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>

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	<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. E., Eliasson, L., Rorsman, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+increases+endocytotic+vesicle+size+and+accelerates+membrane+fission+in+insulin-secreting+INS-1+cells.">Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin-secreting INS-1 cells.</a> Journal of Cell Science. 118, 5911-5920 (2005).</li><li>Zhao, Y., Fang, Q., Straub, S. G., Lindau, M., Sharp, G. 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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 ...

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><tbody><tr><td>Poly-D-Lysine</td><td>Sigma</td><td>P0899</td><td></td></tr><tr><td>DMEM</td><td></td><td>15066024</td><td>Keep out of UV</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium</td><td>Life Technologies</td><td></td><td></td></tr><tr><td>Cover Glass</td><td>Carolina Biological</td><td>633029</td><td>12 mm</td></tr><tr><td>Penicillin Streptomycin</td><td>Life Technologies</td><td>15140122</td><td>100 ml</td></tr><tr><td>Insulin-Trans-Sel-X</td><td>Life Technologies</td><td>51500056</td><td>Only thaw on ICE!</td></tr><tr><td>Papain</td><td>Worthington</td><td>39S11614</td><td></td></tr><tr><td>EPC-7 plus patch amplifier</td><td>HEKA</td><td></td><td></td></tr><tr><td>BNC-2090 data acquisition board</td><td>National Instruments</td><td></td><td></td></tr><tr><td>Igor data acquisition software</td><td>Wavemetrics</td><td></td><td></td></tr><tr><td>P-97 pipette puller</td><td>Sutter Instruments</td><td></td><td></td></tr><tr><td>Microforge</td><td>Scientific Instruments</td><td></td><td></td></tr><tr><td>Borosilicate glass capillaries</td><td>Sutter Instruments</td><td>B150-110-10</td><td>Outer diameter: 1.5 mm&lt;br /&gt;
Inner diameter: 1.10 mm&lt;br/&gt;
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 ...

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

Research

JoVE Journal

Neuroscience

8.8K 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 Adrenal Chromaffin Cell Isolation

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex.  The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting. This protocol illustrates the method used to dissect the adrenals and then isolate the medullary region by stripping away the adrenal cortex. The digestion of the medulla into single chromaffin cells is then demonstrated.

Adrenal medullary chromaffin cell culture systems are extremely useful for the study of excitation-secretion coupling in an in vitro setting.  This ...

Biology

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 ...

Implementing Dynamic Clamp with Synaptic and Artificial Conductances in Mouse Retinal Ganglion Cells

Ganglion cells are the output neurons of the retina and their activity reflects the integration of multiple synaptic inputs arising from specific neural circuits. Patch clamp techniques, in voltage clamp and current clamp configurations, are commonly used to study the physiological properties of neurons and to characterize their synaptic inputs. Although the application of these techniques is highly informative, they pose various limitations. For example, it is difficult to quantify how the precise interactions of excitatory and inhibitory inputs determine response output. To address this issue, we used a modified current clamp technique, dynamic clamp, also called conductance clamp 1, 2, 3 and examined the impact of excitatory and inhibitory synaptic inputs on neuronal excitability. This technique requires the injection of current into the cell and is dependent on the real-time feedback of its membrane potential at that time. The injected current is calculated from predetermined excitatory and inhibitory synaptic conductances, their reversal potentials and the cell's instantaneous membrane potential. Details on the experimental procedures, patch clamping cells to achieve a whole-cell configuration and employment of the dynamic clamp technique are illustrated in this video article. Here, we show the responses of mouse retinal ganglion cells to various conductance waveforms obtained from physiological experiments in control conditions or in the presence of drugs. Furthermore, we show the use of artificial excitatory and inhibitory conductances generated using alpha functions to investigate the responses of the cells.

This video article illustrates the set-up, the procedures to patch cell bodies and how to implement dynamic clamp recordings from ganglion cells in whole-mount mouse retinae. This technique allows the investigation of the precise contribution of excitatory and inhibitory synaptic inputs, and their relative magnitude and timing to neuronal spiking.

Ganglion cells are the output neurons of the  retina and their activity reflects the integration of multiple synaptic inputs  arising from specific neural ...

