Name: one-channel cell-attached patch-clamp recording
Uploaded: 2014-06-09T14:45:00
Description: Watch this Scientific Journal Video about One-channel Cell-attached Patch-clamp Recording at JoVE.com

इस काम F31NS086765 (KAC) द्वारा समर्थित किया गया था, F31NS076235 (एमएपी), और R01 NS052669 (GKP) और EIA9100012. लेखकों विशेषज्ञता और आणविक जीव विज्ञान और टिशू कल्चर के साथ सहायता के लिए Eileen Kasperek धन्यवाद; और जेसन मायर्स जल्दी प्रीफ्रंटल cortical न्यूरॉन्स से प्राप्त आंकड़ों को साझा करने के लिए.

1. सेल संस्कृति और प्रोटीन अभिव्यक्ति <ol><li> HEK293 कोशिकाओं 10% भ्रूण गोजातीय सीरम (FBS) के साथ पूरक DMEM में monolayer संस्कृति में, ATCC द्वारा निष्पादित मार्ग भी शामिल है जो अंश 22 और 40 के बीच (ATCC संख्या सीआरएल-1573), और 5% सीओ 2 म...

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Sakmann and E Neher. , (1995).</li></ol>

आयन चैनल क्षेत्र में अनुसंधान का एक महत्वपूर्ण क्षेत्र को खोलने या चैनल के gating तंत्र चैनल की ओर जाता है कि घटनाओं के अनुक्रम को समझने के लिए समर्पित है. सबसे चैनलों के लिए, इस प्रक्रिया जटिल है और एक macroscopic ?...

