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-Selenium-Supplementation) to 100 ml with DMEM.</li>
	<li>Prepare enzyme solution by mixing 250 ml of DMEM, 2.5 ml of 100 mM CaCl2, and 2.5 ml of 50 mM EDTA.</li>
	<li>Prepare inactivation solution by mixing 225 ml of DMEM, 25 ml of FBS, 625 mg of albumin, and 625 mg trypsin inhibitor.</li>
	<li>Prepare a Locke&#8217;s Solution of 154 mM NaCl, 5.6 mM KCl, 5.0 mM HEPES, 3.6 mM NaHCO3, 5.6 mM glucose. Adjust the pH to 7.3 with NaOH.</li>
	<li>Prepare a poly-d-lysine (PDL) solution of 50 mg/ml. Use a final concentration of 1 mg/ml to coat coverslips.</li>
	<li>Prepare an extracellular solution of 140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES-NaOH, and 10 mM glucose. Adjust the pH to 7.3 with NaOH with an osmolarity of ~310 nmol/kg.</li>
	<li>Prepare a pipette solution of 50 mM NaCl, 100 mM TEACl, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, and 10 mM HEPES. Adjust pH to 7.3 with NaOH with an osmolarity of ~290 nmol/kg.</li>
</ol>
2. Adrenal Gland Isolation
<ol>
	<li>Autoclave a pair of large and small dissection scissors and two pairs of forceps. Wear gloves during the whole process and place the dissecting tray on an ice bucket.</li>
	<li>Use mice taken at Day 0-3. Euthanize animals with a pressurized CO2 tank followed by decapitation.</li>
	<li>Utilize the smaller set of scissors to cut down the back of the pup following the spine from cervical to caudal region. Be sure to cut superficially under the skin and not pierce into the body cavity.</li>
	<li>Next, peel skin away from spine so that musculature is exposed to have a clear view of the spinal column. From here, make a transectional cut at the cervical spinal cord and then make two parallel cuts along the sides of the spine.</li>
	<li>With one pair of forceps holding the main body of the animal, use the other pair of forceps to grab spinal column and peel back spine till kidneys are exposed.</li>
	<li>At this moment, visualize the adrenal glands on the top of the kidneys. Then carefully place the two glands from each animal into a conical tube filled with Locke&#8217;s solution. 
	NOTE: Sometimes the glands will stick to the forceps. If this is the case, gently use another pair of forceps to aid in the removal of the glands from the forceps and into the Locke&#8217;s solution. The glands can be kept in this condition for up to 2 hr.</li>
</ol>
3. Tissue Digestion
<ol>
	<li>In the meantime, bubble the enzyme solution with 5% CO2 + 95% O2 for 15 min prior to use. Ensure that the equilibrated solution turns from a bright pink color into pinkish/orange color.</li>
	<li>Add the papain to the freshly bubbled enzyme solution at a final concentration of 20-25 unit/ml. Incubate each pair of glands from each animal for 40-60 min at 37 &#176;C in the enzyme solution with papain.</li>
	<li>Add 75% of inactivation solution to each tube and incubate at 37 &#176;C for another 10 min. This incubation inactivates the enzyme activity of papain.</li>
	<li>After the 10 min incubation with the inactivation solution, gently transfer the glands into a newly labeled tube and then wash the glands 3x with culture media.</li>
</ol>
4. Cell Dissociation and Plating
<ol>
	<li>After washing, place the two glands into 200 &#181;l of the culture media and then gently titrate the glands up and down through the pipette tip with a 200 &#181;l pipette until there is no longer a large piece of tissue in the solution. 
	NOTE: When titrating, maintain glands at the bottom of the tube with a downward force from the 200 &#181;l pipette tip and engage the tissue gently and slowly. Be certain to use slow, continuous strokes, never pipetting fast or abrupt.</li>
	<li>Add an additional 250 &#181;l culture media to bring the total volume of culture media up to 450 &#181;l.</li>
	<li>Plate 60 &#181;l of this cell-containing solution onto each of seven 12 mm PDL-pre-coated coverslips, which are placed in a 60 x 15 mm culture dish.</li>
	<li>Once plated, place the dish into the incubator that is set to 37 &#176;C and maintained with a steady flow/supply of 5% CO2 for 30 min to ensure an environment for cell viability. After incubation, gently add 5 ml of pre-warmed culture media into the culture dish and return the dish back into the incubator for 24 hr prior to cell-attached recordings. 
	NOTE: Typically, the cells can be used for electrophysiological recordings for up to 4 days.</li>
</ol>
5. Cell Identification and Gigaohm Seal Formation
<ol>
	<li>Place the 12 mm coverslip into extracellular solution. Since the whole adrenal glands are used for cell preparation, mix the chromaffin cells (typically very bright with a brownish/beige coloring and a near perfect circular form (Figure 1) in culture with cortical cells (smaller and dimmer than chromaffin cells).</li>
	<li>Pull the patch pipettes in four stages using a programmable P-97 puller. Dip the pipette tip into a melted wax, which will help to reduce the capacitance of the glass and then fire polish the tip to &#8220;blow-away&#8221; this wax from inside of the pipette tip. 
	NOTE: When filled with patch pipette solution, patch pipettes typically have a resistance of ~2 M&#937;.</li>
	<li>Approach the patch pipette close to the cell within several microns. To achieve a cell-attached configuration, look for any slight cell deformation during the approach of the patch pipette closer to the cell. Apply gentle suction until a G&#937; seal is achieved.</li>
</ol>
6. Cell-Attached Capacitance Recordings
<ol>
	<li>Once a G&#937; seal is revealed, make cell-attached recordings through the use of an EPC-7 plus patch-clamp amplifier and a two-phase analog lock-in amplifier (see attached table of equipment). Apply a sinewave with amplitude of 50 mV (r.m.s.) and frequency of 20 kHz from the lock-in amplifier.</li>
	<li>Set the output filter of the lock-in amplifier at 1 msec time constant and 24 db. Set the phase of the lock-in amplifier such that transient capacitance changes produced by gentle suction pulses appear only in the patch capacitance (Im) trace with no projection into the patch conductance (Re) trace (Figure 2). 
	NOTE: Please see the details for the calibration of the system in reference 19.</li>
	<li>Identify typical capacitance changes by &#8220;downward&#8221; steps (Figure 3). 
	NOTE: A solid recording will be viewed as a &#8220;staircase&#8221; type resemblance with many downward steps, which indicates internalizing single vesicles. The patching process serves as a mechanical stimulation since action current can be typically recorded from most cells.</li>
	<li>Name files as individual folders including date: mm/dd/year and each cell-attached recording as its own individual numbering (xx) within that file.</li>
</ol>

