Samuel L. Pfaff è docente nei Laboratori di espressione genica presso il Salk Institute for Biological Studies e un investigatore della Howard Hughes Medical Institute. Questo lavoro è stato sostenuto dal Christopher e Dana Reeve Foundation. Joe Belcovson, Kent Schnoeker e Mike Sullivan in Risorse Multimediale presso il Salk Institute ha fornito assistenza con la fotografia e montaggio.

1. Preparare artificiale liquido cerebrospinale (aCSF) 1. Per prima cosa preparare un 2L di uno stock di 10X aCSF senza magnesio o di calcio. Reagenti elencati in millimolare. I numeri di catalogo si riferiscono alla Sigma / Aldrich. <table border="1" cellpadding="0"><tbody><tr><td colspan="4"> 2 litri 10X aCSF (senza Mg o Ca) </td></tr><tr><td> Reagente </td><td> mM </td><td> g/2L </td><td> Catalogo </td></tr><tr><td> KCl </td><td> 40 </td><td> 5,96 </td><td> P-9333 </td></tr><tr><td> NaCl </td><td> 1280 </td><td> 149,61 </td><td> S-7653 </td></tr><tr><td> NaHCO 3 </td><td> 210 </td><td> 35,28 </td><td> S-6297 </td></tr><tr><td> NaH 2 PO 4 </td><td> 5 </td><td> 1,38 </td><td> S-9638 </td></tr><tr><td> Glucosio </td><td> 300 </td><td> 108,12 </td><td> D-9434 </td></tr><tr><td colspan="4"> Aggiungere acqua distillata per 2L </td></tr></tbody></table> Ci sarà anche preparare soluzioni 1M di MgSO 4 e CaCl 2 in 50mL di acqua per consentire adeguamenti molarità da esperimento a esperimento e per assicurare il calcio rimane in soluzione. <table border="1" cellpadding="0"><tbody><tr><td colspan="4"> Calcio 1M e degli stock di magnesio </td></tr><tr><td> Reagente </td><td> M </td><td> g/50mL </td><td> Catalogo </td></tr><tr><td> CaCl 2 </td><td> 1 </td><td> 7,35 </td><td> C-5080 </td></tr><tr><td> MgSO 4 </td><td> 1 </td><td> 12,33 </td><td> M-5921 </td></tr></tbody></table> La soluzione aCSF deve essere gasati con CarboGen (95% O2 / 5% CO2) per abbassare il pH prima di aggiungere calcio o il calcio sarà precipitato. <table border="1" cellpadding="0"><tbody><tr><td colspan="2"> 1 litro aCSF </td></tr><tr><td> Reagente </td><td> Volume </td></tr><tr><td> 10X aCSF </td><td> 100 ml </td></tr><tr><td> 1M CaCl 2 </td><td> 1 ml (dissezione) 2 ml (registrazione) </td></tr><tr><td> 1M MgSO 4 </td><td> 1 ml </td></tr><tr><td colspan="2"> Aggiungi ~ 800 mL di acqua distillata, gas con CarboGen per 2 minuti prima di aggiungere calcio </td></tr><tr><td colspan="2"> Aggiungere acqua distillata a 1L. </td></tr></tbody></table> Le pompe e tubi e piatti sezionare devono essere risciacquati prima e dopo l&#39;uso. Dissezione Mentre dissezione, 1 mm di calcio aCSF deve essere continuamente gasati con CarboGen. Il aCSF può essere deviato da una bottiglia al piatto dissezione e pompato dal piatto dissezione alla bottiglia o inviato a rifiuti. Noi usiamo un elastomero (Sylgard, Dow Corning) piatto rivestito di eseguire la dissezione. Il mouse neonatale è rapidamente decapitato con forbici affilate e una incisione fatta con le forbici attraverso il torace e l&#39;addome ventrale. L&#39;animale viene poi svuotato. Il mouse eviscerate è risciacquata con aCSF. L&#39;animale è poi appuntata al piatto dissezione con perni insetti inserito attraverso la arti anteriori e alla base della coda. La colonna vertebrale viene rimossa eseguendo una laminectomia ventrale. La colonna vertebrale è tenuto con una pinza piccola e una lama di piccole forbici è inserito immediatamente dorsale alla colonna vertebrale. La lamina viene tagliato da un lato e poi l&#39;altro mentre leggero sollevamento della colonna vertebrale. Questo avviene per la lunghezza della colonna vertebrale. Il midollo spinale è stato rimosso dal taglio lungo entrambi i lati del midollo spinale per tagliare le radici dei nervi spinali e tagliare le meningi che circonda il midollo spinale. Abbiamo tagliato sui lati destro e sinistro, poi abbiamo tagliato le meningi attaccato al lato dorsale del midollo spinale per liberarlo. Il cavo isolato è poi appuntata al piatto in prossimità della presa aCSF per assicurare una buona ossigenazione se gli animali vengono sezionati aggiuntivi. Il midollo spinale isolato viene trasferito in un piatto di registrazione con un cucchiaio o una spatola. Qui profumato la preparazione con aCSF ossigenato calcio 2mm. Il midollo spinale è appuntato il piatto rivestito in elastomeri e micromanipolatori utilizzato per spostare extracellulare, tutto elettrodi aspirazione radice vicino alle radici. Quando l&#39;elettrodo è vicino alla fine radice libero, delicata aspirazione viene applicata per disegnare la radice nel elettrodo di aspirazione. Questo processo può essere ripetuto per registrare radici contemporaneamente. <img src="/files/ftp_upload/1660/1660fig1.jpg" alt="Figura 1" /> Figura 1. Decapitazione e l&#39;eviscerazione. A. Rapidamente decapitare il mouse con forbici affilate. Contributi più o meno dal tronco cerebrale può essere raggiunto a seconda del livello di taglio. B. Luogo una lama delle forbici nell&#39;apertura della cavità toracica creato dalla decapitazione. Aprire il torace e l&#39;addome ventrale alla base della coda. Rimuovere i visceri e sciacquare con aCSF. <img src="/files/ftp_upload/1660/1660fig2.jpg" alt="Figura 2"/&gt; Figura 2. La rimozione della colonna vertebrale. A. Inserire la lama delle forbici sinistra nello spazio tra la colonna vertebrale e del midollo spinale alla destra del midollo spinale e tagliare le ossa che ventrale del midollo. Quindi inserire la lama delle forbici a destra nello spazio tra la colonna vertebrale e del midollo spinale a sinistra del midollo spinale e taglio. B. Ripetere questo processo mentre si applica una leggera trazione alla colonna vertebrale. Fare attenzione a non forare il midollo spinale, tenendo le forbici con un angolo basso. <img src="/files/ftp_upload/1660/1660fig3.jpg" alt="Figura 3" /> Figura 3. La rimozione del midollo spinale. A. Inserire la lama delle forbici sinistra nello spazio tra il midollo spinale e le ossa a destra del midollo spinale e tagliare le radici spinale e delle meningi che laterale per il cavo. Quindi inserire la lama delle forbici a destra nello spazio tra il midollo spinale e le ossa a sinistra del midollo spinale e tagliare le radici spinale e delle meningi che laterale per il cavo. B. Infine, sollevare delicatamente il midollo rostrale e tagliare le meningi collegare il cavo di dorsale per le lamine. Anche in questo caso non prendere cura di forare il midollo spinale, tenendo le forbici con un angolo basso. Non tagliare le radici troppo vicino al midollo spinale.