Isolation, Fixation, and Immunofluorescence Imaging of Mouse Adrenal Glands

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has a broad spectrum of applications. Detection of antigens allows for characterization and identification of multiple cell types. Located above the kidneys and encapsulated by a layer of mesenchymal cells, the adrenal gland is an endocrine organ composed by two different tissues with different embryological origins, the mesonephric intermediate mesoderm-derived outer cortex and the neural crest-derived inner medulla. The adrenal cortex secretes steroids (i.e., mineralocorticoids, glucocorticoids, sex hormones), whereas the adrenal medulla produces catecholamines (i.e., adrenaline, noradrenaline). While conducting adrenal research, it is important to be able to distinguish unique cells with different functions. Here we provide a protocol developed in our laboratory that describes a series of sequential steps required for obtaining immunofluorescence staining to characterize the cell types of the adrenal gland. We focus first on the dissection of the mouse adrenal glands, the microscopic removal of periadrenal fat followed by the fixation, processing and paraffin embedding of the tissue. We then describe sectioning of the tissue blocks with a rotary microtome. Lastly, we detail a protocol for immunofluorescent staining of adrenal glands that we have developed to minimize both non-specific antibody binding and autofluorescence in order to achieve an optimal signal.

Here we present a method to isolate adrenal glands from mice, fix the tissues, section them, and perform immunofluorescence staining.

Immunofluorescence is a well-established technique for detection of antigens in tissues with the employment of fluorochrome-conjugated antibodies and has ...

Immunology and Infection

Monitoring the Effect of Osmotic Stress on Secretory Vesicles and Exocytosis

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity and the amount of neurotransmitter released from vesicles in single exocytosis events. It has been suggested that the reduction in neurotransmitters expelled is due to alterations in membrane biophysical properties when cells shrink in response to osmotic stress and with assumptions made that secretory vesicles in the cell cytoplasm are not affected by extracellular osmotic stress. Amperometry recording of exocytosis monitors what is released from cells the moment a vesicle fuses with the plasma membrane, but does not provide information on the vesicle content before the vesicle fusion is triggered. Therefore, by combining amperometry recording with other complementary analytical methods that are capable of characterizing the secretory vesicles before exocytosis at cells is triggered offers a broader overview for examining how secretory vesicles and the exocytosis process are affected by osmotic shock. We here describe how complementing amperometry recording with intracellular electrochemical cytometry and transmission electron microscopy (TEM) imaging can be used to characterize alterations in secretory vesicles size and neurotransmitter content at chromaffin cells in relation to exocytosis activity before and after exposure to osmotic stress. By linking the quantitative information gained from experiments using all three analytical methods, conclusions were previously made that secretory vesicles respond to extracellular osmotic stress by shrinking in size and reducing the vesicle quantal size to maintain a constant vesicle neurotransmitter concentration. Hence, this gives some clarification regarding why vesicles, in response to osmotic stress, reduce the amount neurotransmitters released during exocytosis release. The amperometric recordings here indicate this is a reversible process and that vesicle after osmotic shock are refilled with neurotransmitters when placed cells are reverted into an isotonic environment.

Osmotic stress affects exocytosis and the amount of neurotransmitter released during this process. We demonstrate how combining electrochemical methods together with transmission electron microscopy can be used to study the effect of extracellular osmotic pressure on exocytosis activity, vesicle quantal size, and the amount of neurotransmitter released during exocytosis.

Amperometry recording of cells subjected to osmotic shock show that secretory cells respond to this physical stress by reducing the exocytosis activity ...

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

The study of electrophysiological properties of cardiac ion channels with the patch-clamp technique and the exploration of cardiac cellular Ca2+ handling abnormalities requires isolated cardiomyocytes. In addition, the possibility to investigate myocytes from patients using these techniques is an invaluable requirement to elucidate the molecular basis of cardiac diseases such as atrial fibrillation (AF).1 Here we describe a method for isolation of human atrial myocytes which are suitable for both patch-clamp studies and simultaneous measurements of intracellular Ca2+ concentrations. First, right atrial appendages obtained from patients undergoing open heart surgery are chopped into small tissue chunks ("chunk method") and washed in Ca2+-free solution. Then the tissue chunks are digested in collagenase and protease containing solutions with 20 &mu;M Ca2+. Thereafter, the isolated myocytes are harvested by filtration and centrifugation of the tissue suspension. Finally, the Ca2+ concentration in the cell storage solution is adjusted stepwise to 0.2 mM. We briefly discuss the meaning of Ca2+ and Ca2+ buffering during the isolation process and also provide representative recordings of action potentials and membrane currents, both together with simultaneous Ca2+ transient measurements, performed in these isolated myocytes.

Isolation of Human Atrial Myocytes for Simultaneous Measurements of Ca2+ Transients and Membrane Currents

We describe the isolation of human atrial myocytes which can be used for intracellular Ca2+ measurements in combination with electrophysiological patch-clamp studies.

The study of  electrophysiological properties of cardiac ion channels with the patch-clamp  technique and the exploration of cardiac cellular Ca2+ ...