<ol>
	<li>Rettig, J., Neher, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+roles+of+presynaptic+proteins+in+Ca++-triggered+exocytosis.">Emerging roles of presynaptic proteins in Ca++-triggered exocytosis.</a> Science. 298, 781-785 (2002).</li><li>Gong, L. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phosphatidylinositol+phosphate+kinase+type+I+gamma+regulates+dynamics+of+large+dense-core+vesicle+fusion.">Phosphatidylinositol phosphate kinase type I gamma regulates dynamics of large dense-core vesicle fusion.</a> Proceedings of the National Academy of Sciences of the United States of America. 102, 5204-5209 (2005).</li><li>Lindau, M., Alvarez de Toledo, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fusion+pore.">The fusion pore.</a> Biochimica et Biophysica Acta. 1641, 167-173 (2003).</li><li>Albillos, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+exocytotic+event+in+chromaffin+cells+revealed+by+patch+amperometry.">The exocytotic event in chromaffin cells revealed by patch amperometry.</a> Nature. 389, 509-512 (1997).</li><li>Lollike, K., Borregaard, N., 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=Capacitance+flickers+and+pseudoflickers+of+small+granules,+measured+in+the+cell-attached+configuration.">Capacitance flickers and pseudoflickers of small granules, measured in the cell-attached configuration.</a> Biophys J. 75, 53-59 (1998).</li><li>Lollike, K., Borregaard, N., 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=The+exocytotic+fusion+pore+of+small+granules+has+a+conductance+similar+to+an+ion+channel.">The exocytotic fusion pore of small granules has a conductance similar to an ion channel.</a> The Journal of cell biology. 129, 99-104 (1995).</li><li>Dernick, G., Gong, L. W., Tabares, L., Alvarez de Toledo, G., 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=Patch+amperometry:+high-resolution+measurements+of+single-vesicle+fusion+and+release.">Patch amperometry: high-resolution measurements of single-vesicle fusion and release.</a> Nat Methods. 2, 699-708 (2005).</li><li>Neher, E., Marty, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discrete+changes+of+cell+membrane+capacitance+observed+under+conditions+of+enhanced+secretion+in+bovine+adrenal+chromaffin+cells.">Discrete changes of cell membrane capacitance observed under conditions of enhanced secretion in bovine adrenal chromaffin cells.</a> Proceedings of the National Academy of Sciences of the United States of America. 79, 6712-6716 (1982).</li><li>Gong, L. W., de Toledo, G. 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. W., Hafez, I., Alvarez de Toledo, G., 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=Secretory+vesicles+membrane+area+is+regulated+in+tandem+with+quantal+size+in+chromaffin+cells.">Secretory vesicles membrane area is regulated in tandem with quantal size in chromaffin cells.</a> The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 23, 7917-7921 (2003).</li><li>Yao, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+polymerization+does+not+provide+direct+mechanical+forces+for+vesicle+fission+during+clathrin-mediated+endocytosis.">Actin polymerization does not provide direct mechanical forces for vesicle fission during clathrin-mediated endocytosis.</a> The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 33, 15793-15798 (2013).</li><li>Yao, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synaptotagmin+1+is+necessary+for+the+Ca2++dependence+of+clathrin-mediated+endocytosis.">Synaptotagmin 1 is necessary for the Ca2+ dependence of clathrin-mediated endocytosis.</a> The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. 32, 3778-3785 (2012).</li><li>McMahon, H. T., Boucrot, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+mechanism+and+physiological+functions+of+clathrin-mediated+endocytosis.">Molecular mechanism and physiological functions of clathrin-mediated endocytosis.</a> Nature reviews. Molecular Cell Biology. 12, 517-533 (2011).</li><li>Murthy, V. N., De Camilli, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+biology+of+the+presynaptic+terminal.">Cell biology of the presynaptic terminal.</a> Annu Rev Neurosci. 26, 701-728 (2003).</li><li>Granseth, B., Odermatt, B., Royle, S. J., Lagnado, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Clathrin-mediated+endocytosis+is+the+dominant+mechanism+of+vesicle+retrieval+at+hippocampal+synapses.">Clathrin-mediated endocytosis is the dominant mechanism of vesicle retrieval at hippocampal synapses.</a> Neuron. 51, 773-786 (2006).</li><li>Rosenboom, H., 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=Exo-endocytosis+and+closing+of+the+fission+pore+during+endocytosis+in+single+pituitary+nerve+terminals+internally+perfused+with+high+calcium+concentrations.">Exo-endocytosis and closing of the fission pore during endocytosis in single pituitary nerve terminals internally perfused with high calcium concentrations.</a> Proceedings of the National Academy of Sciences of the United States of America. 91, 5267-5271 (1994).</li><li>MacDonald, P. 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The authors declare that they have no competing financial interests.

