Name: methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell
Uploaded: 2014-10-22T14:00:00.000+00:00
Duration: 8 min 24 s
Description: Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

This work is supported by a National Science Foundation award (1145581) to LWG.

NOTA: L&#39;intera procedura è stata condotta in conformità con le linee guida del National Institutes of Health, come approvato dalla cura e uso degli animali Comitato della University of Illinois a Chicago. 1. Soluzioni e Media Cultura Preparati <ol><li> Conservare tutte le soluzioni a -20 ° C per un massimo di sei mesi. Tenere il terreno di coltura a 4 ° C per un massimo di 3 mesi. </li><li> Preparare 100 ml di coltura mescolando 1 ml di soluzione di pen-strep e 1 ml di ITSX (Insulin-transferrina-Selenio-supplementazione) a 100 ml con DMEM. </li><li> Preparare la soluzione enzimatica mescolando 250 ml di DMEM, 2,5 ml di 100 mM CaCl 2, e 2,5 ml di 50 mM EDTA. </li><li> Preparare la soluzione inattivazione mescolando 225 ml di DMEM, 25 ml di FBS, 625 mg di albumina, e 625 mg di inibitore della tripsina. </li><li> Preparare la soluzione di un Locke di 154 mM NaCl, 5.6 mM KCl, 5,0 HEPES mM, 3,6 mM NaHCO3, 5,6 mM di glucosio. Regolare il pH a 7,3 con NaOH. </li><li>Preparare una soluzione di poli-D-lisina (PDL) di 50 mg / ml. Utilizzare una concentrazione finale di 1 mg / ml a lamelle cappotto. </li><li> Preparare una soluzione extracellulare di 140 mM NaCl, KCl 5 mM, 2 mM CaCl 2, 1 mM MgCl 2, 10 mM HEPES-NaOH, e glucosio 10 mM. Regolare il pH a 7,3 con NaOH con una osmolarità di ~ 310 nmol / kg. </li><li> Preparare una soluzione di pipetta di 50 mM NaCl, TEACL 100 mM, KCl 5 mM, 2 mM CaCl 2, 1 mM MgCl 2, e HEPES 10 mM. Regolare il pH a 7,3 con NaOH con una osmolarità di ~ 290 nmol / kg. </li></ol> Isolamento 2. ghiandola surrenale <ol><li> Autoclave un paio di grandi e piccole forbici dissezione e due paia di pinze. Indossare i guanti durante l&#39;intero processo e posizionare il vassoio dissezione su un secchio di ghiaccio. </li><li> Utilizzare i topi prese a Day 0-3. Euthanize animali con una pressione di CO 2 serbatoio seguita da decapitazione. </li><li> Utilizzare il set più piccolo di forbici per tagliare lungo la schiena del cucciolo che segue la colonna vertebrale da cervi<html> cal per caudale regione. Assicuratevi di tagliare superficialmente sotto la pelle e non penetrare nella cavità del corpo. </li><li> Avanti, pelle a buccia lontano da spina dorsale in modo che la muscolatura sia esposto ad avere una visione chiara della colonna vertebrale. Da qui, fare un taglio transectional al midollo spinale cervicale e poi fare due tagli paralleli lungo i lati della colonna vertebrale. </li><li> Con un paio di pinze che tengono il corpo dell&#39;animale, utilizzare l&#39;altra coppia di pinze per afferrare colonna vertebrale e buccia indietro della colonna vertebrale fino reni sono esposti. </li><li> In questo momento, visualizzare le ghiandole surrenali sulla parte superiore dei reni. Poi posizionare accuratamente i due ghiandole di ogni animale in un tubo conico riempito con soluzione di Locke. NOTA: A volte le ghiandole si attacchi alle pinze. Se questo è il caso, utilizzare delicatamente un altro paio di pinze per aiutare nella rimozione delle ghiandole delle pinze e verso la soluzione di Locke. Le ghiandole possono essere mantenuti in questa condizione per un massimo di 2 ore. </li></ol>e &quot;&gt; 3. Tissue digestione <ol><li> Nel frattempo, bolla soluzione enzimatica con 5% di CO 2 + 95% O 2 per 15 minuti prima dell&#39;uso. Assicurarsi che la soluzione equilibrata si trasforma da un colore rosa brillante nel colore rosa / arancio. </li><li> Aggiungere la papaina alla soluzione enzimatica appena bolle ad una concentrazione finale di 20-25 unità / ml. Incubare ciascuna coppia di ghiandole di ciascun animale per 40-60 minuti a 37 ° C nella soluzione enzimatica con papaina. </li><li> Aggiungere 75% di soluzione di inattivazione a ciascuna provetta e incubare a 37 ° C per altri 10 min. Questo incubazione inattiva l&#39;attività enzimatica della papaina. </li><li> Dopo i 10 minuti di incubazione con la soluzione di inattivazione, trasferire delicatamente le ghiandole in una provetta nuova etichetta e poi lavare le ghiandole 3x con la cultura dei media. </li></ol> 4. cellulare Dissociazione e placcatura <ol><li> Dopo il lavaggio, posizionare i due ghiandole in 200 ml di media cultura e poi titolare delicatamente le ghiandole egiù attraverso la punta