जैविक झिल्ली भर में तेजी से संचार सामान्यतः चैनल के रूप में भेजा oligomeric ताकना गठन झिल्ली प्रोटीन, पर लगभग विशेष रूप से निर्भर करता है. ये प्रोटीन सक्रियण संकेतों, gating तंत्र, और प्रवाहकत्त्व गुणों में व्यापक रूप से भिन्न होते हैं. जिसका pores आयनों को चयनात्मक हैं चैनल प्रोटीन आयन चैनल के रूप में वर्गीकृत कर रहे हैं; उनके सक्रियण झिल्ली भर आयनिक धाराओं पैदा करता है, और अपनी प्रतिक्रिया electrophysiologic तकनीक का उपयोग कर वास्तविक समय में उच्च संकल्प के साथ दर्ज किया जा सकता है. सक्रियण संकेतों एकाग्रता ढ़ाल, मैकेनिकल और इलेक्ट्रिकल बलों, और तापमान सहित रासायनिक और भौतिक आदानों की एक व्यापक सरणी अवधि; इस प्रकार, आगे ligand में आयन चैनल वर्गीकृत, gated mechanosensitive, वोल्टेज gated, या गर्मी संवेदनशील प्रकार के. इस अनुच्छेद में, प्रोटोकॉल पैच दबाना तकनीक का उपयोग कर, एक ligand gated चैनल, NMDA रिसेप्टर, और एक mechanosensitive चैनल, PIEZO1 से एक चैनल गतिविधि को रिकॉर्ड करने के लिए वर्णित हैं.0; पैच दबाना इलेक्ट्रोफिजियोलॉजी एकल अणुओं 1, 2 के अवलोकन की अनुमति के लिए पर्याप्त रूप से संवेदनशील पहली और सबसे व्यापक रूप से इस्तेमाल किया प्रयोगात्मक विधि है. इस अति सुंदर संवेदनशीलता के अलावा, यह बेहद electrophysiologic रिकॉर्डिंग करने के लिए उत्तरदायी जैविक तैयारी का विस्तार किया गया है और यह भी बरकरार झिल्ली में आयन चैनल के अवलोकन की अनुमति दी है. वोल्टेज clamping और वर्तमान रिकॉर्डिंग दोनों एक ही इलेक्ट्रोड के साथ पूरा कर रहे हैं, क्योंकि सबसे पहले, यह छोटे कोशिकाओं या झिल्ली पैच भर में संकेत रिकॉर्ड करने के लिए इस्तेमाल किया जा सकता है. तकनीक आयन चैनल मेंढक की मांसपेशियों, मछली electroplaques, या विद्रूप विशाल axons 3, 4, बल्कि वे transmembrane संकेतन तंत्र की देशव्यापी फिक्स्चर का प्रतिनिधित्व करते हैं और विश्वविद्यालय या की सभी सेलुलर झिल्ली प्रकार के आंतरिक कर रहे हैं कि के उत्तेजनीय झिल्ली तक ही सीमित नहीं रहे हैं कि पता चला बहुकोशिकीय जीव, और भी intracellular झिल्ली को. आयातantly, बस एक अक्षुण्ण सेल करने के लिए एक गिलास विंदुक संलग्न द्वारा transmembrane धाराओं रिकॉर्ड करने की क्षमता उनके पैतृक undisrupted झिल्ली में आयन चैनल से गतिविधि को रिकॉर्ड करने के लिए अभूतपूर्व अवसर प्रदान किया. इस प्रकार, इस प्रोटोकॉल में वर्णित है जो सेल संलग्न पैच दबाना तकनीक, मिनट के दसियों या उनके पैतृक वातावरण में लंबे समय तक के लिए लगातार आयन चैनलों की गतिविधि की निगरानी परमिट. सामान्य थर्मल उतार चढ़ाव के तहत, आयन चैनल प्रोटीन सहित सभी प्रोटीन,, पक्ष श्रृंखला आंदोलनों और पूरे के repositioning द्वारा प्रतिनिधित्व बहुत धीमी है, कम लगातार परिवर्तन से सबसे अधिक संभावना प्रतिनिधित्व सबसे तेज और सबसे लगातार rearrangements के साथ, एक व्यापक समय के पैमाने पर संरचनात्मक परिवर्तन से गुजरना डोमेन या सब यूनिटों, या पोस्ट translational संशोधनों या प्रोटीन, प्रोटीन बातचीत 5, 6 के द्वारा कुछ मामलों में. एक अणु के द्वारा उत्पन्न की गतिविधि की लंबी अवधि के अवलोकन समारोह को समझने में मदद कर सकते हैंराष्ट्रीय बरकरार शारीरिक झिल्ली में आयन चैनल की गतिशीलता और मनाया अणु का परिचालन तंत्र के बारे में महत्वपूर्ण जानकारी प्रदान करता है. सेल प्रकार के और विकास के चरणों में आयन चैनल, देशी झिल्ली में आयन चैनल की आणविक संरचना के बारे में ज्ञान की विविधता की बढ़ती समझ के विपरीत अभी भी सीमित है. सभी आयन चैनल multimeric प्रोटीन होते हैं और देशी आयन चैनल का बहुमत अक्सर विविध प्रवाहकत्त्व और gating संपत्तियों के साथ साथ है, जो विस्तृत आणविक विविधता, के प्रोटीन के उत्पादन यूनिटों के कई प्रकार से एकत्र कर लें. इस कारण से, परिभाषित आणविक संरचना के आयन चैनल heterologous प्रणाली में अभिव्यक्ति पर अध्ययन कर रहे हैं. विशेष रूप से, अमर मानव भ्रूण गुर्दे की कोशिकाओं 7 का एक प्रतिरूप लाइन हैं जो HEK293 कोशिकाओं, पुनः संयोजक आयन चैनल का heterologous अभिव्यक्ति के लिए पसंदीदा प्रणाली के रूप में व्यापक स्वीकृति प्राप्त की. आदमी के बीचआयन चैनल इलेक्ट्रोफिजियोलॉजी के लिए चुनाव प्रणाली के रूप में HEK293 कोशिकाओं बुलंद है कि y फायदे आसानी और संवर्धन की खरीदने की क्षमता हैं और लंबे समय रहते स्थिर संस्कृतियों को बनाए रखने, बाद translational तह, प्रसंस्करण और स्तनधारी प्रोटीन की तस्करी से बाहर ले जाने के लिए उनकी क्षमता, और कई मामलों में , उनके कम स्तर या ब्याज 7, 8 चैनल के लिए अंतर्जात अभिव्यक्ति की भी अभाव. पुनः संयोजक आयन चैनल व्यक्त करने और HEK293 कोशिकाओं में उनके कार्यात्मक गुणों का अध्ययन संरचना समारोह आयन चैनल के गुणों के साथ ही आयन चैनल isoforms और देशी ऊतक में उनकी भूमिकाओं के विशिष्ट गुणों के बारे में जानकारी प्राप्त करने के लिए एक मूल्यवान दृष्टिकोण होना जारी है. इस आलेख में वर्णित प्रोटोकॉल HEK293 कोशिकाओं में व्यक्त पुनः संयोजक आयन चैनलों के लिए और देशी आयन चैनलों के लिए समान रूप से अच्छी तरह से लागू किया जा सकता है. संक्षेप में, अपने अभूतपूर्व क्षमता के माध्यम से पैच दबाना तकनीक, संकेत हल करने के लिएएक अणु से है, आज तक, एकल अणुओं के व्यवहार के अवलोकन के लिए सबसे प्रत्यक्ष विधि बनी हुई है. अपने सेल संलग्न मोड में, पैच दबाना रिकॉर्डिंग एक अणु के लिए जब किया, आयन चैनल के आपरेशन में असाधारण अंतर्दृष्टि प्रदान कर सकते हैं, जो लंबे समय से अवलोकन अवधि की अनुमति देता है. नीचे एक आयन चैनल प्रोटीन युक्त सेल संलग्न पैच से उच्च संकल्प वर्तमान रिकॉर्डिंग प्राप्त करने के लिए एक प्रोटोकॉल प्रस्तुत किया है.

