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

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

NOTE: The entire procedure was conducted in accordance with the guidelines of the National Institutes of Health, as approved by the Animal Care and Use Committee of the University of Illinois at Chicago.
1. Solutions and Culture Media Preparations
<ol>
	<li>Keep all the solutions at -20 &#176;C for up to six months. Keep the culture media at 4 &#176;C for up to 3 months.</li>
	<li>Prepare 100 ml of culture media by mixing 1 ml of pen-strep solution and 1 ml of ITSX (Insulin-Transferrin-Selen...

<ol>
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A., Lindau, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exocytotic+catecholamine+release+is+not+associated+with+cation+flux+through+channels+in+the+vesicle+membrane+but+Na++influx+through+the+fusion+pore.">Exocytotic catecholamine release is not associated with cation flux through channels in the vesicle membrane but Na+ influx through the fusion pore.</a> Nature Cell Biology. 9, 915-922 (2007).</li><li>Gong, L. 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Cell-attached capacitance measurements require several critical steps in order to successfully obtain recordings with high quality: 1) viable and healthy cells prepared from adrenal glands; 2) PDL coating of the coverslips; 3) gigaohm seal formation; 4) noise level of the system; and 5) phase correction.
For animal surgery, modifications one can potentially make is to adjust the surgical approach to best suit dexterity and to prevent damage during dissection. Additionally, sufficient practice ...

Synaptic transmission is mediated by exocytosis of neurotransmitter-containing synaptic vesicles, and these vesicles must undergo local endocytic recycling within the nerve terminal to maintain neuronal communication in the long term. Given the essential role of synaptic transmission in the brain, understanding the molecular machinery that constitutes the synaptic vesicle cycle is an essential foundation towards a better comprehension in cellular communication as a whole. Among cell model systems, the adrenal chromaffin cell has provided some of the most definitive insight into the molecular machinery underlying synaptic vesicle recycling. Exocytosis, the final step in neurotransmitter release, has been immensely studied and examined through the use of the adrenal chromaffin cell1,2. In fact, most of the molecular players that orchestrate the formation, targeting, docking, and fusion of secretory granules have been identified due to application of diverse techniques in chromaffin cells1. Furthermore, by providing an opportunity to allow for single-vesicle resolution of the protein machinery involved in exocytosis, the chromaffin cell remains a powerful model to address the questions of vesicle fusion3.
Cell-attached capacitance measurements were first utilized in resolving single-vesicle fusion during exocytosis3. Exocytosis of vesicles as small as ~60 nm in diameter have been demonstrated to be detected by cell membrane admittance measurements with the patch clamp technique in the cell attached configuration4-7. Admittance is defined as a measure of how easily a circuit or device will allow a current to flow; it is the inverse of impedance. Thus, admittance measurements provide an understanding of the membrane capacitance. This is accomplished by the incorporation of the vesicular membrane into the plasma membrane; this incorporation reveals changes in surface area8. Each fusing vesicle causes a stepwise increase in membrane capacitance9,10. Additionally, this admittance measurement provides the membrane conductance and the fusion pore conductance during an exocytotic event3. As this technique has provided a unique tool at identifying single-vesicle kinetics during exocytosis, our lab has recently applied this concept to detect endocytosis of single vesicles11,12.
Our specific interest is clathrin-mediated endocytosis (CME), which has been considered as a fundamental housekeeping component in many cells13 and as a main pathway for synaptic vesicle endocytosis in neuronal terminals14,15. CME is known to be biologically important, however, its kinetics remain not well understood due to technical limitations in monitoring singular endocytic events. Given the similarities in exocytic mechanisms between chromaffin cells and neurons1, it is plausible that the fission mechanisms in chromaffin cells may likely apply to synaptic vesicle endocytosis in neurons. The cell-attached capacitance measurements have been utilized to monitor individual endocytic events and analyze the fission kinetics, which most methods are unable to resolve. In our cell-attached recordings, a sine wave at 20 kHz is superimposed over the holding potential, and the output current is separated into membrane conductance in one channel and membrane capacitance in the other channel from a two-phase lock-in amplifier16-18. From the changes in the membrane conductance and capacitance, one can calculate the kinetics of the fission-pore, which likely corresponds to the tubular membrane neck that connects the internalizing vesicle to the plasma membrane prior to vesicle pinch-off. Collectively, this technique gives us the opportunity to examine the regulatory mechanisms of vesicle fission during CME.

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			<td height="61" style="height:61px;width:88px;">Name of Material/ Equipment</td>
			<td style="width:88px;">Company</td>
			<td style="width:88px;">Catalog Number</td>
			<td style="width:88px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="43" rowspan="2" style="height:43px;width:88px;">Poly-D-Lysine</td>
			<td rowspan="2" style="width:88px;">Sigma</td>
			<td rowspan="2" style="width:88px;">P0899</td>
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		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:88px;">DMEM&#160;&#160;</td>
			<td style="width:88px;"></td>
			<td rowspan="2" style="width:88px;">15066024</td>
			<td rowspan="2" style="width:88px;">Keep out of UV</td>
		</tr>
		<tr height="81">
			<td height="81" style="height:81px;width:88px;">Dulbecco&#8217;s Modified Eagle Medium</td>
			<td style="width:88px;">Life Technologies</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Cover Glass</td>
			<td style="width:88px;">Carolina Biological</td>
			<td style="width:88px;">633029</td>
			<td style="width:88px;">12mm</td>
		</tr>
		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">Penicillin Streptomycin</td>
			<td rowspan="2" style="width:88px;">Life Technologies</td>
			<td rowspan="2" style="width:88px;">15140122</td>
			<td rowspan="2" style="width:88px;">100mL</td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Insulin-Trans-Sel-X</td>
			<td style="width:88px;">Life Technologies</td>
			<td style="width:88px;">51500056</td>
			<td style="width:88px;">Only thaw on ICE!</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:88px;">Papain</td>
			<td style="width:88px;">Worthington</td>
			<td style="width:88px;">39S11614</td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="34">
			<td height="56" rowspan="2" style="height:57px;width:88px;">EPC-7 plus patch amplifier</td>
			<td rowspan="2" style="width:88px;">HEKA</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BNC-2090 data acquisition board</td>
			<td rowspan="2" style="width:88px;">National Instruments</td>
			<td rowspan="2" style="width:88px;"></td>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;"></td>
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		<tr height="61">
			<td height="61" style="height:61px;width:88px;">Igor data acquisition software</td>
			<td style="width:88px;">Wavemetrics</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">P-97 pipette puller</td>
			<td style="width:88px;">Sutter Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Microforge</td>
			<td style="width:88px;">Scientific Instruments</td>
			<td style="width:88px;"></td>
			<td style="width:88px;"></td>
		</tr>
		<tr height="60">
			<td height="161" rowspan="3" style="height:161px;width:88px;">Borosilicate glass capillaries</td>
			<td rowspan="3" style="width:88px;">Sutter Instruments</td>
			<td rowspan="3" style="width:88px;">B150-110-10</td>
			<td style="width:88px;">Outer diameter-1.5 mm</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:88px;">Inner diameter 1.10 mm</td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:88px;">Length- 10 cm</td>
		</tr>
	</tbody>
</table>

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

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

The cell viability and the quality of gigaohm seal are critical in determining the quality of the cell-attached capacitance recordings. Therefore, it is critical to procure an effective and efficient cell culture prior to electrophysiological recordings, and typical viable cells are illustrated in Figure 1. Practice and time will be helpful in achieving a gigaohm seal with high quality. If one can clearly see cell deformation when the patch pipette is approaching the cell as described in protocol Step 5....

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

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

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

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

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