Anal. Chem. 1994,66, 3031-3035
Amperometric Monitoring of Stimulated Catecholamine Release from Rat Pheochromocytoma (PC12) Cells at the Zeptomole Level Ta Kung Chen, Guoan Luo, and Andrew 0. Ewlng' 152 Davey Laboratory, Department of Chemistry, The Pennsylvania State Universiw, University Park, Pennsylvania 16802
Catecholamine release from rat pheochromocytoma (PC12) cells has been observed at zeptomole levels using dc-amperometric detection with carbon fiber microelectrodes. Time resolved individual exocytic events from PC12 cells have been recorded and analyzed with 1.2 ms time resolution. The average area under 1912 current transients from 13 PC12 cells correspondsto 190 zmol(ll4 300 molecules per release event). The average width at half-heightof these current transients is 9.3 ms, in agreement with the time frame of exocytosis. The detection limit of the method reported here is as low as 31 zmol. This is the first report of direct electrochemical observationof quantal release from PC12 cells. The successful application of this electrochemical scheme to monitor catecholamines released from small vesicles also suggests that it may be possible to apply this technique to monitor quantal release from synaptic vesicles.
The development of electrochemical techniques for the study of vesicular release from single cells provides a new and powerful tool for the study of chemical signaling in the nervous system. Investigation of exocytosis of catecholamines1-' and insulin*from cultured bovine adrenomedullary cells and human pancreatic @-cells,respectively, have been reported recently using these techniques. However, the application of these techniques to mammalian neurons has not been demonstrated. This is due in part to the extremely minute amount of chemical released during vesicular exocytosis as well as the small and complex working environment in even a simplified system like nerve cell cultures. It has been estimated that only 1 X 105-2 X lo6 acetylcholine molecules or 100-200 quanta are released from an impulse at the nerve terminal of neuromuscular junction^.^ Assuming this is a close estimation and each ~~~
T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Vivcros, 0. H.; Dilibcrto, E. M., Jr.; Viveros, 0. H. Proc. Natl. Acad. Sei. U S A . 1991, 88, 10754-
(1) Wightman, R. M.; Jankowski, J. A.; Kennedy, R.
10758. (2) Leszczyszyn, D. J.; Jankowski, J. A,; Viveros, 0. H.; Diliberto, E. M., Jr.; Near, J. A.; Wightman, R. M.J . Biol. Chem. 1990, 265, 14736-14737. (3) Ciolkowski,E. L.;Cooper, B. R.; Jankowski, J.A.; Jorgenson,J. M.; Wightman, M. R. J. Am. Chem. SOC.1992, 114, 2815-2821.
(4) Schroeder, T. J.; Jankowski, J. A,; Kawagoe, K. T.; Wightman, R. M. Anal. Chem. 1992,64, 3077-3083. ( 5 ) Jankowski, J. A.; Schrocder, T. J.; Holz, R. W.; Wightman, R. M. J . B i d . Chem. 1992, 267, 18329-18335. (6) Chow, R. H.; von Rilden, L.;Neher, E. Nature 1992, 356, 60-63. (7) Zhou, R.; Luo, G.; Ewing, A. G. J. Neurosci. 1994, 14. 2402-2407. (8) Kennedy, R. T.; Huang, L.; Atkinson, M. A.; Dush, P. Anal. Chem. 1993, 65, 1882-1887. 0003-2700/94/0366-3031$04.50/0
0 1994 American Chemical Society
