di rasoio è che le fette della retina incorrere meno danni in superficie. Con una pipetta di aspirazione è facile da rimuovere questi danni e le cellule bersaglio sane appena sotto la superficie che conservano la lor...

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Micropipette Puller</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>P-97</td>
<td>&nbsp;</td>
<tr>
<td>Borosilicate glass</td>
<td>&nbsp;</td>
<td>Sutter Instruments</td>
<td>B120-69-10</td>
<td>&nbsp;</td>
<tr>
<td>Polyethylene tubing</td>
<td>&nbsp;</td>
<td>Intramedic</td>
<td>PE205</td>
<td>&nbsp;</td>
<tr>
<td>Agar</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A7002</td>
<td>&nbsp;</td>
<tr>
<td>Low gelling temp agarose</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A0701</td>
<td>&nbsp;</td>
<tr>
<td>Ames' Medium</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A1420</td>
<td>&nbsp;</td>
<tr>
<td>Penicillin-Streptomycin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P0781</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

patch clamp recordings from mouse retinal neurons in a dark-adapted slice preparation

La nostra esperienza visiva è iniziata quando il pigmento visivo nel nostro fotorecettori della retina assorbe i fotoni di energia luminosa e avvia una cascata di eventi intracellulari che portano alla chiusura del ciclico-nucleotide-gated canali nella membrana cellulare. Conseguente cambiamento di potenziale di membrana porta a sua volta alla riduzione della quantità del rilascio di neurotrasmettitore da entrambi i terminali asta e cono sinaptica. Per misurare come il cambiamento di luce evocati del potenziale di membrana dei fotorecettori porta ad attività a valle nella retina, gli scienziati hanno effettuato registrazioni elettrofisiologiche da preparazioni fetta della retina per 1,2 decenni. In passato queste fette sono state tagliate manualmente con una lama di rasoio sul tessuto retinico che è collegato a carta da filtro, in questi ultimi anni un altro metodo di taglio è stato sviluppato per cui tessuto retinico è incorporato in agar a bassa temperatura di gelificazione e affettati in soluzione fredda con un vibrante microtomo 3,4. Questa preparazione produce fette della retina con danni superficiali e meno risposte luce evocata molto robusto. Qui documentare come questa procedura può essere fatta sotto la luce a infrarossi per evitare lo sbiancamento del pigmento visivo.

Watch this Scientific Journal Video about Patch Clamp Recordings from Mouse Retinal Neurons in a Dark-adapted Slice Preparation at JoVE.com

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

Qui si descrive una procedura per la generazione di dark-adattato fette della retina del mouse per le registrazioni elettrofisiologiche.

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 ...

patch-clamp-recordings-from-mouse-retinal-neurons-dark-adapted-slice

Research

JoVE Journal

Neuroscience

14.3K Views.  University of Southern California. Qui si descrive una procedura per la generazione di dark-adattato fette della retina del mouse per le registrazioni elettrofisiologiche.

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

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

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target muscle. This is a direct consequence of the accessibility to both cell types and ability to visually distinguish the single segmental CaP motor-neuron on the basis of morphology and location. This video demonstrates the microscopic methods used to identify a CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type. Identification of the CaP motor-neuron type is confirmed by either dye filling or by the biophysical features such as action potential waveform and cell input resistance. Motor-neuron recordings routinely last for one hour permitting long-term recordings from multiple different target muscle cells. Control over the motor-neuron firing pattern enables measurements of the frequency-dependence of synaptic transmission at the neuromuscular junction. Owing to a large quantal size and the low noise provided by whole cell voltage clamp, all of the unitary events can be resolved in muscle. This feature permits study of basic synaptic properties such as release properties, vesicle recycling, as well as synaptic depression and facilitation. The advantages offered by this in vivo preparation eclipse previous neuromuscular model systems studied wherein the motor-neurons are usually stimulated by extracellular electrodes and the muscles are too large for whole cell patch clamp. The zebrafish preparation is amenable to combining electrophysiological analysis with a wide range of approaches including transgenic lines, morpholino knockdown, pharmacological intervention and in vivo imaging. These approaches, coupled with the growing number of neuromuscular disease models provided by mutant lines of zebrafish, open the door for new understanding of human neuromuscular disorders.

