Tutti i membri della Washington University di St. Louis Cardiac molecolare Engineering Lab. Un Burroughs Welcome Fondo Premio alla Carriera al Interface scientifico - 1010299 (JS).

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L&#39;ovocita vaselina tensione gap clamp cut-open consente una rapida risoluzione dei dati, a basso rumore, aumentato il controllo sulla soluzione interna e la composizione soluzione esterna, e registrazioni stabili nel relativamente lunghi protocolli 19. Questi vantaggi impostate questa tecnica a parte il voltage clamp a due elettrodi standard e le tecniche di patch clamp. Sebbene attrezzature specializzate è necessaria e il protocollo è relativamente difficile, molto pochi problemi si verificano quando i...

Tecniche tensione di serraggio specializzati permettono la registrazione di correnti ioniche a potenziali di membrana controllate. Ampiamente usato voltage clamp a due elettrodi (TEVC) e tecniche di patch clamp forniscono informazioni elettrofisiologiche attendibili sulle proprietà di molti canali ionici. Tuttavia, entrambi questi metodi hanno inconvenienti che impediscono l&#39;acquisizione di dati affidabili per i canali del sodio voltaggio-dipendenti veloci e altri canali attivanti veloce nelle membrane come quelle di ovociti di Xenopus. I laboratori Bezanilla e Stefani quindi sviluppato la tecnica del clamp di tensione gap vaselina cut-open (COVG) per ovociti 2. La tecnica è stata ampiamente applicata per registrare, Na +, K + e Ca 2 + canali 3-8. Durante la registrazione COVG, una membrana dell&#39;oocita-proteina eterologa esprimere è diviso in tre regioni. I dati corrente ionica viene registrata dalla regione superiore del ovocita comebagno circonda la regione superiore è fissata ad un potenziale di comando, che può essere cambiata facilmente e rapidamente. Le guardie regione del Medio contro le correnti di dispersione di essere bloccato allo stesso potenziale come la regione top 9. La regione inferiore è dove apertura ovocita (cut-open) avviene mediante l&#39;utilizzo di una soluzione di saponina o una cannula. Chimica o apertura manuale della membrana nella regione inferiore permette il controllo del potenziale interno, che viene fissato al terreno, e rende l&#39;interno della cellula contigua con la soluzione camera inferiore. Perfusione di soluzioni nella camera inferiore può regolare le proprietà dell&#39;ambiente interno, mentre lo scambio di soluzione nella camera superiore altera l&#39;ambiente esterno. <img alt="Figura 1" fo:content-width="5in" fo:src="/files/ftp_upload/51040/51040fig1highres.jpg" src="/files/ftp_upload/51040/51040fig1.jpg" width="500px" /> Figura 1. Ovociti Cut-Open Voltage-Clamp Bath Diagramma di installazione. (A) Topdown vista di tre bagni separati l&#39;uno dall&#39;altro. Le dimensioni delle camere per COVG vengono visualizzati sulla figura. (B) Vista laterale della configurazione bagni in posizione di prova. <a href="http://www.jove.com/files/ftp_upload/51040/51040fig1highres.jpg" target="_blank">Clicca qui per vedere l&#39;immagine ingrandita</a> . I vantaggi della tecnica COVG includono basso rumore corrente (1 nA a 3 kHz), il controllo della composizione ionica del supporto esterno, la capacità di modulare il supporto interno, risoluzione temporale veloce (20-100 costante msec tempo di decadimento della capacità transitorio), e le registrazioni stabili per diverse ore 9. Gli svantaggi sono che richiede attrezzature specializzate ed è più difficile da eseguire rispetto a due serraggio tensione elettrodo (TEVC) 10. Mentre l&#39;approccio COVG richiede attrezzature altamente specializzate e elementi procedurali complessi, può consentire l&#39;acquisizione di Valudati elettrofisiologici grado. Questi dati, come gating correnti con cinetica veloce e correnti di coda 4, può essere registrata senza alcuni dei problemi associati con altri protocolli tensione di chiusura comprese canale run-down. Piccole modifiche alla configurazione COVG possono consentire l&#39;uso di regolatori di temperatura e morsetto di tensione fluorometria (VCF). L&#39;inclusione di tensione morsetto elementi fluorometria all&#39;interno dell&#39;assembly COVG può aumentare output di dati conferendo la possibilità di monitorare cambiamenti conformazionali proteiche e contemporaneamente la registrazione corrente 11-13.

