Name: facial transplants in xenopus laevis embryos
Uploaded: 2014-03-26T13:00:00
Description: Watch this Scientific Journal Video about Facial Transplants in Xenopus laevis Embryos at JoVE.com

Vi takker Radek Sindelka for hans hjelp, og Cas Bresilla for å bistå med frosk driften og embryo forberedelse. Dette arbeidet ble finansiert av NIH via tilskuddet R01DE021109 til HLS Laura Jacox ble finansiert av Herschel Smith Graduate Fellowship ved Harvard University og en F30 individuell stipendet F30DE022989-01 gjennom NIDCR.

En. Forbereder Reagenser <ol><li> 10x MBS: Forbered en L av 10x Modified Barth Saline (MBS) løsning 17. Se Tabell 1, reagenser, ingredienser og instruksjoner. Bruk destillert vann for alle løsninger. Bland i et begerglass under anvendelse av en rørestav, inntil fullstendig oppløsning. Alle løsningene bør bli gjort ved romtemperatur. </li><li> 1x MBS: Fortynn 100 ml 10x MBS-løsning i 900 ml destillert vann for å lage en L av 1x MBS. Til 0,7 ml med 1 M CaCl 2 løsning. </li...

<ol>
	<li>Gorlin, R. J., Cohen, M., Levin, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Syndromes of the head and neck. , (1990).</li><li>Trainor, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Craniofacial+birth+defects:+The+role+of+neural+crest+cells+in+the+etiology+and+pathogenesis+of+Treacher+Collins+syndrome+and+the+potential+for+prevention.">Craniofacial birth defects: The role of neural crest cells in the etiology and pathogenesis of Treacher Collins syndrome and the potential for prevention.</a> Am. J. Med. Gen. A. 152, 2984-2994 (2010).</li><li>Dickinson, A. J., Sive, H. 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+the+primary+mouth+in+Xenopus+laevis.">Development of the primary mouth in Xenopus laevis.</a> Dev. Bio. 295, 700-713 (2006).</li><li>Dickinson, A. J., Sive, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Positioning+the+extreme+anterior+in+Xenopus:+cement+gland,+primary+mouth+and+anterior+pituitary.">Positioning the extreme anterior in Xenopus: cement gland, primary mouth and anterior pituitary.</a> Sem. Cell Dev. Bio. 18, 525-533 (2007).</li><li>Dickinson, A. J., Sive, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Wnt+antagonists+Frzb-1+and+Crescent+locally+regulate+basement+membrane+dissolution+in+the+developing+primary+mouth.">The Wnt antagonists Frzb-1 and Crescent locally regulate basement membrane dissolution in the developing primary mouth.</a> Dev. 136, 1071-1081 (2009).</li><li>Gilbert, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Developmental Biology. , (2010).</li><li>Borchers, A., Epperlein, H. H., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+assay+system+to+study+migratory+behavior+of+cranial+neural+crest+cells+in+Xenopus.">An assay system to study migratory behavior of cranial neural crest cells in Xenopus.</a> Dev. Genes Evol. 210, 217-222 (2000).</li><li>Grunz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homoiogenetic+neural+inducing+activity+of+the+presumptive+neural+plate+of+Xenopus+laevis.">Homoiogenetic neural inducing activity of the presumptive neural plate of Xenopus laevis.</a> Dev. Growth Differ. 32, 583-589 (1990).</li><li>Servetnick, M., Grainger, 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=Changes+in+neural+and+lens+competence+in+Xenopus+ectoderm:+evidence+for+an+autonomous+developmental+timer.">Changes in neural and lens competence in Xenopus ectoderm: evidence for an autonomous developmental timer.</a> Dev. Bio. 112, 177-188 (1991).</li><li>Servetnick, M., Grainger, 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=Homeogenetic+neural+induction+in+Xenopus.">Homeogenetic neural induction in Xenopus.</a> Dev. Bio. 147, 73-82 (1991).</li><li>Couly, G., Coltey, P., Le Douarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+triple+origin+of+skull+in+higher+vertebrates:+a+study+in+quail-chick+chimeras.">The triple origin of skull in higher vertebrates: a study in quail-chick chimeras.</a> Dev. 117, 409-429 (1993).</li><li>Couly, G. F., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+early+neural+primordium+in+quail-chick+chimeras+:+I.">Mapping of the early neural primordium in quail-chick chimeras : I.</a> Developmental relationships between placodes, facial ectoderm. 110, 422-439 (1985).</li><li>Couly, G. F., Le Douarin, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+of+the+early+neural+primordium+in+quail-chick+chimeras+II.+The+prosencephalic+neural+plate+and+neural+folds:+Implications+for+the+genesis+of+cephalic+human+congenital+abnormalities.">Mapping of the early neural primordium in quail-chick chimeras II. The prosencephalic neural plate and neural folds: Implications for the genesis of cephalic human congenital abnormalities.</a> Dev. Bio. 120, 198-214 (1987).</li><li>Lievre, A. L., Le Douarin, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+early+development+of+cranial+sensory+ganglia+and+the+potentialities+of+their+component+cells+studied+in+quail-chick+chimeras.">The early development of cranial sensory ganglia and the potentialities of their component cells studied in quail-chick chimeras.</a> Dev. Bio. 94, 291-310 (1982).</li><li>Kennedy, A., Dickinson, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Median+facial+clefts+in+Xenopus+laevis:+roles+of+retinoic+acid+signaling+and+homeobox+genes.">Median facial clefts in Xenopus laevis: roles of retinoic acid signaling and homeobox genes.</a> Dev. Bio. 365, 229-240 (2012).</li><li>Trainor, P., Tam, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cranial+paraxial+mesoderm+and+neural+crest+of+the+mouse+embryo-+codistribution+in+the+craniofacial+mesenchyme+but+distinct+segregation+in+the+branchial+arches.">Cranial paraxial mesoderm and neural crest of the mouse embryo- codistribution in the craniofacial mesenchyme but distinct segregation in the branchial arches.</a> Dev. 121, 2569-2582 (1995).</li><li>Sive, H. L., Grainger, R. M., Harland, 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=.">.</a> Early Development of Xenopus laevis. , (2000).</li><li>Tandon, P., Showell, C., Christine, K., Conlon, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morpholino+injection+in+Xenopus.">Morpholino injection in Xenopus.</a> Methods Mol. Biol. 843, 29-46 (2012).</li><li>Nieuwkoop, P. D., Faber, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Normal Table of Xenopus laevis (Daudin). , (1994).</li></ol>