Larval zebrafish represent the first vertebrate model system to allow simultaneous patch clamp recording from a spinal motor-neuron and target skeletal muscle. This video demonstrates the microscopic methods used to identify a segmental CaP motor-neuron and target muscle cells as well as the methodologies for recording from each cell type.

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.  ...

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 ...

Simultaneous Whole-cell Recordings from Photoreceptors and Second-order Neurons in an Amphibian Retinal Slice Preparation

One of the central tasks in retinal neuroscience is to understand the circuitry of retinal neurons and how those connections are responsible for shaping the signals transmitted to the brain. Photons are detected in the retina by rod and cone photoreceptors, which convert that energy into an electrical signal, transmitting it to other retinal neurons, where it is processed and communicated to central targets in the brain via the optic nerve. Important early insights into retinal circuitry and visual processing came from the histological studies of Cajal1,2 and, later, from electrophysiological recordings of the spiking activity of retinal ganglion cells - the output cells of the retina3,4.
A detailed understanding of visual processing in the retina requires an understanding of the signaling at each step in the pathway from photoreceptor to retinal ganglion cell. However, many retinal cell types are buried deep in the tissue and therefore relatively inaccessible for electrophysiological recording. This limitation can be overcome by working with vertical slices, in which cells residing within each of the retinal layers are clearly visible and accessible for electrophysiological recording.
Here, we describe a method for making vertical sections of retinas from larval tiger salamanders (Ambystoma tigrinum). While this preparation was originally developed for recordings with sharp microelectrodes5,6, we describe a method for dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells in which we manipulate the photoreceptor's membrane potential while simultaneously recording post-synaptic responses in horizontal or bipolar cells. The photoreceptors of the tiger salamander are considerably larger than those of mammalian species, making this an ideal preparation in which to undertake this technically challenging experimental approach. These experiments are described with an eye toward probing the signaling properties of the synaptic ribbon - a specialized synaptic structure found in a only a handful of neurons, including rod and cone photoreceptors, that is well suited for maintaining a high rate of tonic neurotransmitter release7,8 - and how it contributes to the unique signaling properties of this first retinal synapse.

We describe the preparation of thin retinal slices from aquatic tiger salamanders (Ambystoma tigrinum) and explain how we use these slices to study synaptic processing in the retina by obtaining dual whole-cell voltage clamp recordings from photoreceptors and second-order horizontal and bipolar cells.

Registrazioni simultanee a cellule di fotorecettori e dei neuroni di secondo ordine in un anfibio Retinal Fetta Preparazione

One of the central tasks in retinal neuroscience  is to understand the circuitry of retinal neurons and how those connections are  responsible for shaping ...

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 ...

Recording Light-evoked Postsynaptic Responses in Neurons in Dark-adapted, Mouse Retinal Slice Preparations Using Patch Clamp Techniques

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. Retinal neurons in the network comprise of numerous subtypes. More than 10 subtypes of bipolar cells, ganglion cells, and amacrine cells have been identified by morphological studies. Multiple subtypes of retinal neurons are thought to encode distinct features of visual signaling, such as motion and color, and form multiple neural pathways. However, the functional roles of each neuron in visual signal processing are not fully understood. The patch clamp method is useful to address this fundamental question. Here, a protocol to record light-evoked synaptic responses in mouse retinal neurons using patch clamp recordings in dark-adapted conditions is provided. The mouse eyes are dark-adapted O/N, and retinal slice preparations are dissected in a dark room using infrared illumination and viewers. Infrared light does not activate mouse photoreceptors and thus preserves their light responsiveness. Patch clamp is used to record light-evoked responses in retinal neurons. A fluorescent dye is injected during recordings to characterize neuronal morphological subtypes. This procedure enables us to determine the physiological functions of each neuron in the mouse retina.

We will demonstrate how to prepare retinal slices from the mouse eye and record light responses in retinal neurons. The entire procedure is conducted in dark-adapted conditions.

Registrazione Light-evocato Risposte postsinaptica nei neuroni in Dark-adattati, i preparativi della retina del mouse Slice Utilizzando tecniche di patch clamp

The retina is the gateway to the visual system. To understand visual signal processing mechanisms, we investigate retinal neural network functions. ...

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 ...