della pipetta con una pipetta 200 microlitri fino a quando non vi è più un grosso pezzo di tessuto nella soluzione. NOTA: Quando titolazione, mantenere ghiandole nella parte inferiore del tubo con una forza verso il basso dalla punta della pipetta 200 microlitri e impegnare il tessuto delicatamente e lentamente. Essere certi di utilizzare lenti, continui colpi, mai pipettaggio veloce o improvviso. </li><li> Aggiungere un ulteriore coltura 250 microlitri per portare il volume totale dei terreni di coltura fino a 450 ml. </li><li> Piatto 60 ml di questa soluzione di cellule contenenti su ciascuno dei sette coprioggetto PDL-pre-rivestite 12 mm, che sono collocati in un piatto di coltura 60 x 15 mm. </li><li> Una volta placcato, mettere il piatto in incubatrice che è impostato a 37 ° C e mantenuta con un costante flusso / fornitura di 5% di CO 2 per 30 minuti al fine di garantire un ambiente per la vitalità cellulare. Dopo l&#39;incubazione, aggiungere delicatamente 5 ml di terreni di coltura pre-riscaldato nel piatto cultura e restituire il piatto di nuovo nel incubatore per 24 ore prima di cellula-addettoregistrazioni d. NOTA: in genere, le cellule possono essere utilizzate per le registrazioni elettrofisiologiche per un massimo di 4 giorni. </li></ol> 5. cellulare Identificazione e Gigaohm Seal Formazione <ol><li> Posizionare il coprioggetto 12 millimetri nella soluzione extracellulare. Dal momento che l&#39;intero ghiandole surrenali sono utilizzati per la preparazione delle cellule, mescolare le cellule cromaffini (in genere molto luminose con un marrone / colorazione beige e una forma quasi perfetta circolare (Figura 1) in coltura con cellule corticali (più piccole e dimmer di cellule cromaffini). </li><li> Tirare le pipette di patch in quattro fasi utilizzando una programmabile P-97 estrattore. Immergere la punta della pipetta in una cera fusa, che contribuirà a ridurre la capacità del vetro e poi sparano lucidare la punta di &quot;blow-away&quot;, questa cera dall&#39;interno della punta della pipetta. NOTA: Quando riempito con una soluzione di patch pipetta, pipette di patch in genere hanno una resistenza di circa 2 MW. </li><li> Avvicinarsi alla pipetta di patch vicino alla cella all&#39;interno diversi microns. Per ottenere una configurazione cell-schiera, cercare qualsiasi lieve deformazione cellula durante l&#39;avvicinamento della pipetta di patch più vicino alla cella. Esercitare una leggera aspirazione fino ad ottenere un sigillo GΩ. </li></ol> 6. cellulari-Attached Recordings di capacità <ol><li> Una volta che un sigillo GΩ si rivela, effettuare registrazioni cellulari-attached attraverso l&#39;uso di un amplificatore EPC-7 plus patch-clamp e un amplificatore lock-in analogico a due fasi (vedi tabella allegata delle attrezzature). Applicare una sinusoide con ampiezza di 50 mV (rms) e la frequenza di 20 kHz dall&#39;amplificatore lock-in. </li><li> Impostare il filtro di uscita dell&#39;amplificatore lock-in a 1 costante di tempo msec e 24 db. Impostare la fase dell&#39;amplificatore lock-in modo tale che variazioni di capacità transitori prodotte da impulsi di aspirazione dolci appaiono solo nella traccia senza proiezione nel tracciato cerotto conduttanza (Ri) cerotto capacità (Im) (Figura 2). NOTA: Si prega di vedere i dettagli per la calibrazione del sistema in reFerence 19. </li><li> Identificare le tipiche variazioni di capacità di passi &quot;verso il basso&quot; (Figura 3). NOTA: Una registrazione solido sarà considerata come una &quot;scala&quot; tipo somiglianza con molti passi verso il basso, che indica interiorizzare singole vescicole. Il processo di patching serve da stimolo meccanico in quanto attuale azione può essere in genere registrato dalla maggior parte delle cellule. </li><li> Nome file come singole cartelle compresa la data: gg / mm / anno e ogni registrazione cell-attached come propria numerazione individuale (xx) all&#39;interno di quel file. </li></ol>

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Misure di capacità cellulari iscritti richiedono diversi passaggi critici al fine di ottenere con successo le registrazioni di alta qualità: 1) cellule vitali e sane preparate dalle ghiandole surrenali; 2) il rivestimento PDL dei coprioggetto; 3) formazione sigillo gigaohm; 4) livello di rumore del sistema; e 5) correzione di fase. Per la chirurgia animali, le modifiche si può potenzialmente fare è quello di regolare l&#39;approccio chirurgico al meglio destrezza vestito e per evitare da...