quantum represents a single vesicular release event, one can calculate that each of the cholinergicsynapticvesiclescontains from 500 to 2 X lo4 (0.8 to 33 zmol) acetylcholine molecules. Wightman and co-worker~l-~ have observed current transients at microelectrodes corresponding to the single vesicular release of epinephrine (E) and norepinephrine (NE) from isolated bovine adrenal medullary chromaffin cells. Each catecholaminergic vesicle in these cells contains an average of 3.7 X lo6 (6.1 amol) releasablecatecholaminemolecules.1~5 The amount of releasable catecholamines in a synaptic vesicle is expected to be less because of the smaller size (40-100 nml0 average diameter vs 400 nm for chromaffin cellsll). If the catecholamine concentration is similar in all vesicles, the average amount of releasable catecholamine in a synaptic vesicle is about 1.1 X 103-5.8 X lo4 molecule (or 1.8-96.4 zmol).12 It is clear that a very low detection limit is needed in order to monitor vesicular release at the synaptic area. Mammalian neurons are very difficult to work with in the laboratory because mammalian nerve cells stop proliferation once they have matured. For this reason, cell lines and primary cell cultures which have properties similar to nerve cells are commonly used in the research laboratory for neuroscientific research. Greene and Tischler'3 established a nerve growth factor (NGF) responding clone line of rat pheochromocytoma (PC 12) cells in 1976. This clone cell line possesses many of the physiologicalproperties of primary cultures of sympathetic ganglion neuron^^^-^^ and has been studied as a model for the developing sympathetic nerve.16 PC12 cells (with or without NGF treatment) can synthesize, store, and release catecholamines in a manner similar to sympathetic ganglion neur0ns.13-~~ They are more similar to sympathetic ganglion neurons than to chromaffin cells, because they contain more dopamine (DA) than norepinephrine (NE) (the NE/DA ratio varies from 0.003 to 0.53)17,18with no detectable level of epinephrine (E). ~~
(9) Shepherd, G. M. Neurobiology, 2nd ed.; Oxford: New York,1988; Chapter 2, pp 35-38. (10) Shepherd, G. M. Neurobiology, 2nd ed.; Oxford: New York,1988; Chapter 4, pp 79-80. (11) Coupland, R. E. Nature 1968, 217, 384-388. (12) Dahlstrh, A.; Hbggendal, J.; HBkfelt, T.Acta Physiol. Scand. 1966, 67, 289-294. (13) Greene, L. A.; Tischlcr, A. S. Proc. Natl. Acad. Sei. U.S.A. 1976,73,24242428. (14) Greene, L. A.; Rein, G. Brain Res. 1977, 138, 521-528. (15) Schubert, D.; LaCorbiere, M.; Klier, F. G.; Steinbach, J. H. Brain Res. 1980, 190,67-79. (16) Wagner, J. A. J. Neurochem. 1985, 45, 1244-1253. (17) Clift-OGrady, L.; Linstedt, A. D.; Lowe, A. W.; Grote, E.; Kelly, R. B. J. Cell Biol. 1990, 110, 1693-1703. (18) Takashima, A.; Koike, T. Biochim. Biophys. Acta 1985,847, 101-107.
AnaMicalChemistry, Vot. 66,No. 19, October 1, 1994 3031
Most importantly, the PC12 cell vesicles may be considered valid analogues of brain synaptic vesicles for four criteria: size, density, protein composition, and endocytotic origin.17 Wagner16 has also reported that secretory vesicles in PC12 cells have a mean radius of 74 nm. This suggests that PC12 cells can be used as models of neurons to further understand exocytosis at the synaptic junction. This paper describes an amperometric system developed to monitor individual vesicular release from single nerve cells. The nerve cell model system used in this study is from a locally maintained rat pheochromocytoma cells line (PC 12) without NGF treatment. Amperometry with 1.2 ms time resolution and a detection limit of 31 zmol has been used to monitor single exocytotic events from neuronal-like PC12 cells.