Kritiske trinn og begrensninger: Den EAD ansikt transplantasjon prosedyren er tid og arbeidsintensivt. Det krever øvelse, stødige hender, og fingerferdighet å perfeksjonere. Ansiktet transplantasjon protokollen er avhengig av forskerens evne til effektivt å fjerne og transplantasjon vev. Hvis man tar for lang tid å sette transplantasjon inn i vertens ansikt, vil verts ansiktet begynner å trekke seg sammen og helbrede. Tang kan brukes til å delikat utvide ansiktsregionen. Imidlertid, hvis større sår samme...

For å forstå mekanismene bak kraniofaciale misdannelser 1-2, viktige vev og deres signal bidrag under kraniofaciale utvikling må identifiseres. I frosken Xenopus laevis, en del av ansiktet, inkludert munn og nesebor skjema fra &quot;Extreme Anterior Domain&quot; (EAD), hvor ektoderm og endoderm er direkte sammenstilt 3-4. Den EAD fungerer også som et signalsenter å påvirke omkringliggende vev, inkludert kranie neural crest, som danner kjevene og andre ansikts regioner fem. For å identifisere genene som bidrar til EAD funksjon, ble et &quot;ansikt transplantasjon&quot; teknikken utviklet, hvor vevet er transplantert fra en donor til en verts embryo, etter fjerning av de tilsvarende verts region. Etter transplantasjon, fører ansikts utvikling vurderes. Således er effekten av tap av funksjon (LOF) eller gevinst av funksjon (GOF) for et bestemt gen i EAD analysert lokalt, der resten av head og kropp er sammensatt av villtype vev. Den gjensidige transplantasjon kan utføres, hvor vill type vev er transplantert inn embryoer med global LOF eller GOF i bestemte gener. Transplantasjon har blitt hyppig brukt i Xenopus og dama studerer seks. For eksempel har Xenopus transplantasjon adressert homogenetic nevrale induksjon, linse og nevrale kompetanse, og neural crest migrasjon 7-10. Vaktel-chick kimert pode har analysert utviklingen av fremre nevrale plate, anterior neural rygg, neural crest, og kraniebein 11-14. Dette er den første transplantasjon teknikk for studier av kraniofaciale utvikling i Xenopus. Denne teknikken har vist en roman rolle for Wnt hemmere Frzb1 og Crescent i å regulere basalmembran formasjonen i presumptive munnen fem. Xenopus laevis er en ideell modell for studier av kraniofaciale utvikling som embryoer er store, utvikle eksternt, ennd ansiktet er lett synlig, slik at micromanipulation og avbildning av utvikling. Mekanismene bak ansikts utvikling vises bevart, noe som indikerer at funn som er gjort i frosken gi innsikt i menneskelig utvikling 4,15-16.