<table border="0" cellpadding="0" cellspacing="0" width="515">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">External Solution</td>
			<td style="width:131px;">Brand</td>
			<td style="width:72px;">Catalog Number</td>
			<td style="width:124px;">[Final], weight, or volume</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">N-methyl-D-glucamine (NMDG)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M2004</td>
			<td style="width:124px;">25mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">MES Sodium Salt</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M5057</td>
			<td style="width:124px;">90mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">20mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Calcium hydroxide</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">239232</td>
			<td style="width:124px;">2mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">MES Hydrate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8250</td>
			<td style="width:124px;">variable (pH to 7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Internal Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">N-methyl-D-glucamine (NMDG)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M2004</td>
			<td style="width:124px;">105mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">MES Sodium Salt</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M5057</td>
			<td style="width:124px;">10mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">20mM</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:189px;">Ethylene glycol-bis(2-aminoethylether)-N,N,N&#39;,N&#39;-tetraacetic acid (EGTA)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">E4378</td>
			<td style="width:124px;">2mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">MES Hydrate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8250</td>
			<td style="width:124px;">variable (pH to 7.4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Depolarizing Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">KCl</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">221473</td>
			<td style="width:124px;">110mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Magnesium chloride</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8266</td>
			<td style="width:124px;">1.5mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Calcium Chloride</td>
			<td style="width:131px;">Caisson</td>
			<td style="width:72px;">C021</td>
			<td style="width:124px;">0.8mM</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">10mM</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Pipet Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">KCl</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">221473</td>
			<td style="width:124px;">3M</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:189px;">Saponin Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Saponin</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">47036</td>
			<td style="width:124px;">0.125g</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Internal Solution</td>
			<td style="width:131px;">See above</td>
			<td style="width:72px;"></td>
			<td style="width:124px;">50mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Agar Bridge Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">N-methyl-D-glucamine (NMDG)</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M2004</td>
			<td style="width:124px;">100ml of 1M</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">HEPES</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">H75030</td>
			<td style="width:124px;">1.2g</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">MES Hydrate</td>
			<td style="width:131px;">Sigma-Aldrich</td>
			<td style="width:72px;">M8250</td>
			<td style="width:124px;">variable (pH to 7.4)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">Granulated Agar</td>
			<td style="width:131px;">Research Products International</td>
			<td style="width:72px;">A20250</td>
			<td align="right" style="width:124px;">3%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;"></td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">NMDG Storage Solution</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:189px;">NMDG, HEPES, MES Hydrate solution</td>
			<td style="width:131px;">see above</td>
			<td style="width:72px;"></td>
			<td style="width:124px;">40ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:189px;">Water</td>
			<td style="width:131px;"></td>
			<td style="width:72px;"></td>
			<td style="width:124px;">60ml</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:144px;">Catalog Number</td>
			<td style="width:285px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">High Performance Oocyte Clamp</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">CA-1B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Data Acquisition System</td>
			<td style="width:148px;">Axon CNS</td>
			<td style="width:144px;">&#160;Digidata 1440A</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Oscilloscope</td>
			<td style="width:148px;">Tektronix&#160;</td>
			<td style="width:144px;">TDS 210</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Rack Power Filter</td>
			<td style="width:148px;">APC&#160;</td>
			<td style="width:144px;">G5</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">Heating/Cooling Bath Temperature Controller</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">HCC-100A</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">PC</td>
			<td style="width:148px;">Dell</td>
			<td style="width:144px;">Optiplex 990</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">pCLAMP 10.3 Voltage Clamp Software</td>
			<td style="width:148px;">Molecular Devices, LLC</td>
			<td style="width:144px;">pCLAMP10.3</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">TMC Vibration Control TableTop Platform</td>
			<td style="width:148px;">TMC</td>
			<td style="width:144px;">64 SERIES</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">TMC Vibration Control Air Table</td>
			<td style="width:148px;">TMC</td>