<ol>
	<li>Myers, C. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cholinergic+input+is+required+during+embryonic+development+to+mediate+proper+assembly+of+spinal+locomotor+circuits.">Cholinergic input is required during embryonic development to mediate proper assembly of spinal locomotor circuits.</a> Neuron. 46, 37-49 (2005).</li><li>Goulding, M., Pfaff, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+circuits+that+generate+simple+rhythmic+behaviors+in+vertebrates.">Development of circuits that generate simple rhythmic behaviors in vertebrates.</a> Curr Opin Neurobiol. 15 (1), 14-20 (2005).</li><li>Gallarda, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segregation+of+axial+motor+and+sensory+pathways+via+heterotypic+trans-axonal+signaling.">Segregation of axial motor and sensory pathways via heterotypic trans-axonal signaling.</a> Science. 320, (2008).</li><li>Gosgnach, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=V1+spinal+neurons+regulate+the+speed+of+vertebrate+locomotor+outputs.">V1 spinal neurons regulate the speed of vertebrate locomotor outputs.</a> Nature. 440 (7081), 215-219 (2006).</li><li>Lanuza, G. M., Gosgnach, S., Pierani, A., Jessell, T. M., Goulding, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+identification+of+spinal+interneurons+that+coordinate+left-right+locomotor+activity+necessary+for+walking+movements.">Genetic identification of spinal interneurons that coordinate left-right locomotor activity necessary for walking movements.</a> Neuron. 42 (3), 375-386 (2004).</li><li>Jiang, Z., Carlin, K. P., Brownstone, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+functionally+mature+mouse+spinal+cord+preparation+for+the+study+of+spinal+motor+networks.">An in vitro functionally mature mouse spinal cord preparation for the study of spinal motor networks.</a> Brain Res. 816 (2), 493-499 (1999).</li><li>Ziskind-Conhaim, L., Gao, B. X., Hinckley, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ethanol+dual+modulatory+actions+on+spontaneous+postsynaptic+currents+in+spinal+motoneurons.">Ethanol dual modulatory actions on spontaneous postsynaptic currents in spinal motoneurons.</a> J Neurophysiol. 89 (2), 806-813 (2003).</li><li>Tabak, J., Rinzel, J., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+activity-dependent+network+depression+in+the+expression+and+self-regulation+of+spontaneous+activity+in+the+developing+spinal+cord.">The role of activity-dependent network depression in the expression and self-regulation of spontaneous activity in the developing spinal cord.</a> J Neurosci. 21 (22), 8966-8978 (2001).</li><li>Chub, N., Mentis, G. Z., O'Donovan, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chloride-sensitive+MEQ+fluorescence+in+chick+embryo+motoneurons+following+manipulations+of+chloride+and+during+spontaneous+network+activity.">Chloride-sensitive MEQ fluorescence in chick embryo motoneurons following manipulations of chloride and during spontaneous network activity.</a> J Neurophysiol. 95 (1), 323-330 (2006).</li></ol>