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 on locating and removing the adrenal glands is imperative as cells that will be utilized for electrophysiological recordings come exclusively from the medulla of the adrenal gland. For enzyme digestion, it is critical to equilibrate the enzyme solution by bubbling the solution with 5% CO2 + 95% O2. Be certain that the tubing connected to the cylinder is sufficient and tight with no leaks. Additionally, be sure that the end in solution is properly placed during the entire 15 min bubbling time; this can be achieved with placing a 20 G needle end that has been blunted at the end of the tubing so as to direct air flow appropriately. Moreover, additional time for this step will not hurt so long as the airflow is not so powerful as to overflow the solution in the tube. Cell titration is another critical component that will take practice. The majority of the cells can be damaged if over-titrated; conversely, if titration is not enough, there will be very few single isolated cells dissociated from the tissues. To effectively titrate, utilize a 200 &#181;l pipette tip to pipette up and down 7-8x, making sure the glands are gently passed through the 200 &#181;l pipette tip as you titrate.
For PDL coating, one can always extend the incubation time of the PDL on the coverslips up to 3 hr to ensure sufficient coating of coverslips. The PDL coating is critical for chromaffin cells to attach to and grow on the coverslips after cell plating. If PDL coating is not sufficient, most of the cells will be detach from the coverslip, which will make the gigaohm seal formation difficult.
Cell-attached recordings can always be modified and improved, as the patching technique is a dedicated process. Gentle and subtle suction is critical in facilitating the gigaohm seal formation when the patch pipette and cell are in contact. Too much suction will destroy the patch tip-cell contact and in extreme situations, the cells can be sucked into the patch pipette. This will be very easily observed on the oscilloscope by changing into a whole cell configuration or by a very minimal resistance returning to a very large one; practice is of the utmost importance to obtain a high quality gigaohm seal formation.
While high seal resistance is critical to minimize the noise level in the cell-attached recordings, the following two steps are also important to reduce the noise level to resolve single endocytic events: (1) In the patch pipette solution, TEACl is utilized to block voltage-gated K+ channels; (2) the pipette tip is coated with sticky wax and the wax inside the pipette tip can be removed by heat polishing.
While the phase adjustment detailed in Protocol step 6.2 is critical to set up the initial phase for the recording, this step may not be ideal. Therefore, it is important to perform phase correction during fission-pore analysis for individual events. At a resting membrane potential of -65 mV, whole-cell recordings show that a typical mouse chromaffin cell has an input capacitance of 5 pF and an input resistance of 500 M&#937;, which calculates the membrane conductance of mouse chromaffin cells as ~0.4 pS/fF. This implies that an endocytic event with a capacitance size of 1 fF will result in a net loss of membrane conductance of ~0.4 pS. This value is negligible compared to a typical 50-100 pS membrane conductance change associated with endocytic fission-pore closure in cell-attached recordings. Therefore, we are confident that our phase correction allows us to adjust the baseline of membrane conductance such that the pre and post fission-pore closure is at the same level; this process may bring &#60;1% error into our data analysis.
In summary, the protocol described here demonstrates how cell-attached capacitance recordings can be utilized to monitor single vesicle endocytosis using mouse chromaffin cells as the model system. This technique allows one to analyze the regulatory mechanisms for vesicle fission during endocytosis.