			<td style="width:144px;"><a name="RANGE!C10">20 Series&#160;</a></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">V1/I Electrode Data Collector</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">part of CA-1B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">MX10L Micromanipulator</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:144px;">MX10L</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Bath/Guard (I/V) Headstage (with appropriate connectors)</td>
			<td style="width:148px;">Dagan</td>
			<td style="width:144px;">part of CA-1B</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Microscope</td>
			<td style="width:148px;">Omano</td>
			<td style="width:144px;">OM2300S-JW11</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Temperature Control Bath</td>
			<td style="width:148px;">Custom or Dagan</td>
			<td style="width:144px;">Custom or HE-204C</td>
			<td style="width:285px;">Custom chamber made from materials from Cool Polymers (D-series). Dagan also provides a prefeabricated stage (HE-204C).</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Custom AgCl Pellet Container</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;">Custom</td>
			<td style="width:285px;">Custom machined</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Ag/AgCl electrode, pellet, 2.0 mm</td>
			<td style="width:148px;">Warner</td>
			<td style="width:144px;">E-206</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">External Oocyte Bath</td>
			<td style="width:148px;">Custom or Dagan</td>
			<td style="width:144px;">Custom or CC-1-T-LB</td>
			<td style="width:285px;">Custom machined or purchased from Dagan</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Internal Oocyte Bath</td>
			<td style="width:148px;">Custom or Dagan</td>
			<td style="width:144px;">Custom or CC-TG-ND</td>
			<td style="width:285px;">Custom machined or purchased from Dagan</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">Capillaries for Agar Bridges and Pulled Electrodes</td>
			<td style="width:148px;">Warner</td>
			<td style="width:144px;">G150T-4</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Rotatable Mounts for the Microscope, Micromanipulator, and Bath</td>
			<td style="width:148px;">Siskiyou</td>
			<td style="width:144px;">SD-1280P</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Fiber-Lite</td>
			<td style="width:148px;">Dolan-Jenner</td>
			<td style="width:144px;">LMI-600</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Regular Bleach</td>
			<td style="width:148px;">Clorox</td>
			<td style="width:144px;">470174-764</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Xenopus laevis Oocytes</td>
			<td style="width:148px;">Nasco</td>
			<td style="width:144px;">LM535M (sexually mature females)</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:149px;">90 Na+ External Solution</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:149px;">10 Na+ Internal Solution</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">3 M KCL</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Saponin</td>
			<td style="width:148px;">Sigma-Aldrich</td>
			<td style="width:144px;">47036</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">NMDG Storage Solution</td>
			<td style="width:148px;">See Solutions sheet</td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">5mL transfer pipets</td>
			<td style="width:148px;">SciMart</td>
			<td style="width:144px;">GS-52</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">Modified KCl electrode injector</td>
			<td style="width:148px;">BD</td>
			<td style="width:144px;">309659</td>
			<td style="width:285px;">Plastic syringe tip melted to allow for injection of solution into electrodes. Alternatively, a Microfil by WPI can be purchased.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Microvaccum</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;">Custom</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">Forceps</td>
			<td style="width:148px;">VWR</td>
			<td style="width:144px;">63040-458</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Oocyte Handling Tools (Pipette Pump)</td>
			<td style="width:148px;">VWR</td>
			<td style="width:144px;">53502-222</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Deionized Water Squirt Bottle</td>
			<td style="width:148px;">VWR</td>
			<td style="width:144px;">16649-911</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Vaseline Petroleum Jelly</td>
			<td style="width:148px;">Fisher Scientific</td>
			<td style="width:144px;">19-086-291&#160;</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;"></td>
			<td style="width:148px;"></td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:149px;">Additional Materials Required for VCF Recordings:</td>
			<td style="width:148px;"></td>
			<td style="width:144px;"></td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:149px;">VCF Microscope</td>
			<td style="width:148px;">Nikon</td>
			<td style="width:144px;">Eclipse FN1</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Nikon CFI APO 40XW NIR Objective</td>
			<td style="width:148px;">Nikon</td>
			<td style="width:144px;">N40X-NIR</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:149px;">X-Y Translator System for Fixed-Stage Upright Microscopes</td>
			<td style="width:148px;">Sutter Instruments</td>
			<td style="width:144px;">MT500-586</td>
			<td style="width:285px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">External VCF Oocyte Bath</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;"></td>
			<td style="width:285px;">Custom machined. The chamber dimensions are 2.7 x 1.9 x 0.4 cm.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:149px;">Internal VCF Oocyte Bath</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;"></td>
			<td style="width:285px;">Custom machined. The chamber dimensions are 1.6 x 1.6 x 0.4 cm.</td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:149px;">Modified Temperature Control Bath</td>
			<td style="width:148px;">Custom</td>
			<td style="width:144px;"></td>
			<td style="width:285px;">Custom chamber made from materials from Cool Polymers (D-series). The chamber dimensions of the modified temperature controller bath are 2.7 x 1.9 x 0.3 cm for the horizontal chamber, and 1 x 2.5 x 0.5 cm for the vertical chamber.</td>
		</tr>
	</tbody>
</table>