CURA DEGLI ANIMALI<br /&gt;<br /Esperimenti&gt; sugli animali sono stati eseguiti in conformità con le linee guida e dei regolamenti previsti dal Salk Institute for Biological Studies AIACUC.

L&#39;isolato midollo spinale neonatale fornisce un metodo di studio trattabili sviluppo1 sistema nervoso, 2. All&#39;interno del midollo spinale lombare di roditori neonatale, centrale circuito generando modello in grado di produrre locomozione fittizia in presenza di neurotrasmettitori. Questo locomozione fittizio costituito da un aumento dell&#39;attività ritmica, scoppi, che vengono prodotte a 0,2-0,5 Hz. Queste esplosioni sono organizzati per la produzione di alternanza destra-sinistra per tutta la lunghezza del c...

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>KCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>P-9333</td>
<td>&nbsp;</td>
<tr>
<td>NaCl	</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>S-7653</td>
<td>&nbsp;</td>
<tr>
<td>NaHCO3</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>S-6297</td>
<td>&nbsp;</td>
<tr>
<td>NaH2PO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>S-9638</td>
<td>&nbsp;</td>
<tr>
<td>Glucose</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>D-9434</td>
<td>&nbsp;</td>
<tr>
<td>CaCl2</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>C-5080</td>
<td>&nbsp;</td>
<tr>
<td>MgSO4</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>M-5921</td>
<td>&nbsp;</td>
<tr>
<td>Large Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>14070-12</td>
<td>&nbsp;</td>
<tr>
<td>Forceps</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11050-10</td>
<td>&nbsp;</td>
<tr>
<td>Fine Scissors</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>15000-10</td>
<td>&nbsp;</td>
<tr>
<td>Insect pins</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>26002-10</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 (Dow-Corning)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1L volumetric flask</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>100mL volumetric flask</td>
<td>&nbsp;</td>
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spinal cord electrophysiology

Il midollo spinale del mouse neonatale è un modello per studiare lo sviluppo della circuiteria neuronale e del movimento motorio. Dimostriamo la dissezione del midollo spinale e la preparazione del bagno di liquido cerebrospinale di registrazione artificiali utilizzati per gli studi locomotore. Una volta sezionato, l&#39;radici nervose del midollo spinale ventrale può essere collegato a un elettrodo per la registrazione dei segnali elettrofisiologici del circuito centrale generando modello all&#39;interno del midollo lombare.

Watch this Scientific Journal Video about Spinal Cord Electrophysiology at JoVE.com

Spinal Cord Electrophysiology

Una dimostrazione dell&#39;isolamento di midollo spinale neonatale del mouse per studi elettrofisiologici.

Spinal Cord Elettrofisiologia

The neonatal mouse spinal cord is a model for studying the development of neural circuitries and locomotor movement. We demonstrate the spinal cord ...