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.

<table border="0" cellpadding="0" cellspacing="0" width="352">
	<tbody>
		<tr height="61">
			<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>
			<td rowspan="2" style="width:88px;"></td>
		</tr>
		<tr height="22">
		</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>
		</tr>
		<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 5.3, there is a better chance in obtaining a high-quality seal. Figure 3 demonstrates a typical recording of membrane capacitance with multiple downwards steps associated with single vesicle endocytosis.
<img alt="Figure 1" src="/files/ftp_upload/52024/52024fig1highres.jpg" /> 
Figure 1. Examples of viable mouse adrenal chromaffin cells 24 hr after cell culture. Arrows point to 3 viable chromaffin cells. <a href="https://www.jove.com/files/ftp_upload/52024/52024fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52024/52024fig2highres.jpg" /> 
Figure 2. Phase adjustment in the cell-attached recordings. With the initial phase setting, gentle suctions cause transient changes in both capacitance (Im) and conductance (Re) traces, and the projections in Re trace are cancelled out by a 23&#176; phase shift. <a href="https://www.jove.com/files/ftp_upload/52024/52024fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/52024/52024fig3highres.jpg" /> 
Figure 3. A typical cell-attached capacitance recording with multiple downward steps, associated with single vesicle endocytosis. <a href="https://www.jove.com/files/ftp_upload/52024/52024fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

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

Universidad San Sebastian

Research

JoVE Journal

Neuroscience

8.7K Views.  University of Illinois at Chicago. 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.

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

Mouse Models of Periventricular Leukomalacia

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature infants and the most common antecedent of cerebral palsy. PVL is characterized by periventricular white matter damage with prominent oligodendroglial injury. Hypoxia/ischemia with or without systemic infection/inflammation are the primary causes of PVL. We use P6 mice to create models of neonatal brain injury by the induction of hypoxia/ischemia with or without systemic infection/inflammation with unilateral carotid ligation followed by exposure to hypoxia with or without injection of the endotoxin lipopolysaccharide (LPS). Immunohistochemistry of myelin basic protein (MBP) or O1 and electron microscopic examination show prominent myelin loss in cerebral white matter with additional damage to the hippocampus and thalamus. Establishment of mouse models of PVL will greatly facilitate the study of disease pathogenesis using available transgenic mouse strains, conduction of drug trials in a relatively high throughput manner to identify candidate therapeutic agents, and testing of stem cell transplantation using immunodeficiency mouse strains.