the xenopus oocyte cut-open vaseline gap voltage-clamp technique with fluorometry

L&#39;ovocita vaselina gap cut-open (COVG) clamp di tensione permette l&#39;analisi delle proprietà elettrofisiologiche e cinetiche dei canali ionici eterologa in oociti. Registrazioni dalla configurazione cut-open sono particolarmente utili per risolvere basse correnti di magnitudo gating, rapida attivazione corrente ionica e disattivazione. I principali vantaggi rispetto al morsetto di tensione a due elettrodi (TEVC) tecnica includono maggiore velocità morsetto, miglior rapporto segnale-rumore, e la capacità di modulare la intracellulare ed extracellulare. Qui, impieghiamo canale del sodio cardiaco umano (HNA 1,5 V), espresso in oociti di Xenopus, per dimostrare la configurazione e protocollo cut-open nonché modifiche necessarie per aggiungere capacità fluorometria morsetto di tensione. Le proprietà dei canali ionici veloci attivanti, come HNA 1,5 V, non possono essere completamente risolti a temperatura ambiente usando TEVC, in which la totalità della membrana dell&#39;oocita è serrato, rendendo difficile il controllo della tensione. Tuttavia, nella tecnica cut-aperto, separazione di solo una piccola porzione della membrana cellulare consente il bloccaggio rapido necessaria per registrare accuratamente cinetica veloce evitando canale run-down associato a tecniche di patch clamp. In concomitanza con la tecnica COVG, cinetica del canale ionico e proprietà elettrofisiologiche possono essere ulteriormente analizzati mediante morsetto fluorometria tensione, in cui il movimento proteina viene monitorato tramite cisteina coniugazione di fluorofori applicate extracellulare, inserimento di proteine ​​fluorescenti geneticamente codificati, o l&#39;incorporazione di amminoacidi non naturali nella regione di interesse 1. Questi dati aggiuntivi produce informazioni cinetiche su voltaggio-dipendenti riarrangiamenti conformazionali della proteina attraverso cambiamenti nel microambiente circostante la molecola fluorescente.

Figura 4 mostra la variazione di permeabilità del ovocita come soluzione saponina viene applicata alla parte inferiore della ovocita. Figura 5 mostra il tasso di conversione soluzione intracellulare per diffusione seguente permeabilizzazione saponina. 20-40 min sono tenuti a venire a uno stato stazionario 2,18. Figura 6A mostra tracce generate dal protocollo di registrazione. La figura mostra correnti ioniche (dopo P/-8 perdita s...

Watch this Scientific Journal Video about The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry at JoVE.com

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

L&#39;approccio gap vaselina cut-aperto viene utilizzato per ottenere bassi registrazioni sonore di correnti ioniche e gating di canali ionici voltaggio-dipendenti espressi in oociti di Xenopus con alta risoluzione di canale cinetiche veloci. Con piccola modifica, morsetto di tensione fluorometria può essere accoppiato al protocollo ovocita cut-open.

Il<em&gt; Xenopus</em&gt; Ovociti Cut-open vaselina Gap Tecnica Voltage-clamp Con fluorometria

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion ...

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Unit 1

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

Root-Locus Method

SCIENTISTS IN ACTION

18.3K Views.  Washington University in St. Louis.  L'approccio gap vaselina cut-aperto viene utilizzato per ottenere bassi registrazioni sonore di correnti ioniche e gating di canali ionici voltaggio-dipendenti espressi in oociti di Xenopus con alta risoluzione di canale cinetiche veloci. Con piccola modifica, morsetto di tensione fluorometria può essere accoppiato al protocollo ovocita cut-open. 

Video: The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

Microinjection of Xenopus Laevis Oocytes

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic transport. Because of its large nucleus and high density of nuclear pore complexes (NPCs), nuclear transport can be easily visualized in the Xenopus oocyte. Much insight into the mechanisms of nuclear import and export has been gained through use of this system (reviewed by Pant&eacute;, 2006). In addition, we have used microinjection of Xenopus oocytes to dissect the nuclear import pathways of several viruses that replicate in the host nucleus. 
Here we demonstrate the cytoplasmic microinjection of Xenopus oocytes with a nuclear import substrate. We also show preparation of the injected oocytes for visualization by thin-sectioning EM, including dissection, dehydration, and embedding of the oocytes into an epoxy embedding resin. Finally, we provide representative results for oocytes that have been microinjected with the capsid of the baculovirus Autographa californica nucleopolyhedrovirus (AcMNPV) or the parvovirus Minute Virus of Mice (MVM), and discuss potential applications of the technique.

Here we demonstrate cytoplasmic microinjection of Xenopus laevis oocytes with a nuclear import substrate, as well as preparation of the injected oocytes for visualization by thin-sectioning electron microscopy.