EXPER I MENTAL SECTION Electrodesand Apparatus. Carbon fiber working electrodes were prepared by a procedure similar to the scheme described by Kelly and Wightman.lg In brief, a 5 pm diameter carbon fiber (Amoco, Greenville, SC) was sealed in a 1.2 mm X 0.68 mm glass capillary (A-M Systems, Everett, WA) by pulling the glass tube with a vertical capillary puller (Ealing, Harvard Apparatus, Edenbridge, KY). The tip of the electrode was then dipped into epoxy (Epo-Tek 301, Epoxy Technology, Billerica, MA) for 30 s and cured at 100 OC for 60 min. The carbon fiber disk was exposed by cutting the end of the glass using a scalpel. Electrodes were polished at an angle near 50° on a micropipet beveler (World Precision Instruments, New Haven, CT), with diamond paste (0.25 pm diameter, Buehler, Lake Bluff, IL) for 2 min. Electrodes were examined under the microscope and tested in 0.1 mM dopamine solution by cyclic voltammetry before use. Locally constructed sodiumsaturated calomel electrodes (SSCE) were used as reference electrodes. Amperometric detection was carried out with an EI-400 potentiostat (Ensman Instrumentation, Bloomington, IN) at 0.65 V vs SSCE. A built-in two-pole low-pass filter in EI-400 was set at 100 Hz; no other electronic filtering device was used. A Gateway 2000 386125 PC computer with 8 MB RAM memory and a commercial interface (Lab Master, Scientific Solutions,Solon, OH) were used for data acquisition, display, and analysis. Amperometric data points were collected and displayed every 1.22 ms (820 Hz). This was limited by the software requirement for real-time data display of the amperometric data obtained. All apparatuses were grounded though a common ground. Shielding with aluminum foil was used where necessary to minimize the electrical noise. Cell Culture and Buffer Solutions. Stock PC 12 cells were obtained from American Type Culture Collection (ATCC, Rockville, MD) and maintained in phenol red free RPMI1640 media supplemented with 10% heat-inactivated horse serum, 5% fetal bovine serum (JRH Bioscience, Lenexa, KS), and 1% of antibiotic (100 units/mL) antimycotic (0.1 mg streptomycin/mL and 0.25 pg/mL) solution, in 8% COZ atmosphere at 37 OC, as described by Greene and Tischler.13 L-Ascorbic acid (0.015 g/L) was added into the culture medium for the first 2 days after each cell propagation. Cells were grown to near confluence in mouse collagen IV coated (5 pg/cm2, Collaborative BiomedicalProducts, Bedford, MA) (19) Kelly, R. S.;Wightman, R. M.Anal. Chim. Acta 1986, 187, 79-87.
3032 AnalyticalChemistry, Vol. 66,No. 19, Ocrober 1, 1994
Falcon cell culture dishes (60 mm) and were washed twice with 4 mL of warm (25-30 "C) unsupplemented media immediately prior to experiments, unless otherwise indicated. Nicotine (1 mM final concentration) was added to a K+elevated balanced salt solution containing 105 mM KC1 and 50 mM NaC1,2 mM CaC12,0.7 mM MgC12, 1 mM NaH2Pod, and 10mM Hepes for single-cellexperiments as chemical stimulant. For the Ca2+-free solution, MgCl2 was used to substitute for CaCl2 in order to maintain the ionic strength. All chemical stimulation solutions contained 0.1% (by weight) fast green dye as a visual aid. Single-cell Experiments. All cell experiments were performed on the stage of an inverted microscope (IM-35, Carl Ziess, Thornwood, NY) at room temperature as shown in Figure 1. The working electrode was placed on a target cell and gently pushed down against the cell body (about 1 pm) with a piezomicropositioner (PCS-750/ 1000, Burleigh, Fishers, NY). Micropipets containing stimulants were positioned 10 pm above the bottom level of the cell culture dish and 150 pm away from the target cell by micropositioners (Mertzhouser). Chemical stimulants were delivered by a pressure pulse from a microinjector (Picospritzer 11, General Valve, Fairfield, NJ) for 6 s. The estimated total injection volume was 30 nllinjection. All experimental data discussed here were obtained from PC12 cells without NGF treatment. To eliminate unwanted effects from extracellular catecholamines and cellular recovery time, only data from the first stimulation were