We established mouse models of periventricular leukomalacia (PVL), the predominant brain injury in premature infants characterized by periventricular white matter lesions. Hypoxia/ischemia with/without systemic infection are the primary causes of PVL. Unilateral carotid ligation and hypoxia exposure with/without lipopolysaccharide injection creates PVL-like lesions in P6 mice.

We describe a protocol for establishing mouse models of periventricular leukomalacia (PVL). PVL is the predominant form of brain injury in premature ...

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. However, because the uterine wall is opaque during early embryogenesis, the ability to target specific parts of the embryo for microinjection is greatly limited. Fortunately, high-frequency ultrasound imaging permits the generation of images that can be used in real time to guide a microinjection needle into the embryonic region of interest. Here we describe the use of such imaging to guide the injection of retroviral vectors into the ventricular system of the mouse forebrain at embryonic day (E) 9.5. This method uses a laparotomy to permit access to the uterine horns, and a specially designed plate that permits host embryos to be bathed in saline while they are imaged and injected. Successful surgeries often result in most or all of the injected embryos surviving to any subsequent time point of interest (embryonically or postnatally). The principles described here can be used with slight modifications to perform injections into the amnionic fluid of E8.5 embryos (thereby permitting infection along the anterior posterior extent of the neural tube, which has not yet closed), or into the ventricular system of the brain at E10.5/11.5. Furthermore, at mid-neurogenic ages (~E13.5), ultrasound imaging can be used direct injection into specific brain regions for viral infection or cell transplantation. The use of ultrasound imaging to guide in utero injections in mice is a very powerful technique that permits the molecular and cellular manipulation of mouse embryos in ways that would otherwise be exceptionally difficult if not impossible.

Ultrasound-Guided Microinjection into the Mouse Forebrain In Utero at E9.5

In utero survival surgery in mice permits the molecular manipulation of gene expression during development. Here we describe the use of high-frequency ultrasound imaging to guide the injection of retroviral vectors into the mouse brain at embryonic day (E) 9.5.

In utero survival surgery in mice permits the molecular manipulation of gene expression during development.  However, because the uterine wall is opaque ...

Multiple-mouse Neuroanatomical Magnetic Resonance Imaging

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and evaluating mouse models of human disease. MRI is an excellent modality for investigating genetically altered animals. It is capable of whole brain coverage, can be used in vivo, and provides multiple contrast mechanisms for investigating different aspects of neuranatomy and physiology. The advent of high-field scanners along with the ability to scan multiple mice simultaneously allows for rapid phenotyping of novel mutations.
Effective mouse MRI studies require attention to many aspects of experiment design. In this article, we will describe general methods to acquire quality images for mouse phenotyping using a system that images mice concurrently in shielded transmit/receive radio frequency (RF) coils in a common magnet (Bock et al., 2003). We focus particularly on anatomical phenotyping, an important and accessible application that has shown a high potential for impact in many mouse models at our imaging centre. Before we can provide the detailed steps to acquire such images, there are important practical considerations for both in vivo brain imaging (Dazai et al., 2004) and ex vivo brain imaging (Spring et al., 2007) that should be noted. These are discussed below.

Magnetic resonance imaging (MRI) has become an increasingly popular tool for examining the phenotype of genetically altered mice. This article illustrates the methods necessary to achieve high-throughput phenotyping of genetically altered mice using multiple-mouse MRI.

The field of mouse phenotyping with magnetic resonance imaging (MRI) is rapidly growing, motivated by the need for improved tools for characterizing and ...