Microiniezione degli ovociti di Xenopus Laevis

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

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Biologia

Fertilization of Xenopus oocytes using the Host Transfer Method

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to generate maternal-effect mutant animals. Additionally, many of the techniques to overexpress or inhibit gene function in organisms such as Xenopus and zebrafish fail to sufficiently target critical maternal signaling pathways, such as Wnt signaling. In Xenopus, manipulating gene function in cultured oocytes and subsequently fertilizing them can ameliorate these problems to some extent. Oocytes are manually defolliculated from donor ovary tissue, injected or treated in culture as desired, and then stimulated with progesterone to induce maturation. Next, the oocytes are introduced into the body cavity of an ovulating host female frog, whereupon they will be translocated through the host's oviduct and acquire modifications and jelly coats necessary for fertilization. The resulting embryos can then be raised to the desired stage and analyzed for the effects of any experimental perturbations. This host-transfer method has been highly effective in uncovering basic mechanisms of early development and allows a wide range of experimental possibilities not available in any other vertebrate model organism.

Fertilization of Xenopus oocytes using the Host Transfer Method

Procedure for fertilizing Xenopus oocytes by the host transfer method.

Fertilizzazione Ovociti di Xenopus Utilizzando il metodo di trasferimento Host

Studying the contribution of maternally inherited molecules to vertebrate early development is often hampered by the time and expense necessary to ...

Mouse Oocyte Microinjection, Maturation and Ploidy Assessment

Mistakes in chromosome segregation lead to aneuploid cells. In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to infertility, miscarriages or developmental disorders like Down syndrome. Haploid gametes form through species-specific developmental programs that are coupled to meiosis. The first meiotic division (MI) is unique to meiosis because sister chromatids remain attached while homologous chromosomes are segregated. For reasons not fully understood, this reductional division is prone to errors and is more commonly the source of aneuploidy than errors in meiosis II (MII) or than errors in male meiosis 1,2. 
In mammals, oocytes arrest at prophase of MI with a large, intact germinal vesicle (GV; nucleus) and only resume meiosis when they receive ovulatory cues. Once meiosis resumes, oocytes complete MI and undergo an asymmetric cell division, arresting again at metaphase of MII. Eggs will not complete MII until they are fertilized by sperm. Oocytes also can undergo meiotic maturation using established in vitro culture conditions 3. Because generation of transgenic and gene-targeted mouse mutants is costly and can take long periods of time, manipulation of female gametes in vitro is a more economical and time-saving strategy. 
Here, we describe methods to isolate prophase-arrested oocytes from mice and for microinjection. Any material of choice may be introduced into the oocyte, but because meiotically-competent oocytes are transcriptionally silent 4,5 cRNA, and not DNA, must be injected for ectopic expression studies. To assess ploidy, we describe our conditions for in vitro maturation of oocytes to MII eggs. Historically, chromosome-spreading techniques are used for counting chromosome number 6. This method is technically challenging and is limited to only identifying hyperploidies. Here, we describe a method to determine hypo-and hyperploidies using intact eggs 7-8. This method uses monastrol, a kinesin-5 inhibitor, that collapses the bipolar spindle into a monopolar spindle 9 thus separating chromosomes such that individual kinetochores can readily be detected and counted by using an anti-CREST autoimmune serum. Because this method is performed in intact eggs, chromosomes are not lost due to operator error.

Oocytes are prone to aneuploidy due to errors in chromosome segregation during meiotic maturation. Aneuploid eggs can cause infertility, miscarriages or developmental disorders like Down syndrome. Here, we describe methods to introduce materials of choice into oocytes and methods to study meiotic maturation and assess ploidy.

Microiniezione degli ovociti mouse, Maturazione e Ploidia valutazione

Mistakes in chromosome segregation lead to aneuploid cells.  In somatic cells, aneuploidy is associated with cancer but in gametes, aneuploidy leads to ...

Single Oocyte Bisulfite Mutagenesis

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for regulating gene transcription1. DNA methylation is an epigenetic modification that acts predominantly as a repressive mark. Through the covalent addition of a methyl group onto cytosines in CpG dinucleotides, it can recruit additional repressive proteins and histone modifications to initiate processes involved in condensing chromatin and silencing genes2. DNA methylation is essential for normal development as it plays a critical role in developmental programming, cell differentiation, repression of retroviral elements, X-chromosome inactivation and genomic imprinting.
One of the most powerful methods for DNA methylation analysis is bisulfite mutagenesis. Sodium bisulfite is a DNA mutagen that deaminates cytosines into uracils. Following PCR amplification and sequencing, these conversion events are detected as thymines. Methylated cytosines are protected from deamination and thus remain as cytosines, enabling identification of DNA methylation at the individual nucleotide level3. Development of the bisulfite mutagenesis assay has advanced from those originally reported4-6 towards ones that are more sensitive and reproducible7. One key advancement was embedding smaller amounts of DNA in an agarose bead, thereby protecting DNA from the harsh bisulfite treatment8. This enabled methylation analysis to be performed on pools of oocytes and blastocyst-stage embryos9. The most sophisticated bisulfite mutagenesis protocol to date is for individual blastocyst-stage embryos10. However, since blastocysts have on average 64 cells (containing 120-720 pg of genomic DNA), this method is not efficacious for methylation studies on individual oocytes or cleavage-stage embryos.
Taking clues from agarose embedding of minute DNA amounts including oocytes11, here we present a method whereby oocytes are directly embedded in an agarose and lysis solution bead immediately following retrieval and removal of the zona pellucida from the oocyte. This enables us to bypass the two main challenges of single oocyte bisulfite mutagenesis: protecting a minute amount of DNA from degradation, and subsequent loss during the numerous protocol steps. Importantly, as data are obtained from single oocytes, the issue of PCR bias within pools is eliminated. Furthermore, inadvertent cumulus cell contamination is detectable by this method since any sample with more than one methylation pattern may be excluded from analysis12. This protocol provides an improved method for successful and reproducible analyses of DNA methylation at the single-cell level and is ideally suited for individual oocytes as well as cleavage-stage embryos.