<table border="0" cellpadding="0" cellspacing="0" width="695">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:189px;">Chemicals</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:128px;">Various</td>
			<td style="width:205px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Borosillicate Glass</td>
			<td>Sutter</td>
			<td>BF-150-86-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Bright field inverted microscope</td>
			<td>Olympus</td>
			<td>1x51</td>
			<td>Nikon also has similar microscopes</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluroescent box</td>
			<td>X-cite</td>
			<td>Series 120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Liquid Light Guide</td>
			<td>X-cite</td>
			<td>OEX-LG15</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Micromanipulator</td>
			<td>Sutter Instruments</td>
			<td>MP-225</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Oscilloscope</td>
			<td>Tektronix</td>
			<td>TDS1001</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Amplifier</td>
			<td>Molecular Devices</td>
			<td colspan="2">Axon Axopatch 200B&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Table</td>
			<td>TMC</td>
			<td align="right">63561</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">NIDAQ card</td>
			<td>National Instruments</td>
			<td>776844-01</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Puller</td>
			<td>Narishige</td>
			<td>PC-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Polisher</td>
			<td>Narishige</td>
			<td>Microforge MF-830</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Faraday Cage</td>
			<td>TMC</td>
			<td align="right">8133306</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Speed Pressure Clamp</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA HSPC</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressue/Vaccuum Pump</td>
			<td>ALA Scientific Instruments</td>
			<td>ALA PV-PUMP</td>
			<td>For HSPC-1</td>
		</tr>
	</tbody>
</table>

one-channel cell-attached patch-clamp recording

आयन चैनल प्रोटीन जैविक झिल्ली भर में तेजी से संचार के लिए यूनिवर्सल उपकरणों रहे हैं. वे उत्पन्न आयनिक प्रवाह के अस्थायी हस्ताक्षर प्रत्येक चैनल प्रोटीन के साथ ही यह उत्पन्न होता है और नियंत्रित किया जाता है जिसके द्वारा तंत्र को आंतरिक गुणों पर निर्भर करता है और वर्तमान अनुसंधान का एक महत्वपूर्ण क्षेत्र का प्रतिनिधित्व करता है. आयन चैनल प्रोटीन का परिचालन गतिशीलता के बारे में सूचना एक ही अणु के द्वारा उत्पादित वर्तमान की लंबी हिस्सों देख द्वारा प्राप्त किया जा सकता है. एक ligand gated आयन चैनल, NMDA रिसेप्टर के लिए एक चैनल सेल संलग्न पैच दबाना वर्तमान रिकॉर्डिंग प्राप्त करने के लिए एक प्रोटोकॉल यहाँ है वर्णित है, cortical न्यूरॉन्स में HEK293 कोशिकाओं में या natively heterologously व्यक्त किया. इसके अलावा mechano संवेदनशील चैनल PIEZO1 का उदाहरण पेश करके ब्याज की अन्य आयन चैनलों के लिए विधि अनुकूलित करने के निर्देश हैं प्रदान की. इस विधि चैनल के प्रवाहकत्त्व गुण और ope के अस्थायी अनुक्रम के बारे में डेटा प्रदान कर सकते हैंइस प्रकार स्वास्थ्य और रोग में उनके कार्यों को समझने में मदद करने, चैनल के सक्रियण तंत्र को बनाने वाली N-बंद रचना.