Research

JoVE Journal

Biology

21.1K Views.  Howard Hughes Medical Institute and Gene Expression Laboratory. Una dimostrazione dell'isolamento di midollo spinale neonatale del mouse per studi elettrofisiologici.

Video: Spinal Cord Electrophysiology

Applying Microfluidics to Electrophysiology

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the microfluidic brain slice device is discussed including how the device docks to standard perfusion chambers and the technique of passive pumping which is used to deliver boluses of neuromodulators to the brain slice.  By simplifying the device design, we are able to achieve a practical solution to the current unmet electrophysiology need of applying multiple neuromodulators across multiple regions of the brain slice.  This is achieved by substituting the standard coverglass substrate of the perfusion chamber with a thin microfluidic device bonded to the coverglass substrate.  This was then attached to the perfusion chamber and small holes connect the open-well of the perfusion chamber to the microfluidic channels buried within the microfluidic substrate.  These microfluidic channels are interfaced with ports drilled into the edge of the perfusion chamber to access and deliver stimulants.  This project represents how the field of microfluidics is transitioning away from proof-of concept device demonstrations and into practical solutions for unmet experimental and clinical needs.

Applicando Microfluidica di Elettrofisiologia

Microfluidics can be integrated with standard electrophysiology techniques to allow new experimental modalities.  Specifically, the motivation for the ...

Ricerca

Biologia

Preparation of Artificial Bilayers for Electrophysiology Experiments

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can be used for many different studies, such as the characterization of membrane-active peptides, the reconstitution of ion channels or investigations on how changes in lipid bilayer properties alter the function of bilayer-spanning channels.  Here, we show how to form a planar bilayer and how to isolate small patches from the bilayer, and in a second video  will also demonstrate a procedure for using gramicidin channels to determine changes in lipid bilayer elastic properties.   We also demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. Here, we demonstrate the individual steps needed to prepare the bilayer chamber, the electrodes and how to test that the bilayer is suitable for single-channel measurements.

Preparazione di doppi strati artificiali per esperimenti Elettrofisiologia

Planar lipid bilayers, also called artificial lipid bilayers, allow you to study ion-conducting channels in a well-defined environment. These bilayers can ...

Combining Peripheral Nerve Grafting and Matrix Modulation to Repair the Injured Rat Spinal Cord

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of communication with the brain. One of the goals of our research is to promote axon regeneration to restore connectivity across the lesion site. To accomplish this we developed a peripheral nerve (PN) grafting technique where segments of sciatic nerve are either placed directly between the damaged ends of the spinal cord or are used to form a bridge across the lesion. There are several advantages to this approach compared to transplantation of other neural tissues; regenerating axons can be directed towards a specific target area, the number and source of regenerating axons is easily determined by tracing techniques, the graft can be used for electrophysiological experiments to measure functional recovery associated with axons in the graft, and it is possible to use an autologous nerve to reduce the possibility of graft rejection. In our lab we have performed both autologous (donor and recipient are the same animal) and heterologous (donor and recipient are different animals) grafts with comparable results. This approach has been used successfully in both acute and chronic injury situations. Regenerated axons that reach the distal end of the PN graft often fail to extend back into the spinal cord, so we use microinjections of chondroitinase to degrade inhibitory molecules associated with the scar tissue surrounding the area of SCI. At the same time we have found that providing exogenous growth and trophic molecules encourages longer distance axonal regrowth into the spinal cord. Several months after transplantation we perform a variety of anatomical, behavioral and electrophysiological tests to evaluate the recovery of function in our spinal cord injured animals. This experimental approach has been used successfully in several spinal cord injury models, at different levels of injury and in different species (mouse, rat and cat). Importantly, the peripheral nerve grafting approach is effective in promoting regeneration by acute and chronically injured neurons.

Traumatic injury to the spinal cord disrupts communication with the brain. To restore lost connectivity we utilize a peripheral nerve graft to provide a substratum for regenerating fibers in combination with neurotrophic factors and matrix-modulating enzymes to remove inhibitory molecules to promote long distance growth.

La combinazione di innesto del nervo periferico e Matrix modulazione di riparare il cavo feriti Rat spinale

Traumatic injury to the spinal cord (SCI) causes death of neurons, disruption of motor and sensory nerve fiber (axon) pathways and disruption of ...