<table border="0" cellpadding="0" cellspacing="0" width="559">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Pasteur pipette</td>
			<td rowspan="3" style="width:101px;">VWR</td>
			<td rowspan="3" style="width:136px;">14672-400</td>
			<td style="width:128px;">Lime Glass&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Size 5 3/4&#8217;&#8217;</td>
			<td style="width:128px;">Cotton Plugged</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;"></td>
			<td style="width:128px;">Disposable</td>
		</tr>
		<tr height="20">
			<td height="41" rowspan="2" style="height:41px;width:195px;">Graduated Transfer Pipette</td>
			<td rowspan="2" style="width:101px;">VWR</td>
			<td rowspan="2" style="width:136px;">16001-180</td>
			<td style="width:128px;">Disposable&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Polyethylene</td>
		</tr>
		<tr height="80">
			<td height="100" rowspan="2" style="height:100px;width:195px;">#5/45 forceps</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools by Dupont medical</td>
			<td rowspan="2" style="width:136px;">11251-35</td>
			<td rowspan="2" style="width:128px;">Angled 45 degrees</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="40">
			<td height="81" rowspan="3" style="height:81px;width:195px;">Standard Pattern Forceps</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">11000-20</td>
			<td style="width:128px;">Straight; serrated tip</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Stainless Steel;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">20cm long</td>
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:195px;">Capillary Tubing (for needles)</td>
			<td rowspan="2" style="width:101px;">FHC</td>
			<td rowspan="2" style="width:136px;">30-30-1</td>
			<td style="width:128px;">Borosil 1.0mm OD x 0.5mm ID/Fiber</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">100mm each</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Cover slip&#160;</td>
			<td rowspan="4" style="width:101px;">VWR</td>
			<td style="width:136px;">48393 252&#160;</td>
			<td style="width:128px;">24x60mm&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">micro cover glass</td>
			<td style="width:136px;">or&#160;</td>
			<td style="width:128px;">or&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">(for glass bridges)</td>
			<td style="width:136px;">48393 230</td>
			<td style="width:128px;">24x40mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">No.1.5</td>
			<td style="width:136px;"></td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Ficoll 400</td>
			<td style="width:101px;">Sigma-Aldrich</td>
			<td style="width:136px;">F9378</td>
			<td style="width:128px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:195px;">Needle Puller&#160;</td>
			<td style="width:101px;">Sutter Instrument Co</td>
			<td rowspan="3" style="width:136px;">Needle Puller: discontinued Filament: FB300B</td>
			<td rowspan="3" style="width:128px;">The most similar, currently available&#160; needle puller is the P-97. For filaments, use Sutter 3.00mm square box filaments, 3.0mm wide.</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:195px;">Model P-80</td>
			<td style="width:101px;">Flaming / Brown micropipette puller</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">(discontinued)</td>
			<td style="width:101px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Stereomicroscope</td>
			<td rowspan="2" style="width:101px;">Zeiss</td>
			<td rowspan="2" style="width:136px;"></td>
			<td rowspan="2" style="width:128px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:195px;">Zeiss Stemi 1000</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:195px;">Stereomicroscope Lighting by Fostec</td>
			<td style="width:101px;">Fostec</td>
			<td style="width:136px;"></td>
			<td style="width:128px;">Use a light box with 2 fiberoptic arms.&#160;&#160;</td>
		</tr>
		<tr height="60">
			<td height="81" rowspan="2" style="height:81px;width:195px;">Nickel Plated Pin Holder</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">26018-17</td>
			<td style="width:128px;">Jaw Opening Diameter: 0 to 1mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;">Length: 17cm</td>
		</tr>
		<tr height="60">
			<td height="101" rowspan="3" style="height:101px;width:195px;">Moria Nickel Plated Pin Holder</td>
			<td rowspan="3" style="width:101px;">Fine Science Tools</td>
			<td rowspan="3" style="width:136px;">26016-12</td>
			<td style="width:128px;">Jaw opening Diameter: 0 to 1mm</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:128px;">Length: 12cm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:128px;"></td>
		</tr>
		<tr height="40">
			<td height="60" rowspan="2" style="height:60px;width:195px;">Tungsten Needles</td>
			<td rowspan="2" style="width:101px;">Fine Science Tools</td>
			<td rowspan="2" style="width:136px;">10130-05</td>
			<td rowspan="2" style="width:128px;">0.125mm Rod diameter</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:195px;">Van Aken Plastalina</td>
			<td rowspan="2" style="width:101px;">Blick&#160;</td>
			<td rowspan="2" style="width:136px;">#33268-2981</td>
			<td rowspan="2" style="width:128px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">Modeling Clay- white, red or yellow</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:195px;">mMessage mMashine SP6 or T7 Kit</td>
			<td style="width:101px;">Ambion</td>
			<td style="width:136px;">AM1340</td>
			<td style="width:128px;"></td>
		</tr>
	</tbody>
</table>