Bisulfite mutagenesis is the gold standard for analyzing DNA methylation. Our modified protocol allows for DNA methylation analysis at the single-cell level and was specifically designed for individual oocytes. It can also be used for cleavage-stage embryos.

Singolo ovociti Mutagenesi bisolfito

Epigenetics encompasses all heritable and reversible modifications to chromatin that alter gene accessibility, and thus are the primary mechanisms for ...

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage checkpoint signaling as a surveillance mechanism to sense DNA damage and direct cellular responses to DNA damage. There are several groups of proteins called sensors, transducers and effectors involved in DNA damage checkpoint signaling (Figure 1). In this complex signaling pathway, ATR (ATM and Rad3-related) is one of the major kinases that can respond to DNA damage and replication stress. Activated ATR can phosphorylate its downstream substrates such as Chk1 (Checkpoint kinase 1). Consequently, phosphorylated and activated Chk1 leads to many downstream effects in the DNA damage checkpoint including cell cycle arrest, transcription activation, DNA damage repair, and apoptosis or senescence (Figure 1). When DNA is damaged, failing to activate the DNA damage checkpoint results in unrepaired damage and, subsequently, genomic instability. The study of the DNA damage checkpoint will elucidate how cells maintain genomic integrity and provide a better understanding of how human diseases, such as cancer, develop.
Xenopus laevis egg extracts are emerging as a powerful cell-free extract model system in DNA damage checkpoint research. Low-speed extract (LSE) was initially described by the Masui group1. The addition of demembranated sperm chromatin to LSE results in nuclei formation where DNA is replicated in a semiconservative fashion once per cell cycle.
The ATR/Chk1-mediated checkpoint signaling pathway is triggered by DNA damage or replication stress 2. Two methods are currently used to induce the DNA damage checkpoint: DNA damaging approaches and DNA damage-mimicking structures 3. DNA damage can be induced by ultraviolet (UV) irradiation, &gamma;-irradiation, methyl methanesulfonate (MMS), mitomycin C (MMC), 4-nitroquinoline-1-oxide (4-NQO), or aphidicolin3, 4. MMS is an alkylating agent that inhibits DNA replication and activates the ATR/Chk1-mediated DNA damage checkpoint 4-7. UV irradiation also triggers the ATR/Chk1-dependent DNA damage checkpoint 8. The DNA damage-mimicking structure AT70 is an annealed complex of two oligonucleotides poly-(dA)70 and poly-(dT)70. The AT70 system was developed in Bill Dunphy's laboratory and is widely used to induce ATR/Chk1 checkpoint signaling 9-12.
Here, we describe protocols (1) to prepare cell-free egg extracts (LSE), (2) to treat Xenopus sperm chromatin with two different DNA damaging approaches (MMS and UV), (3) to prepare the DNA damage-mimicking structure AT70, and (4) to trigger the ATR/Chk1-mediated DNA damage checkpoint in LSE with damaged sperm chromatin or a DNA damage-mimicking structure.

Study of the DNA Damage Checkpoint using Xenopus Egg Extracts

Xenopus egg extract is a useful model system to investigate the DNA damage checkpoint. This protocol is for the preparation of Xenopus egg extracts and DNA damage checkpoint inducing reagents. These techniques are adaptable to a variety of DNA damaging approaches in the study of the DNA damage checkpoint signaling.

Studio del danno al DNA con Checkpoint<em&gt; Xenopus</em&gt; Uovo Estratti

On a daily basis, cells are subjected to a variety of endogenous and environmental insults. To combat these insults, cells have evolved DNA damage ...