Trasmissione sinaptica è mediata da esocitosi delle vescicole sinaptiche, contenenti neurotrasmettitori, e queste vescicole deve subire riciclaggio endocitica locale all&#39;interno del nervo terminale per mantenere la comunicazione neuronale a lungo termine. Dato il ruolo essenziale della trasmissione sinaptica nel cervello, la comprensione del macchinario molecolare che costituisce il ciclo delle vescicole sinaptiche è un fondamento essenziale verso una migliore comprensione nella comunicazione cellulare nel suo complesso. Tra sistemi modello delle cellule, la cellula cromaffini surrenale ha fornito alcune delle l&#39;intuizione più definitiva nel meccanismo molecolare alla base riciclo delle vescicole sinaptiche. Esocitosi, la fase finale di rilascio di neurotrasmettitore, è stato immensamente studiata ed esaminata attraverso l&#39;uso della cella cromaffini surrenale 1,2. In realtà, la maggior parte dei giocatori molecolari che orchestrano la formazione, il targeting, attracco, e la fusione dei granuli secretori sono stati identificati grazie alla applicazione di divtecniche di Erse nelle cellule cromaffini 1. Inoltre, fornendo l&#39;opportunità di consentire la risoluzione di una singola vescicola dei macchinari proteina coinvolta nella esocitosi, la cella cromaffini rimane un modello potente per affrontare le questioni della fusione delle vescicole 3. Misure di capacità cellulari iscritti sono stati utilizzati nella risoluzione di singolo-fusione delle vescicole durante esocitosi 3. Esocitosi di vescicole piccole come ~ 60 nm di diametro è stato dimostrato di essere rilevato da membrana cellulare misurazioni ammissione con la tecnica del patch clamp in configurazione cell allegato 4-7. Accettazione è definita come una misura di quanto facilmente un circuito o un dispositivo consentirà una corrente di flusso; è l&#39;inverso di impedenza. Pertanto, le misure di ammissione forniscono una comprensione della capacità di membrana. Questo si ottiene l&#39;incorporazione della membrana vescicolare nella membrana plasmatica; questa incorporazione rivela cambiamenti di superficiezona 8. Ogni vescicola fusione provoca un aumento graduale di capacità di membrana 9,10. Inoltre, questa misura ammettenza fornisce la conduttanza della membrana e la fusione dei pori conduttanza durante un evento esocitotico 3. Poiché questa tecnica ha fornito uno strumento unico di identificare cinetica singoli-vescicole durante esocitosi, il nostro laboratorio ha recentemente applicato questo concetto per rilevare endocitosi di singole vescicole 11,12. Il nostro interesse specifico è clatrina-mediata endocitosi (CME), che è stato considerato come una componente fondamentale di pulizia in molte cellule 13 e come via principale per endocitosi delle vescicole sinaptiche nei terminali neuronali 14,15. CME è conosciuto per essere biologicamente importante, tuttavia, la sua cinetica non restino ben capito a causa di limitazioni tecniche di monitoraggio degli eventi endocitiche singolari. Date le somiglianze nei meccanismi di esocitosi tra le cellule cromaffini e neuroni 1, è plausible che i meccanismi di fissione nelle cellule cromaffini può probabilmente applicarsi a endocitosi delle vescicole sinaptiche nei neuroni. Le misure di capacità di cella-attached sono stati utilizzati per monitorare singoli eventi endocitiche e analizzare la cinetica di fissione, che la maggior parte dei metodi non sono in grado di risolvere. Nelle nostre registrazioni cellulari-attached, un&#39;onda sinusoidale a 20 kHz si trova sopra il potenziale di detenzione, e la corrente di uscita è suddiviso in membrana conduttanza in un canale e capacità di membrana in un altro canale da una a due fasi amplificatore lock-in 16 18. Dai cambiamenti nella conduttanza di membrana e capacità, si può calcolare la cinetica di fissione poro, che probabilmente corrispondente al collo membrana tubolare che collega la vescicola internalizzazione alla membrana plasmatica prima vescicola pinch-off. Collettivamente, questa tecnica ci dà l&#39;opportunità di esaminare i meccanismi regolatori della vescicola fissione durante CME.