used for analysis. All errors are reported as standard error of the mean (SEM). Data Treatment. A locally written program was used to acquire, store, display, and analyze data. Voltageoutput from the potentiostat was first digitized by an AID converter and then displayed as current vs time and stored in RAM memory for later analysis. Peak identification was carried out by using the starting point of four consecutivedata points with a positive slopegreater than 1.4 times the slope from 18 different 60 Hz noise waves as starting point of a peak. The first point of four consecutivedata points with a positive slope immediately after a negative slope or the value of the starting point of the peak, whichever was larger, and was used to mark the end of the peak. Only peaks with baseline width between 9 and 29 ms (about 99% of the peaks identified) were considered due to the software limitation. The number of molecules corresponding to the area under each current transient was calculated from the charge, Q = nNF,where n was assumed to be 2 (for catecholamines), N was the number of moles, and F was Faraday's constant (96 485 Clequiv). The charge, Q, for each current transient was obtained as the sum of the areas under each set of two sequential data points in the current transient by assuming a straight line between points as the upper limit and the baselineas the lower limit. For a transient having 12.2 ms width, 10 data points were obtained. ChemicalsandMaterials. All chemicals and materials were obtained from Sigma Chemical Co. (St. Louis, MO) and used without further purification. All buffers were prepared, adjusted to pH 7.4 in locally prepared doubly distilled water (Corning Mega-Pure MP-3A purification system, Corning, NY), and sterilized by filtering through 0.2 pm membrane filter units (Nalgene, Rochester, NY).
Figure 1. Experimental setup for PC12 cell experiments. The cell used is marked with an arrow. A carbon fiber working electrode (A) was placed on the cell surface and gently pushed down about 1 pm to increase the amperometric signal observed during the exocytosis. Micropipets (B, C) filled with chemical stimulants were placed about 150 pm away from the cell and about 10 pm above the bottom of the cell dish.
RESULTS AND DISCUSSION Amperometric Detection of CatecholaminesReleased from PC12 Cells. Until recently, the primary dynamic evidence for quantal release at single hormonal or nerve cells has been obtained indirectly by measuring cell membrane capacitance changes.6+20In 1990,Wightman and co-workersdemonstrated for the first time that microelectrodescould be used to directly monitor catecholaminesreleased in singlequanta from adrenal cells in culture.2 Each granule (vesicle) in an adrenal cell contains 5-1 0 amol of catecholamine (norepinephrine, epinephrine, or In contrast, catecholamine vesicles in synaptic terminals typically contain estimated amounts of neurotransmitter from 1.8 to 96.4 zmol.12 A model system that contains small vesicular quantities is needed. The PC12 cell line provides an excellent neuronal model system for development of methods to monitor synaptic quantal release. PC 12 cells share many physiological properties with primary cultures of sympathetic ganglion n e u r o n ~ . ~In~ addition, J~ it is relatively easy to maintain the PC12 cell line in the laboratory. Finally, secretory vesicles in PC12 cells contain dopamine and norepinephrine and have sizes and properties similar to those in dopaminergic n e u r o n ~ . ~ ~ J To ~ +detect ~~-*~ quantal release from single PC12 cells, a carbon fiber electrode is placed on the cell in culture and the cell is bathed with approximately 30 nL of a solution containing nicotine and elevated potassium chloride. For initial experiments, this solution assured cell stimulation by affecting any available (20) (21) (22) (23)
Neher, E.; Marty, A. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 6712-6716. Greene, L. A.; Rein, G. J . Neurochem. 1978, 30, 549-555. Baizer, L.; Weiner, N. J . Neurochem. 1985, 44, 495-501. Courtney, N . D.; Howlett, A. C.; Westfall, T. C. LiJe Sci. 1991, 48, 16711678.