Confocal Microscopy to Measure Three Modes of Fusion Pore Dynamics in Adrenal Chromaffin Cells

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal microscopy was used in combination with patch-clamp recording to detect three fusion modes in primary culture bovine adrenal chromaffin cells. The three fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving shrinkage of the fusion-generated &#937;-shape profile until it merges completely at the plasma membrane.
To detect these fusion modes, the plasma membrane was labeled by overexpressing mNeonGreen attached with the PH domain of phospholipase C &#948; (PH-mNG), which binds to phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) at the cytosol-facing leaflet of the plasma membrane; vesicles were loaded with the fluorescent false neurotransmitter FFN511 to detect vesicular content release; and Atto 655 was included in the bath solution to detect fusion pore closure. These three fluorescent probes were imaged simultaneously at ~20-90 ms per frame in live chromaffin cells to detect fusion pore opening, content release, fusion pore closure, and fusing vesicle size changes. The analysis method is described to distinguish three fusion modes from these fluorescence measurements. The method described here can, in principle, apply to many secretory cells beyond chromaffin cells.

This protocol describes a confocal imaging technique to detect three fusion modes in bovine adrenal chromaffin cells. These fusion modes include 1) close-fusion (also called kiss-and-run), involving fusion pore opening and closure, 2) stay-fusion, involving fusion pore opening and maintaining the opened pore, and 3) shrink-fusion, involving fused vesicle shrinkage.

Dynamic fusion pore opening and closure mediate exocytosis and endocytosis and determine their kinetics. Here, it is demonstrated in detail how confocal ...

Whole Cell Electrophysiology of Primary Cultured Murine Enterochromaffin Cells

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory cells, EC cells sense luminal stimuli and convert them into serotonin (5-hyroxytryptamine, 5-HT) release events. However, the electrophysiology of these cells is poorly understood because they are difficult to culture and to identify. The method presented in this paper outlines primary EC cell cultures optimized for single cell electrophysiology. This protocol utilizes a transgenic cyan fluorescent protein (CFP) reporter to identify mouse EC cells in mixed primary cultures, advancing the approach to obtaining high-quality recordings of whole cell electrophysiology in voltage- and current-clamp modes.

Enterochromaffin (EC) cells comprise a small subset of gastrointestinal epithelial cells. EC cells are electrically excitable and release serotonin, yet difficulties in culturing and identifying EC cells have limited physiological studies. The method presented here establishes a primary culture model amenable to examination of single EC cells by electrophysiology.

Enterochromaffin (EC) cells in the gastrointestinal (GI) epithelium constitute the largest subpopulation of enteroendocrine cells. As specialized sensory ...

Camera-based Measurements of Intracellular [Na+] in Murine Atrial Myocytes

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the sarcolemmal Na+/Ca2+ exchanger, the behavior of voltage-gated Na+ channels and the Na+,K+-ATPase. Intracellular Ca2+ signaling is altered in atrial diseases such as atrial fibrillation. While many of the mechanisms underlying altered intracellular Ca2+ homeostasis are characterized, the role of [Na+]i and its dysregulation in atrial pathologies is poorly understood. [Na+]i in atrial myocytes increases in response to increasing stimulation rates. Responsiveness to external field stimulation is therefore crucial for [Na+]i measurements in these cells. In addition, the long preparation (dye-loading) and experiment duration (calibration) require an isolation protocol that yields atrial myocytes of exceptional quality. Due to the small size of mouse atria and the composition of the intercellular matrix, the isolation of high quality adult murine atrial myocytes is difficult. Here, we describe an optimized Langendorff-perfusion based isolation protocol that consistently delivers a high yield of high quality atrial murine myocytes.
Sodium-binding benzofuran isophthalate (SBFI) is the most commonly used fluorescent Na+ indicator. SBFI can be loaded into the cardiac myocyte either in its salt form through a glass pipette or as an acetoxymethyl (AM) ester that can penetrate the myocyte&#8217;s sarcolemmal membrane. Intracellularly, SBFI-AM is de-esterified by cytosolic esterases. Due to variabilities in membrane penetration and cytosolic de-esterification each cell has to be calibrated in situ. Typically, measurements of [Na+]i using SBFI whole-cell epifluorescence are performed using a photomultiplier tube (PMT). This experimental set-up allows for only one cell to be measured at one time. Due to the length of myocyte dye loading and the calibration following each experiment data yield is low. We therefore developed an EMCCD camera-based technique to measure [Na+]i. This approach permits simultaneous [Na+]i measurements in multiple myocytes thus significantly increasing experimental yield.

The intracellular Na+ concentration ([Na+]i) in cardiac myocytes is altered during cardiac diseases. [Na+]i is an important regulator of intracellular Ca2+. We introduce a novel approach to measure [Na+]i in freshly isolated murine atrial myocytes using an electron multiplying charged coupled device (EMCCD) camera and a rapid, controllable illuminator.

Intracellular sodium concentration ([Na+]i) is an important regulator of intracellular Ca2+. Its study provides insight into the activation of the ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...

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

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