Semi-automated Optical Heartbeat Analysis of Small Hearts

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our Semi-automatic Optical Heartbeat Analysis (SOHA) uses a novel movement detection algorithm that is able to detect cardiac movements associated with individual contractile and relaxation events. The program provides a host of physiologically relevant readouts including systolic and diastolic intervals, heart rate, as well as qualitative and quantitative measures of heartbeat arrhythmicity. The program also calculates heart diameter measurements during both diastole and systole from which fractional shortening and fractional area changes are calculated. Output is provided as a digital file compatible with most spreadsheet programs. Measurements are made for every heartbeat in a record increasing the statistical power of the output. We demonstrate each of the steps where user input is required and show the application of our methodology to the analysis of heart function in all three genetically tractable heart models.

We have developed a Semi-automated Optical Heartbeat Analysis method (SOHA) for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts. We demonstrate the application of our methodology to the analysis of heart function in fruit fly and embryonic mouse hearts.

Semiautomatici Analisi Heartbeat ottica di piccoli cuori

We have developed a method for analyzing high speed optical recordings from Drosophila, zebrafish and embryonic mouse hearts (Fink, et. al., 2009). Our ...

Exploring Arterial Smooth Muscle Kv7 Potassium Channel Function using Patch Clamp Electrophysiology and Pressure Myography

Contraction or relaxation of smooth muscle cells within the walls of resistance arteries determines the artery diameter and thereby controls flow of blood through the vessel and contributes to systemic blood pressure. The contraction process is regulated primarily by cytosolic calcium concentration ([Ca2+]cyt), which is in turn controlled by a variety of ion transporters and channels. Ion channels are common intermediates in signal transduction pathways activated by vasoactive hormones to effect vasoconstriction or vasodilation. And ion channels are often targeted by therapeutic agents either intentionally (e.g. calcium channel blockers used to induce vasodilation and lower blood pressure) or unintentionally (e.g. to induce unwanted cardiovascular side effects).
Kv7 (KCNQ) voltage-activated potassium channels have recently been implicated as important physiological and therapeutic targets for regulation of smooth muscle contraction. To elucidate the specific roles of Kv7 channels in both physiological signal transduction and in the actions of therapeutic agents, we need to study how their activity is modulated at the cellular level as well as evaluate their contribution in the context of the intact artery.
The rat mesenteric arteries provide a useful model system. The arteries can be easily dissected, cleaned of connective tissue, and used to prepare isolated arterial myocytes for patch clamp electrophysiology, or cannulated and pressurized for measurements of vasoconstrictor/vasodilator responses under relatively physiological conditions. Here we describe the methods used for both types of measurements and provide some examples of how the experimental design can be integrated to provide a clearer understanding of the roles of these ion channels in the regulation of vascular tone.

Measurements of Kv7 (KCNQ) potassium channel activity in isolated arterial myocytes (using patch clamp electrophysiological techniques) in parallel with measurements of constrictor/dilator responses (using pressure myography) can reveal important information about the roles of Kv7 channels in vascular smooth muscle physiology and pharmacology.

Esplorare arteriosa Muscolo liscio KV7 Funzione canale del potassio con morsetto Elettrofisiologia Patch e Myography pressione

Contraction  or relaxation of smooth muscle cells within the walls of resistance arteries  determines the artery diameter and thereby controls flow of ...

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify and explore the function of these important proteins. However, classical preparations, such as planar bilayers, limit the manipulations and experiments that can be performed on the reconstituted channel and its membrane environment. The more cell-like structure of giant liposomes permits traditional patch-clamp experiments without sacrificing control of the lipid environment.
Electroformation is an efficient mean to produce giant liposomes &gt;10 &mu;m in diameter which relies on the application of alternating voltage to a thin, ordered lipid film deposited on an electrode surface. However, since the classical protocol calls for the lipids to be deposited from organic solvents, it is not compatible with less robust membrane proteins like ion channels and must be modified. Recently, protocols have been developed to electroform giant liposomes from partially dehydrated small liposomes, which we have adapted to protein-containing liposomes in our laboratory.
We present here the background, equipment, techniques, and pitfalls of electroformation of giant liposomes from small liposome dispersions. We begin with the classic protocol, which should be mastered first before attempting the more challenging protocols that follow. We demonstrate the process of controlled partial dehydration of small liposomes using vapor equilibrium with saturated salt solutions. Finally, we demonstrate the process of electroformation itself. We will describe simple, inexpensive equipment that can be made in-house to produce high-quality liposomes, and describe visual inspection of the preparation at each stage to ensure the best results.

Reconstituting functional membrane proteins into giant liposomes of defined composition is a powerful approach when combined with patch-clamp electrophysiology. However, conventional giant liposome production may be incompatible with protein stability. We describe protocols for producing giant liposomes from pure lipids or small liposomes containing ion channels.