In vivo Electroporation of Developing Mouse Retina

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge. Gene targeting to generate constitutive or conditional loss of function knockouts remains cost and labor intensive, as well as time consuming. Adding to these challenges, retina expressed genes may have essential roles outside the retina leading to unintended confounds when using a knockout approach. Furthermore, the ability to ectopically express a gene in a gain of function experiment can be extremely valuable when attempting to identify a role in cell fate specification and/or terminal differentiation.
We present a method for the rapid and efficient incorporation of DNA plasmids into the neonatal mouse retina by electroporation. The application of short electrical impulses above a certain field strength results in a transient increase in plasma membrane permeability, facilitating the transfer of material across the membrane 1,2,3,4. Groundbreaking work demonstrated that electroporation could be utilized as a method of gene transfer into mammalian cells by inducing the formation of hydrophilic plasma membrane pores allowing the passage of highly charged DNA through the lipid bilayer 5. Continuous technical development has resulted in the viability of electroporation as a method for in vivo gene transfer in multiple mouse tissues including the retina, the method for which is described herein 6, 7, 8, 9, 10. 
DNA solution is injected into the subretinal space so that DNA is placed between the retinal pigmented epithelium and retina of the neonatal (P0) mouse and electrical pulses are applied using a tweezer electrode. The lateral placement of the eyes in the mouse allows for the easy orientation of the tweezer electrode to the necessary negative pole-DNA-retina-positive pole alignment. Extensive incorporation and expression of transferred genes can be identified by postnatal day 2 (P2). Due to the lack of significant lateral migration of cells in the retina, electroporated and non-electroporated regions are generated. Non-electroporated regions may serve as internal histological controls where appropriate. 
Retinal electroporation can be used to express a gene under a ubiquitous promoter, such as CAG, or to disrupt gene function using shRNA constructs or Cre-recombinase. More targeted expression can be achieved by designing constructs with cell specific gene promoters. Visualization of electroporated cells is achieved using bicistronic constructs expressing GFP or by co-electroporating a GFP expression construct. Furthermore, multiple constructs may be electroporated for the study of combinatorial gene effects or simultaneous gain and loss of function of different genes. Retinal electroporation may also be utilized for the analysis of genomic cis-regulatory elements by generating appropriate expression constructs and deletion mutants. Such experiments can be used to identify cis-regulatory regions sufficient or required for cell specific gene expression 11. Potential experiments are limited only by construct availability.

In vivo Electroporation of Developing Mouse Retina

A method for the incorporation of plasmid DNA into murine retinal cells for the purpose of performing either gain- or loss of function studies in vivo is presented. This method capitalizes on the transient increase in permeability of cell plasma membranes induced by the application of an external electrical field.

The functional characterization of genes expressed during mammalian retinal development remains a significant challenge.  Gene targeting to generate ...

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate retina. Two classes of retinal neurons, photoreceptors and bipolar cells, accomplish this by using ribbon-type active zones, which enable sustained and high-throughput neurotransmitter release over long time periods. ON-type mixed bipolar cell (Mb) terminals in the goldfish retina, which depolarize to light stimuli and receive mixed rod and cone photoreceptor input, are suitable for the study of ribbon-type synapses both due to their large size (~10-12 &mu;m diameter) and to their numerous lateral and reciprocal synaptic connections with amacrine cell dendrites. Direct access to Mb bipolar cell terminals in goldfish retinal slices with the patch-clamp technique allows the measurement of presynaptic Ca2+ currents, membrane capacitance changes, and reciprocal synaptic feedback inhibition mediated by GABAA and GABAC receptors expressed on the terminals. Presynaptic membrane capacitance measurements of exocytosis allow one to study the short-term plasticity of excitatory neurotransmitter release 14,15. In addition, short-term and long-term plasticity of inhibitory neurotransmitter release from amacrine cells can also be investigated by recordings of reciprocal feedback inhibition arriving at the Mb terminal 21. Over short periods of time (e.g. ~10 s), GABAergic reciprocal feedback inhibition from amacrine cells undergoes paired-pulse depression via GABA vesicle pool depletion 11. The synaptic dynamics of retinal microcircuits in the inner plexiform layer of the retina can thus be directly studied.
The brain-slice technique was introduced more than 40 years ago but is still very useful for the investigation of the electrical properties of neurons, both at the single cell soma, single dendrite or axon, and microcircuit synaptic level 19. Tissues that are too small to be glued directly onto the slicing chamber are often first embedded in agar (or placed onto a filter paper) and then sliced 20, 23, 18, 9. In this video, we employ the pre-embedding agar technique using goldfish retina. Some of the giant bipolar cell terminals in our slices of goldfish retina are axotomized (axon-cut) during the slicing procedure. This allows us to isolate single presynaptic nerve terminal inputs, because recording from axotomized terminals excludes the signals from the soma-dendritic compartment. Alternatively, one can also record from intact Mb bipolar cells, by recording from terminals attached to axons that have not been cut during the slicing procedure. Overall, use of this experimental protocol will aid in studies of retinal synaptic physiology, microcircuit functional analysis, and synaptic transmission at ribbon synapses.