संयोजक NMDA रिसेप्टर्स NMDA रिसेप्टर्स बाँध और दो सह agonists के सहवर्ती कार्रवाई का जवाब: ग्लूटामेट और ग्लाइसिन. वे GluN1 सब यूनिटों और GluN2 सब यूनिटों बंधन दो ग्लूटामेट बंधन दो ग्लाइसिन के heterotetramers के ?...

Watch this Scientific Journal Video about One-channel Cell-attached Patch-clamp Recording at JoVE.com

One-channel Cell-attached Patch-clamp Recording

यहाँ वर्णित सेल संलग्न पैच दबाना तकनीक के साथ एक आयन चैनल से वर्तमान रिकॉर्डिंग की लंबी हिस्सों प्राप्त करने के लिए एक प्रक्रिया है. इस विधि, वास्तविक समय में अवलोकन के लिए अनुमति देता है, जैविक संकेत कायम करना है कि खुले करीब चैनल रचना के पैटर्न. इन आंकड़ों अबाधित जैविक झिल्लियों में चैनल गुणों के बारे में सूचित करें.

एक चैनल सेल संलग्न पैच दबाना रिकॉर्डिंग

Ion channel proteins are universal devices for fast communication across biological membranes. The temporal signature of the ionic flux they generate ...

one-channel-cell-attached-patch-clamp-recording

Research

JoVE Journal

Biology

Methods for Patch Clamp Capacitance Recordings from the Calyx

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. Electrical recordings from the presynaptic terminal allow the measurement of action potentials, calcium channel currents, vesicle fusion (exocytosis) and subsequent membrane uptake (endocytosis). The fusion of vesicles containing neurotransmitter causes the vesicle membrane to be added to the cell membrane of the calyx. This increase in the amount of cell membrane is measured as an increase in capacitance. The subsequent reduction in capacitance indicates endocytosis, the process of membrane uptake or removal from the calyx membrane. Endocytosis, is necessary to maintain the structure of the calyx and it is also necessary to form vesicles that will be filled with neurotransmitter for future exocytosis events. Capacitance recordings at the calyx of Held have made it possible to directly and rapidly measure vesicular release and subsequent endocytosis in a mammalian CNS nerve terminal. In addition, the corresponding postsynaptic activity can be simultaneously measured by using paired recordings. Thus a complete picture of the presynaptic and postsynaptic electrical activity at a central nervous system synapse is achievable using this preparation. Here, the methods for slice preparation, morphological features for identification of calyces of Held, basic patch clamping techniques, and examples of capacitance recordings to measure exocytosis and endocytosis are presented.

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal.

Calyx से पैच दबाना समाई रिकॉर्डिंग के लिए तरीके

We demonstrate the basic techniques for presynaptic patch clamp recording at the calyx of Held, a mammalian central nervous system nerve terminal. ...

Patch Clamp Recording of Ion Channels Expressed in  Xenopus Oocytes

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological measurement of single or multiple ion channels in cells. This technique can be applied to ion channels in both their native environment and expressed in heterologous cells, such as oocytes harvested from the African clawed frog, Xenopus laevis. Here, we describe the well-established technique of patch clamp recording from Xenopus oocytes. This technique is used to measure the properties of expressed ion channels either in populations (macropatch) or individually (single-channel recording). We focus on techniques to maximize the quality of oocyte preparation and seal generation. With all factors optimized, this technique gives a probability of successful seal generation over 90 percent. The process may be optimized differently by every researcher based on the factors he or she finds most important, and we present the approach that have lead to the greatest success in our hands.

This is intended as an introduction to patch clamp recording from Xenopus laevis oocytes. It covers vitelline membrane removal, formation of a gigaohm seal (gigaseal), and the optional conversion of the patch to the outside-out topology.

Xenopus oocytes में व्यक्त आयन चैनल के पैच दबाना रिकॉर्डिंग

Since its development by Sakmann and Neher 1, 2, the patch clamp has become established as an extremely useful technique for electrophysiological ...