Giant Preparazione liposomi per Immagini e di patch-clamp elettrofisiologia

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

Introduction to Solid Supported Membrane Based Electrophysiology

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated sensor chip and a phosphatidylcholine monolayer on top. This assembly is mounted into a cuvette system containing the reference electrode, a chlorinated silver wire.
After adsorption of membrane fragments or proteoliposomes containing the membrane protein of interest, a fast solution exchange is used to induce the transport activity of the membrane protein. In the single solution exchange protocol two solutions, one non-activating and one activating solution, are needed. The flow is controlled by pressurized air and a valve and tubing system within a faraday cage.
The kinetics of the electrogenic transport activity is obtained via capacitive coupling between the SSM and the proteoliposomes or membrane fragments. The method, therefore, yields only transient currents. The peak current represents the stationary transport activity. The time dependent transporter currents can be reconstructed by circuit analysis.
This method is especially suited for prokaryotic transporters or eukaryotic transporters from intracellular membranes, which cannot be investigated by patch clamp or voltage clamp methods.

Here we present an electrophysiological method based on solid supported membranes with focus on its applications for the characterization of electrogenic membrane transporters.

Introduzione alla membrana supportata Solid Sulla Elettrofisiologia

The electrophysiological method we present is based on a solid supported membrane (SSM) composed of an octadecanethiol layer chemisorbed on a gold coated ...

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter implantation. The technical limitation of this surgical technique lies in the finesse of the hands. The injection is rapid, especially for a trained experimenter, and since tissue disruption with this technique is minimal, repeated injections are possible; moreover immune reaction to foreign tools (e.g. catheter) does not occur, thereby giving a better and more specific read out of spinal cord modulation. Since the application of the substance is largely limited to the target region of the spinal cord, drugs do not need to be applied in large dosages, and more importantly unwanted effects on other tissue, as observed with a systemic delivery, could be circumvented1,2. Moreover, we combine this technique with in vivo transfection of nucleic acid with the help of polyethylenimine (PEI) reagent3, which provides tremendous versatility for studying spinal functions via delivery of pharmacological agents as well as gene, RNA, and protein modulators.

In Vivo SiRNA Transfection and Gene Knockdown in Spinal Cord via Rapid Noninvasive Lumbar Intrathecal Injections in Mice

This report describes a simple and rapid technique of intrathecal needle puncture for a localized transfection of siRNA in the lumbar spinal cord in mouse under short lasting light anesthesia.

In Vivo trasfezione di siRNA e Gene Knockdown in Spinal Cord via rapida non invasiva lombare spinale iniezioni nei topi

This report describes a step-by-step guide to the technique of acute intrathecal needle injections in a noninvasive manner, i.e. independent of catheter ...

Cystometric and External Urethral Sphincter Measurements in Awake Rats with Implanted Catheter and Electrodes Allowing for Repeated Measurements

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually performed as terminal analysis under urethane anesthesia. It is well known that anesthetic drugs can influence bladder function. Hence, the aim of this technique is to perform cystometric measurements of the urinary bladder and external urethral sphincter in lightly restrained awake rats. For this purpose, a bladder catheter is implanted into the bladder dome. Subsequently, two electrodes are implanted bilateral to the external urethral sphincter and a ground electrode is sutured to a non-responsive skeletal muscle. The bladder catheter and the three electrodes are finally tunneled subcutaneously to the neck region and affixed to a harness. With this technique, the lower urinary tract can be measured at multiple time points in the same animal to assess lower urinary tract function. The main application of this technique is the follow-up of simultaneous urinary bladder and external urethral sphincter function in awake healthy rats and after induction of a disease or injury. Moreover, subsequent lower urinary tract monitoring can be performed during evaluation of the disease/injury and to monitor treatment efficacy.

This protocol first describes the surgical procedure of the permanent implantation of a urinary bladder catheter combined with external urethral sphincter electrodes, and second, the measurement of the function of the urinary bladder and external urethral sphincter in implanted awake animals.

Cistometrici ed esterni misure dello sfintere uretrale in ratti svegli con catetere impiantato ed elettrodi consentendo misurazioni ripetute

Lower urinary tract function is mainly assessed by means of cystometric bladder function analysis in rodents. Conventional cystometries are usually ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

Applicazione di misura di elettrofisiologia di studiare l'attività dei trasportatori di Electro-neutro

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...