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Spermatozoa are male reproductive cells especially designed to reach, recognize and fuse with the egg. To perform these tasks, sperm cells must be prepared to face a constantly changing environment and to overcome several physical barriers. Being in essence transcriptionally and translationally silent, these motile cells rely profoundly on diverse signaling mechanisms to orient themselves and swim in a directed fashion, and to contend with challenging environmental conditions during their journey to find the egg. In particular, Ca2+-mediated signaling is pivotal for several sperm functions: activation of motility, capacitation (a complex process that prepares sperm for the acrosome reaction) and the acrosome reaction (an exocytotic event that allows sperm-egg fusion). The use of fluorescent dyes to track intracellular fluctuations of this ion is of remarkable importance due to their ease of application, sensitivity, and versatility of detection. Using one single dye-loading protocol we utilize four different fluorometric techniques to monitor sperm Ca2+ dynamics. Each technique provides distinct information that enables spatial and/or temporal resolution, generating data both at single cell and cell population levels.

Measuring Intracellular Ca2+ Changes in Human Sperm using Four Techniques: Conventional Fluorometry, Stopped Flow Fluorometry, Flow Cytometry and Single Cell Imaging

Intracellular Ca2+ dynamics are very important in sperm physiology and Ca2+-sensitive fluorescent dyes constitute a versatile tool to study them. Population experiments (fluorometry and stopped flow fluorometry) and single cell experiments (flow cytometry and single cell imaging) are used to track spatio-temporal [Ca2+] changes in human sperm cells.

Misurare intracellulare Ca<sup&gt; 2 +</sup&gt; Modifiche di sperma umano utilizzando quattro tecniche: fluorometria convenzionale, si fermò fluorometria Flow, Citometria a flusso singolo e l&#39;imaging cellulare

Spermatozoa  are male reproductive cells especially designed to reach, recognize and fuse  with the egg. To perform these tasks, sperm cells must be ...

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including by mechanical forces. Certain TRPV4 mutations cause complex hereditary bone or neuronal pathologies in human. Wild-type or mutant TRPV4 transgenes are commonly expressed in cultured mammalian cells and examined by Fura-2 fluorometry and by electrodes. In terms of the mechanism of mechanosensitivity and the molecular bases of the diseases, the current literature is confusing and controversial. To complement existing methods, we describe two additional methods to examine the molecular properties of TRPV4. (1) Rat TRPV4 and an aequorin transgene are transformed into budding yeast. A hypo-osmtic shock of the transformant population yields a luminometric signal due to the combination of aequorin with Ca2+, released through the TRPV4 channel. Here TRPV4 is isolated from its usual mammalian partner proteins and reveals its own mechanosensitivity. (2) cRNA of TRPV4 is injected into Xenopus oocytes. After a suitable period of incubation, the macroscopic TRPV4 current is examined with a two-electrode voltage clamp. The current rise upon removal of inert osmoticum from the oocyte bath is indicative of mechanosensitivity. The microAmpere (10-6 to 10-4 A) currents from oocytes are much larger than the subnano- to nanoAmpere (10-10 to 10-9 A) currents from cultured cells, yielding clearer quantifications and more confident assessments. Microscopic currents reflecting the activities of individual channel proteins can also be directly registered under a patch clamp, in on-cell or excised mode. The same oocyte provides multiple patch samples, allowing better data replication. Suctions applied to the patches can activate TRPV4 to directly assess mechanosensitivity. These methods should also be useful in the study of other types of TRP channels.

Yeast Luminometric and Xenopus Oocyte Electrophysiological Examinations of the Molecular Mechanosensitivity of TRPV4

To complement commonly used methods to study TRPV4&#8217;s, two methods are described: Its mechanosensitivity can be studied by Ca2+-aequorin luminometry in transgenic yeast upon hypo-osmotic challenge. It can also be examined in TRPV4-RNA injected Xenopus oocytes by whole-cell two-electrode voltage clamp or patch clamp in on-cell or excised mode.

Lievito luminometrici e<em&gt; Xenopus</em&gt; ovocita elettrofisiologiche Esami del mechanosensitivity molecolare del TRPV4

TRPV4 (Transient Receptor Potentials, vanilloid family, type 4) is widely expressed in vertebrate tissues and is activated by several stimuli, including ...