Patch-clamp Capacitance Measurements and Ca2+ Imaging at Single Nerve Terminals in Retinal Slices

Here we describe a protocol for the preparation of agar-embedded retinal slices that are suitable for electrophysiology and Ca2+ imaging. This method allows one to study ribbon-type synapses in retinal microcircuits using direct patch-clamp recordings of single presynaptic nerve terminals.

Visual stimuli are detected and conveyed over a wide dynamic range of light intensities and frequency changes by specialized neurons in the vertebrate ...

Methods for Intravenous Self Administration in a Mouse Model

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages of using an IVSA paradigm to study the reinforcing properties of drugs of abuse such as cocaine include the fact that the drug is self-administered instead of experimenter-administered, the schedule of reinforcement can be altered, and accurate measurement of the quantities of drug consumed as well as the timing and pattern of IV injections can be obtained. Furthermore, the intravenous route of administration avoids potential confounds related to first pass metabolism or taste, and produces rapid increases in blood and brain drug levels. As outlined in this video, intravenous self-administration can be obtained without prior food restriction or prior drug training following careful catheter placement during surgery and meticulous daily catheter flushing and maintenance. Experimental procedures outlined in this paper include a description of animal housing and acclimation methods, operant training using sweetened milk solutions, and catheter implantation surgery.

The intravenous self-administration (IVSA) paradigm is considered to be the gold standard in examining the reinforcing properties of drugs of abuse in rodents. This manuscript outlines the experimental procedures and surgical techniques necessary to obtain reliable IVSA data. In particular, meticulous catheter implantation and maintenance are highlighted.

Animal models have been developed to study the reinforcing effects of drugs, including the intravenous self-administration (IVSA) paradigm. The advantages ...

Methods for the Modulation and Analysis of NF-&#954;B-dependent Adult Neurogenesis

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult hippocampus has been viewed as a very static anatomical region of the mammalian brain. However, recent findings have demonstrated that the dentate gyrus of the hippocampus is an area of tremendous plasticity in adults, involving not only modifications of existing neuronal circuits, but also adult neurogenesis. This plasticity is regulated by complex transcriptional networks, in which the transcription factor NF-&#954;B plays a prominent role. To study and manipulate adult neurogenesis, a transgenic mouse model for forebrain-specific neuronal inhibition of NF-&#954;B activity can be used.
In this study, methods are described for the analysis of NF-&#954;B-dependent neurogenesis, including its structural aspects, neuronal apoptosis and progenitor proliferation, and cognitive significance, which was specifically assessed via a dentate gyrus (DG)-dependent behavioral test, the spatial pattern separation-Barnes maze (SPS-BM). The SPS-BM protocol could be simply adapted for use with other transgenic animal models designed to assess the influence of particular genes on adult hippocampal neurogenesis. Furthermore, SPS-BM could be used in other experimental settings aimed at investigating and manipulating DG-dependent learning, for example, using pharmacological agents.

Methods for the Modulation and Analysis of NF-&amp;#954;B-dependent Adult Neurogenesis

Methods for the manipulation and analysis of NF-&#954;B-dependent adult hippocampal neurogenesis are described. A detailed protocol is presented for a dentate gyrus-dependent behavioral test (termed the spatial pattern separation-Barnes maze) for the investigation of cognitive outcome in mice. This technique should also help enable investigations in other experimental settings.

The hippocampus plays a pivotal role in the formation and consolidation of episodic memories, and in spatial orientation. Historically, the adult ...

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological techniques, in the understanding of the underlying pathophysiology1. Here we describe a method to perform electrophysiological studies on mouse sciatic nerves in vivo.
The animals are anesthetized with isoflurane in order to ensure analgesia for the tested mice and undisturbed working environment during the measurements that take about 30 min/animal. A constant body temperature of 37 &#176;C is maintained by a heating plate and continuously measured by a rectal thermo probe2. Additionally, an electrocardiogram (ECG) is routinely recorded during the measurements in order to continuously monitor the physiological state of the investigated animals.
Electrophysiological recordings are performed on the sciatic nerve, the largest nerve of the peripheral nervous system (PNS), supplying the mouse hind limb with both motoric and sensory fiber tracts. In our protocol, sciatic nerves remain in situ and therefore do not have to be extracted or exposed, allowing measurements without any adverse nerve irritations along with actual recordings. Using appropriate needle electrodes3 we perform both proximal and distal nerve stimulations, registering the transmitted potentials with sensing electrodes at gastrocnemius muscles. After data processing, reliable and highly consistent values for the nerve conduction velocity (NCV) and the compound motor action potential (CMAP), the key parameters for quantification of gross peripheral nerve functioning, can be achieved.