nicotinic receptors and by direct membrane depolarization by elevated potassium chloride. This stimulus results in multiple current transients that appear to be due to the oxidation of catecholamines from individual exocytotic events (Figure 2). Each vesicle contains approximately 200 zmol of catecholamine. In order to detect zeptomole levels of DA and NE released from PC12 cells, oxidation current owing to the transmitters has to be maximized while system noise is minimized. To increase the signal level, the working electrode is gently pushed against the cell surface to minimize the volume between the cell surface and the electrode. The applied potential and physical force of the working electrodedo not seem to interefere with normal cellular function,as unstimulated vesicular release has only been observed occasionally as random current transients of very low frequency (less than 1 Hz). These transients could result from small amounts of stimulant leaking from the injection pipet. Hence, control experiments have been carried out by placing an electrode in contact with a cell when no injection pipet is present. No current transients have been observed in these experiments (n = 3 cells). Interference noise has been minimized by isolating and grounding all key electronic components of the system. In the best cases, the peak-to-peak noise has been limited to 0.6 pA with a two-pole low-pass filter (rolloff point at 100 Hz). Assuming a peak width of 10 ms and a peak height of 1.2 pA (2 X p p noise) for a current transient resulting from a singleexocytoticevent, this leads to a total charge of 6 fC, correspondingto a detection limit of 3 1 zmol for catecholamines. The detection limit can be further improved if more sophisticated noise reduction schemes are employed. Time-resolved amperometric signals AnalyticalChemistry, Vol. 66, No. 19, October 1, 1994
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a.
3.8 pA
3.8 pA
L 87 s
36.6 c
b.
t
3.8 pA
1 36.6 s
C.
I 1
Flguro 2. Amperograms of individual vesicular exocytosis from PC 12 cells. (a) Amperogram of a control experiment with the tip of the working electrode 200 pm from the cell. (b) Amperogram of vesicular exocytosis induced by bathing a cell wLh 1 mM nicotine in 105 mM K+ balanced salt solution while the electrode was placed on top of the cell. (c)Enlargement of a 1 s period of the amperogram from the first stimulation In Figure 2b. Data dlsplayed correspond to the time period near the middle of the first baseline disturbance of Flgure 2b. Traces a and b were obtained from computer screen dumps: each prlnted line represents 600 data points. Arrows represent the time at which stlmulatlons were applied.
from cell experiments can be displayed in real time and saved into RAM for later processing. The real-time display ability also provides a nondestructive means to identify individual cells in the cell cultures or tissue preparations that contain easily oxidized amines as neurotransmitters. Ca2+Dependence of CatecholamineExocytosis from PC12 Cells. Free calcium ions are considered to play an obligatory role in cellular exocytosis events. Cultured PC12 cells stop secreting transmitter in a Ca2+-freemedia.14*21 To investigate the Ca2+ dependence of vesicular release observed with amperometry, PC12 cell cultures were washed and bathed in Caz+-free balanced salt solution immediately before the experiments. When PC 12 cells in Ca2+-freeculture preparations are stimulated with an injection of Ca2+-free 105 mM K+ balanced salt solution (Figure 3, injections 1 and 3), and with an injection of 12 mM Ca2+ in 105 mM K+ solution (Figure 3, injections 2 and 4), oxidation current transients are only observed following the stimulation with Ca2+ present. Hence, the released substance appears to result from exocytosis.9.14,22,23 Discussion of Catecholamine Exocytosis from PC12 Cells. Bathing PC12 cells in either elevated K+solutions2lor nicotine (10 pM-1 mM) solutions14evokes DA and NE release from PC12 cells in culture. The nicotine-induced secretion of catecholamines in PC 12 cell cultures gradually slows down 3034 Analyfical Chemistry, Voi. 66,No. 19, October 1, 1994
t
2
t
3
t
4
Flgure 3. Ca2+dependence of release from PC12 d s . Amperogram of slngleveslcular release froma PC12dbathedInCa2+-freabalanced salt solution. Arrows and numbers represent times and sequence of Stimulation. Ca2+-free105 mM K+ balanced salt solution was applied at stimulations labeled 1 and 3; 12 mM Ca2+ In 105 mM K+ balanced saR solution was applied at stimulations labeled 2 and 4. The amperogramshown here was obtained froma computer screen dump: each prlnted line represents 1420 data points.