Pressure-polishing Pipettes for Improved Patch-clamp Recording

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording. We first developed this method for fabricating pipettes suitable for recording from small (&lt;3 m) neuronal cell bodies. The basic principal is similar to glass-blowing and combines air pressure and heat to modify the shape of patch pipettes prepared by a conventional micropipette puller. It can be applied to so-called soft (soda lime) and hard (borosilicate) glasses. Generally speaking, pressure polishing can reduce pipette resistance by 25% without decreasing the diameter of the tip opening (Goodman and Lockery, 2000). It can be applied to virtually any type of glass and requires only the addition of a high-pressure valve and fitting to a microforge. This technique is essential for recording from ultrasmall cells (&lt;5 m) and can also improve single-channel recording by minimizing pipette resistance. The blunt shape is also useful for perforated-patch clamp recording since this tip shape results in a larger membrane bleb available for perforation.

This is a guide to modifying the shape of glass micropipettes. Specifically, by using heat and air pressure the taper is widened without increasing the tip opening, leading to lower pipette resistance. This is critical to obtain low noise recordings of small cells but is useful in many applications.

बेहतर पैच दबाना रिकॉर्डिंग के लिए दबाव चमकाने pipettes

Pressure-polishing is a method for shaping glass pipettes for patch-clamp recording.  We first developed this method for fabricating pipettes suitable for ...

Dissecting and Recording from The C. Elegans Neuromuscular Junction

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale. This complex process requires the coordinated activity of many pre- and post-synaptic proteins to ensure appropriate synaptic connectivity, conduction of electrical signals, targeting and priming of secretory vesicles, calcium sensing, vesicle fusion, localization and function of postsynaptic receptors and finally, recycling mechanisms. As neuroscientists it is our goal to elucidate which proteins function in each of these steps and understand their mechanisms of action. Electrophysiological recordings from synapses provide a quantifiable read out of the underlying electrical events that occur during synaptic transmission. By combining this technique with the powerful array of molecular and genetic tools available to manipulate synaptic proteins in C. elegans, we can analyze the resulting functional changes in synaptic transmission. 
The C. elegans NMJs formed between motor neurons and body wall muscles control locomotion, therefore, mutants with uncoordinated locomotory phenotypes (known as unc s) often perturb synaptic transmission at these synapses 1. Since unc mutants are maintained on a rich supply of a bacterial food source, they remain viable as long as they retain some pharyngeal pumping ability to ingest food. This, together with the fact that C. elegans exist as hermaphrodites, allows them to pass on mutant progeny without the need for elaborate mating behaviors. These attributes, coupled with our recent ability to record from the worms NMJs 2,3,7 make this an excellent model organism in which to address precisely how unc mutants impact neurotransmission. 
The dissection method involves immobilizing adult worms using a cyanoacrylic glue in order to make an incision in the worm cuticle exposing the NMJs. Since C. elegans adults are only 1 mm in length the dissection is performed with the use of a dissecting microscope and requires excellent hand-eye coordination. NMJ recordings are made by whole-cell voltage clamping individual body wall muscle cells and neurotransmitter release can be evoked using a variety of stimulation protocols including electrical stimulation, light-activated channel-rhodopsin-mediated depolarization 4 and hyperosmotic saline, all of which will be briefly described.

Application of electrophysiology to accessible synapses provides a quantifiable measure of synaptic activity, useful in analyzing synaptic mutants. This article describes a dissection method used to expose the neuromuscular junctions (NMJ) of Caenorhabditis elegans (C. elegans) and briefly discusses some of the uses to which this preparation can be applied.

सी एलिगेंस Neuromuscular जंक्शन से विदारक और रिकॉर्डिंग

Neurotransmission is the process by which neurons transfer information via chemical signals to their post-synaptic targets, on a rapid time scale.   This ...

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Electrophysiological रिकॉर्डिंग में ड्रोसोफिला भ्रूण

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Ambulatory ECG Recording in Mice

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although the surface ECG is utilized for short-term measurements of waveform intervals, it is not practical for long-term studies of heart rate variability or the capture of rare episodes of arrhythmias. Implantable ECG telemeters offer the advantages of simple surgical implantation, long-term recording of electrograms in ambulatory mice, and scalability with simultaneous recordings of multiple animals. Here, we present a step-by-step guide to the implantation of telemeters for ambulatory ECG recording in mice. Careful adherence to aseptic technique is required for favorable survival results with the possibility of implantation and recording from weeks to months. Thus, implantable ECG telemetry is a valuable tool for detection of critical information on cardiac electrophysiology in ambulatory animal models such as the mouse. 