<table><tbody><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P0899</td><td></td></tr><tr><td>DMEM</td><td></td><td>15066024</td><td>Keep out of UV</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Cover Glass</td><td>Carolina Biological</td><td>633029</td><td>12 mm</td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies</td><td>15140122</td><td>100 ml</td></tr><tr><td>Insulin-Trans-Sel-X</td><td>Life Technologies</td><td>51500056</td><td>Only thaw on ICE!</td></tr><tr><td>Papain</td><td>Worthington</td><td>39S11614</td><td></td></tr><tr><td>EPC-7 plus patch amplifier</td><td>HEKA</td><td></td><td></td></tr><tr><td>BNC-2090 data acquisition board</td><td>National Instruments</td><td></td><td></td></tr><tr><td>Igor data acquisition software</td><td>Wavemetrics</td><td></td><td></td></tr><tr><td>P-97 pipette puller</td><td>Sutter Instruments</td><td></td><td></td></tr><tr><td>Microforge</td><td>Scientific Instruments</td><td></td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Sutter Instruments</td><td>B150-110-10</td><td>Outer diameter: 1.5 mm&lt;br /&gt;
Inner diameter: 1.10 mm&lt;br/&gt;
Length: 10 cm</td></tr></tbody></table>

methods for cell-attached capacitance measurements in mouse adrenal chromaffin cell

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion known as exocytosis that mediates synaptic transmission. Subsequent retrieval of synaptic vesicles is necessary to generate new neurotransmitter-filled vesicles in a process identified as endocytosis. During exocytosis, fusing vesicle membranes will result in an increase in surface area and subsequent endocytosis results in a decrease in the surface area. Here, our lab demonstrates a basic introduction to cell-attached capacitance recordings of single endocytic events in the mouse adrenal chromaffin cell. This type of electrical recording is useful for high-resolution recordings of exocytosis and endocytosis at the single vesicle level. While this technique can detect both vesicle exocytosis and endocytosis, the focus of our lab is vesicle endocytosis. Moreover, this technique allows us to analyze the kinetics of single endocytic events. Here the methods for mouse adrenal gland tissue dissection, chromaffin cell culture, basic cell-attached techniques, and subsequent examples of individual traces measuring singular endocytic event are described.

La vitalità delle cellule e la qualità di tenuta gigaohm sono critici nel determinare la qualità delle registrazioni capacità della cella collegata. Pertanto, è fondamentale per procurare un coltura cellulare efficace ed efficiente prima di registrazioni elettrofisiologiche, e le cellule vitali tipiche sono illustrate nella figura 1. Practice e il tempo sarà utile nel raggiungimento di un sigillo gigaohm con alta qualità. Se si può vedere chiaramente deformazione cella quando la pipetta di patch...

Watch this Scientific Journal Video about Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell at JoVE.com

Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Metodi per cellulari-attached Misure di capacità in mouse surrenale cromaffini cellulare

Neuronal transmission is an integral part of cellular communication within the brain. Depolarization of the presynaptic membrane leads to vesicle fusion ...

methods-for-cell-attached-capacitance-measurements-mouse-adrenal

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Neuroscience

8.8K Views.  University of Illinois at Chicago. After exocytosis, fused plasma membrane is retrieved through a process known as endocytosis. This mechanism reforms new synaptic vesicles for the next round of release. Individual endocytic events are captured and analyzed through the use of the cell-attached capacitance recordings in mouse adrenal chromaffin cells.

Video: Methods for Cell-attached Capacitance Measurements in Mouse Adrenal Chromaffin Cell

Mouse Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

Topo surrenale cromaffini cella di isolamento

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

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Biologia

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Patch-clamp Misure di capacità e Ca 2 + Imaging terminazioni nervose della retina singola in Fette

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Neuroscienze

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Implementazione morsetto dinamico con conduttanze sinaptiche e artificiale nel topo cellule gangliari della retina

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Isolamento, fissazione e immunofluorescenza Imaging delle ghiandole adrenali Mouse

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

Immunologia e infezioni

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

L'effetto di Stress osmotico su vescicole secretorie ed esocitosi di monitoraggio

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

Isolamento dei miociti atriali umane per misure simultanee di Ca<sup&gt; 2 +</sup&gt; Transitori e correnti di membrana

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

Microscopia confocale per misurare tre modalità di dinamica dei pori di fusione nelle cellule cromaffini surrenali

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

A cellula intera elettrofisiologia di cellule enterocromaffini murino coltura primaria

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes.
Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte&#8217;s sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

The intracellular Na+ concentration ([Na+]i) in cardiac myocytes is altered during cardiac diseases. [Na+]i is an important regulator of intracellular Ca2+. We introduce a novel approach to measure [Na+]i in freshly isolated murine atrial myocytes using an electron multiplying charged coupled device (EMCCD) camera and a rapid, controllable illuminator.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Isolamento di miociti atriali e ventricolari murini di alta qualità per misurazioni simultanee di transitori Ca2+ e corrente di calcio di tipo L

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

Metodi per cellulari-attached Misure di capacità in mouse surrenale cromaffini cellulare

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