In Vivo Electrophysiological Measurements on Mouse Sciatic Nerves

Measurements of nerve conduction properties in vivo exemplify a powerful tool to characterize various animal models of neuromuscular diseases. Here, we present an easy and reliable protocol by which electrophysiological analysis on sciatic nerves of anesthetized mice can be performed.

Electrophysiological studies allow a rational classification of various neuromuscular diseases and are of help, together with neuropathological ...

Live Imaging of Mitosis in the Developing Mouse Embryonic Cortex

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the developing cerebral cortex, abnormal mitosis of neural progenitors can cause defects in brain size and function. Hence, there is a critical need for tools to understand the mechanisms of neural progenitor mitosis. Cortical development in rodents is an outstanding model for studying this process. Neural progenitor mitosis is commonly examined in fixed brain sections. This protocol will describe in detail an approach for live imaging of mitosis in ex vivo embryonic brain slices. We will describe the critical steps for this procedure, which include: brain extraction, brain embedding, vibratome sectioning of brain slices, staining and culturing of slices, and time-lapse imaging. We will then demonstrate and describe in detail how to perform post-acquisition analysis of mitosis. We include representative results from this assay using the vital dye Syto11, transgenic mice (histone H2B-EGFP and centrin-EGFP), and in utero electroporation (mCherry-&#945;-tubulin). We will discuss how this procedure can be best optimized and how it can be modified for study of genetic regulation of mitosis. Live imaging of mitosis in brain slices is a flexible approach to assess the impact of age, anatomy, and genetic perturbation in a controlled environment, and to generate a large amount of data with high temporal and spatial resolution. Hence this protocol will complement existing tools for analysis of neural progenitor mitosis.

Neural progenitor mitosis is a critical parameter of neurogenesis. Much of our understanding of neural progenitor mitosis is based on analysis of fixed tissue. Live imaging in embryonic brain slices is a versatile technique to assess mitosis with high temporal and spatial resolution in a controlled environment.

Although of short duration, mitosis is a complex and dynamic multi-step process fundamental for development of organs including the brain. In the ...

A Behavioral Screen for Heat-Induced Seizures in Mouse Models of Epilepsy

Transgenic mouse models have proved to be powerful tools in studying various aspects of human neurological disorders, including epilepsy. The SCN1A-associated genetic epilepsies comprise a wide spectrum of seizure disorders with incomplete penetrance and clinical variability. SCN1A mutations can result in a large variety of seizure phenotype ranging from simple, self-limited fever-associated febrile seizures (FS), moderate-level genetic epilepsy with febrile seizures plus (GEFS+) to more severe Dravet Syndrome (DS). Although FS are commonly seen in children below 6-7 years of age who do not have genetic epilepsy, FS in GEFS+ patients continue to occur into adulthood. Traditionally, experimental FS have been induced in mice by exposing the animal to a stream of dry air or heating lamps, and the rate of change in body temperature is often not well controlled. Here, we describe a custom-built heating chamber, with a plexiglass front, that is fitted with a digital temperature controller and a heater-equipped electric fan, which can send heated forced air into the test arena in a temperature-controlled manner. The body temperature of a mouse placed in the chamber, monitored through a rectal probe, can be increased to 40-42 &#176;C in a reproducible manner by increasing the temperature inside the chamber. Continual visual monitoring of the animals during the heating period demonstrates induction of heat-induced seizures in mice carrying an FS mutation at a body temperature that does not elicit behavioral seizures in wild-type litter mates. Animals can be easily removed from the chamber and placed on a cooling pad to rapidly return body temperature to normal. This method provides for a simple, rapid, and reproducible screening protocol for the occurrence of heat-induced seizures in epilepsy mouse models.

The goal of the method is to screen for hyperthermia or heat-induced seizures in mouse models. The protocol describes the use of a custom-built chamber with continuous monitoring of the body temperature to determine whether elevated body temperature leads to seizures.