Telemetric ECG has emerged as an essential tool in evaluating animal models for cardiac arrhythmias and sudden cardiac death. Here, we present a stepwise guide to telemetric ECG recordings for application in long-term ambulatory ECG monitoring in mice.

चूहे में चल ईसीजी रिकॉर्डिंग

Telemetric ECG recording in mice is essential to understanding the mechanisms behind arrhythmias, conduction disorders, and sudden cardiac death. Although ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may also be used to study the structure-function relationship for other ion channels and neurotransmitter receptors1. BK channels are widely expressed in different tissues and have been implicated in many physiological functions, including regulation of smooth muscle contraction, frequency tuning of inner hair cells and regulation of neurotransmitter release2-6. BK channels are activated by membrane depolarization and by intracellular Ca2+ and Mg2+ 6-9. Therefore, the protocol is designed to control both the membrane voltage and the intracellular solution. In this protocol, messenger RNA of BK channels is injected into Xenopus laevis oocytes (stage V-VI) followed by 2-5 days of incubation at 18&deg;C10-13. Membrane patches that contain single or multiple BK channels are excised with the inside-out configuration using patch clamp techniques10-13. The intracellular side of the patch is perfused with desired solutions during recording so that the channel activation under different conditions can be examined. To summarize, the mRNA of BK channels is injected into Xenopus laevis oocytes to express channel proteins on the oocyte membrane; patch clamp techniques are used to record currents flowing through the channels under controlled voltage and intracellular solutions.

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Ionic current of BK channels is recorded using patch clamp techniques. BK channels are expressed in Xenopus oocytes by injecting messenger RNA. The intracellular solution during patch clamp recordings is controlled by a perfusion system.

पैच और आयन चैनल के अध्ययन के लिए दबाना छिड़काव तकनीक में व्यक्त Xenopus Oocytes

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

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.

धमनी चिकनी पेशी Kv7 पोटेशियम चैनल फंक्शन तलाश पैच दबाना इलैक्ट्रोफिजियोलॉजी और दबाव Myography का उपयोग

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.

इमेजिंग और पैच दबाना इलैक्ट्रोफिजियोलॉजी के लिए विशालकाय Liposome तैयारी

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

Reconstitution of a Transmembrane Protein, the Voltage-gated Ion Channel, KvAP, into Giant Unilamellar Vesicles for Microscopy and Patch Clamp Studies

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow GUVs must be modified in order to form GUVs containing functional transmembrane proteins. This article describes two dehydration-rehydration methods &#8212; electroformation and gel-assisted swelling &#8212; to form GUVs containing the voltage-gated potassium channel, KvAP. In both methods, a solution of protein-containing small unilamellar vesicles is partially dehydrated to form a stack of membranes, which is then allowed to swell in a rehydration buffer. For the electroformation method, the film is deposited on platinum electrodes so that an AC field can be applied during film rehydration. In contrast, the gel-assisted swelling method uses an agarose gel substrate to enhance film rehydration. Both methods can produce GUVs in low (e.g., 5 mM) and physiological (e.g., 100 mM) salt concentrations. The resulting GUVs are characterized via fluorescence microscopy, and the function of reconstituted channels measured using the inside-out patch-clamp configuration. While swelling in the presence of an alternating electric field (electroformation) gives a high yield of defect-free GUVs, the gel-assisted swelling method produces a more homogeneous protein distribution and requires no special equipment.

The reconstitution of the transmembrane protein, KvAP, into giant unilamellar vesicles (GUVs) is demonstrated for two dehydration-rehydration methods &#8212; electroformation, and gel-assisted swelling. In both methods, small unilamellar vesicles containing the protein are fused together to form GUVs that can then be studied by fluorescence microscopy and patch-clamp electrophysiology.

एक transmembrane प्रोटीन के पुनर्गठन, माइक्रोस्कोपी और पैच दबाना अध्ययन के लिए विशालकाय unilamellar vesicles में वोल्टेज-गेटेड आयन चैनल, KvAP,

Giant Unilamellar Vesicles (GUVs) are a popular biomimetic system for studying membrane associated phenomena. However, commonly used protocols to grow ...