facial transplants in xenopus laevis embryos

Kraniofaciale misdannelser oppstår hos 1 av hver 700 levendefødte, men etiologi er sjelden kjent på grunn av begrenset forståelse av kraniofaciale utvikling. For å identifisere hvor signalveier og vev opptre under fordelingen av utviklings ansiktet, har en &quot;ansiktstransplantasjon&quot; teknikken er utviklet i embryoer fra frosken Xenopus laevis. En region av presumptive ansiktsservietter (det &quot;Extreme Anterior Domain&quot; (EAD)) blir fjernet fra en donor embryo ved tailbud stadium, og transplanteres til en verts embryo av samme fase, hvorfra det tilsvarende region er blitt fjernet. Dette kan brukes til å generere et kimært ansikt der verten eller donorvev har en tap eller vinning av funksjon i et gen, og / eller omfatter en avstamning etikett. Etter at helbredelse er resultatet av utviklingen overvåkes, og indikerer rollene til signalveien inne i donor-eller liggende vertsvevet. Xenopus er et verdifullt modell for ansikts utvikling, idet ansikts-området er stort og lett enccessible for micromanipulation. Mange embryoer kan analyseres i løpet av en kort tidsperiode, siden utviklingen skjer raskt. Funn i frosken er relevant for menneskelig utvikling, siden kraniofaciale prosesser vises konservert mellom Xenopus og pattedyr.

Transplantert vev skal settes helt inn i vertshodet etter transplantasjon, som vist i figur 3A, og har en glass-broen hensiktsmessig plassert på embryo ansikt, som vist i figur 2BC. Den transplanterte donorvevet må være riktig dimensjonert for verten åpning, for transplantasjon for å være vellykket. Den EAD vevet bør ikke stikker ut fra hodet, i en hvilken som helst måte, som vist i figurene 3B og 3C. I tillegg bør ansikt transplantasjon ikke r...

Watch this Scientific Journal Video about Facial Transplants in Xenopus laevis Embryos at JoVE.com

Facial Transplants in Xenopus laevis Embryos

Ansikts transplantasjoner i<em&gt; Xenopus laevis</em&gt; Embryo

En teknikk for å transplantere &quot;Extreme Anterior Domain&quot; ansikts vev mellom Xenopus laevis embryoer har blitt utviklet. Vev kan flyttes fra en genekspresjon bakgrunn inn i en annen, slik at undersøkelse av lokale krav til kraniofacial utvikling og for signalisering interaksjoner mellom ansikts regioner.

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Research

JoVE Journal

Biology

Facial Transplants in Xenopus laevis Embryos

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Obtaining Eggs from Xenopus laevis Females

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

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

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 ...