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The initiation step, which involves the assembly of the ribosomal subunits at the mRNA initiation codon, involved initiation factor including eIF4G1. Defects in this rate limiting step of translation are linked to diverse disorders. To study the potential consequences of such deregulations, Xenopus laevis oocytes constitute an attractive model with high degrees of conservation of essential cellular and molecular mechanisms with human. In addition, during meiotic maturation, oocytes are transcriptionally repressed and all necessary proteins are translated from preexisting, maternally derived mRNAs. This inexpensive model enables exogenous mRNA to become perfectly integrated with an effective translation. Here is described a protocol for assessing translation with a factor of interest (here eIF4G1) using stored maternal mRNA that are the first to be polyadenylated and translated during oocyte maturation as a physiological readout. At first, mRNA synthetized by in vitro transcription of plasmids of interest (here eIF4G1) are injected in oocytes and kinetics of oocyte maturation by Germinal Vesicle Breakdown detection is determined. The studied maternal mRNA target is the serine/threonine-protein-kinase mos. Its polyadenylation and its subsequent translation are investigated together with the expression and phosphorylation of proteins of the mos signaling cascade involved in oocyte maturation. Variations of the current protocol to put forward translational defects are also proposed to emphasize its general applicability. In light of emerging evidence that aberrant protein synthesis may be involved in the pathogenesis of neurological disorders, such a model provides the opportunity to easily assess this impairment and identify new targets.

Xenopus laevis as a Model to Identify Translation Impairment

Protein synthesis control occurs mainly at the translation initiation step, deficiencies in which are linked to diverse disorders. To better understand their etiology, we described here a protocol using Xenopus laevis oocytes assessing the translation of mos transcript in the presence of a mutant of translation initiation factor eIF4G1.

Xenopus laevis come un modello per individuare Traduzione Impairment

Protein synthesis is a fundamental process to gene expression impacting diverse biological processes notably adaptation to environmental conditions. The ...

Analysis of the Gap Junction-dependent Transfer of miRNA with 3D-FRAP Microscopy

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents in the treatment of several diseases. The development of new, innovative strategies for miRNA/siRNA therapy is based on an extensive knowledge of the underlying mechanisms. Recent data suggest that small RNAs are exchanged between cells in a gap junction-dependent manner, thereby inducing gene regulatory effects in the recipient cell. Molecular biological techniques and flow cytometric analysis are commonly used to study the intercellular exchange of miRNA. However, these methods do not provide high temporal resolution, which is necessary when studying the gap junctional flux of molecules. Therefore, to investigate the impact of miRNA/siRNA as intercellular signaling molecules, novel tools are needed that will allow for the analysis of these small RNAs at the cellular level. The present protocol describes the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) microscopy to elucidating the gap junction-dependent exchange of miRNA molecules between cardiac cells. Importantly, this straightforward and non-invasive live-cell imaging approach allows for the visualization and quantification of the gap junctional shuttling of fluorescently labeled small RNAs in real time, with high spatio-temporal resolution. The data obtained by 3D-FRAP confirm a novel pathway of intercellular gene regulation, where small RNAs act as signaling molecules within the intercellular network.

Here, we describe the application of three-dimensional fluorescence recovery after photobleaching (3D-FRAP) for the analysis of the gap junction-dependent shuttling of miRNA. In contrast to commonly applied methods, 3D-FRAP allows for the quantification of the intercellular transfer of small RNAs in real time, with high spatio-temporal resolution.

Analisi del trasferimento di gig Junction-dependent di miRNA con la microscopia 3D-FRAP

Small antisense RNAs, like miRNA and siRNA, play an important role in cellular physiology and pathology and, moreover, can be used as therapeutic agents ...

Application of Passive Head Motion to Generate Defined Accelerations at the Heads of Rodents

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular mechanisms behind the beneficial effects of exercise are poorly understood. Many physical workouts, particularly those classified as aerobic exercises such as jogging and walking, produce impulsive forces at the time of foot contact with the ground. Therefore, it was speculated that mechanical impact might be implicated in how exercise contributes to organismal homeostasis. For testing this hypothesis on the brain, a custom-designed ''passive head motion'' (hereafter referred to as PHM) system was developed that can generate vertical accelerations with controlled and defined magnitudes and modes and reproduce mechanical stimulation that might be applied to the heads of rodents during treadmill running at moderate velocities, a typical intervention to test the effects of exercise in animals. By using this system, it was demonstrated that PHM recapitulates the serotonin (5-hydroxytryptamine, hereafter referred to as 5-HT) receptor subtype 2A (5-HT2A) signaling in the prefrontal cortex (PFC) neurons of mice. This work provides detailed protocols for applying PHM and measuring its resultant mechanical accelerations at rodents' heads.

The present protocol describes a custom-designed ''passive head motion'' system, which reproduces mechanical accelerations at rodents' heads generated during their treadmill running at moderate velocities. It allows dissecting mechanical factors/elements from the beneficial effects of physical exercise.

Applicazione del movimento passivo della testa per generare accelerazioni definite alle teste dei roditori

Exercise is widely recognized as effective for various diseases and physical disorders, including those related to brain dysfunction. However, molecular ...