to undetectable levels within 5 min. Catecholamine secretion returns in smaller amounts after cells are allowed to rest for 20 minor morel4in a normal environment. Since this secretory behavior of PC12 cells is Ca2+ dependent, it is generally believed that the catecholamines detected are released via e~ocytosis.1~.~~ However, to the best of our knowledge, no direct chemical evidence of vesicular exocytosis from PC 12 cells has been reported. Amperometric detection has been used to monitor chemically induced quantal transmitter release from PC12 cells. In these experiments, chemical stimulation of PC12 cells is achieved by localized perfusion (30 nL totalvolumeestimated) of 1 mM nicotine in 105 mM KCl balanced salt solution for 6 s. A control experiment has been carried out by positioning the electrode near the bottom of the cell culture dish about 200 pm away from any PC12 cells. When the electrode tip is bathed with 30 nL of stimulation solution, a slow change in the amperometric response is observed (Figure 2a). Figure 2b shows a typical compressed amperogram for three consecutive stimulations obtained with the electrode positioned on top of a PC12 cell. Narrow current transients are clearly observed at times corresponding to the first two chemical stimulations. Only background current is observed following a third cell stimulation. It appears that thevesicles responsible for the release of catecholamines are depleted with multiple stimulations on this time scale. This is in agreement with data from transmitter release experiments conducted with whole cell culture ~reparati0ns.l~ Because catecholamines (DA and NE) are the only known electrochemically active neurotransmitters present at significant levels in PC12 cell cultures,13-15 the easily oxidized substances responsible for the current transients observed have been tentatively identified as catecholamines. Figure 2c shows a 1 s section (820 data points) of the amperogram corresponding roughly to the time period when the largest current transient was observed during the first stimulation. A series of spikes can be seen on the amperogram during the 1 s period. The shape of each transit is similar to those observed from chromaffin cells by Wightman et a1.l except the average charge for each transient is significantly smaller. Each individual current transient appears to correspond to a single vesicular release Individual current transients from PC12 cell secretion have an average width at half-height equal to 9.3 ms (SEM = 0.1,
"I
corresponding to less than 330 zmol (200 000 molecules). Based on the previously estimated vesicular radius (74 nm),15 the average vesicular transmitter concentration in PC12 cells is estimated to be 0.11 M.
n
, , , ,
1
0
,
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0.63
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Attomole per Vesicle Flgure 4. Frequency distribution of the amount of catecholamine contained in individual current transients from single ceiis. The bins are divided into 66 zmol intervals. Numbers on the x-axis represent the mean of each interval. Data shown here were obtained from the first stimulation of 13 individual cells; 1912 spikes have been included.
n = 1912 from 13 individual cells). Nearly all (99%) of the current transients observed have baseline widths between 10 and 28 ms, suggesting that the size of secretory vesicles in PC12 cells is relatively uniform. Figure 4 shows a histogram of the amount of catecholamine released per vesicular event for exocytosis from 13 individual PC12 cells. The skewed histogram is consistent with data obtained from bovine chromaffin cells.'J The average amount of catecholamines released per vesicle from PC12 cells following stimulation with nicotine and K+ is 190 zmol (1 14 300 molecules, from 1912 spikes, SEM = 3.5 zmol). Thedistribution of vesicular quantities is relatively narrow with 85% of current transients
CONCLUSIONS A high-sensitivityamperometric detection scheme has been described to monitor current transients from catecholamine exocytosis from single monoaminergic cells. When this detection method is applied to PC12 cells, a dopaminergic nerve cell model, chemically induced exocytic release can be successfully monitored. Quantal release from PC12 cells is Ca*+ dependent, indicating that the release is the result of calcium-dependent exocytosis. The evidencethat the observed current transients represent exocytosisfrom individual vesicles includes the small amount of neurotransmitter released, the shape of the current transients, the shape of the histogram of frequency vs size of release events, and the time scale of the release events ( 4 0 ms average). Calculations assuming that only single vesicles are observed indicate that each vesicle has an average of 190 zmol (SEM = 3.7 zmol) of releasable transmitter. The detection limit in this system has been calculated as low as 31 zmol. ACKNOWLEDGMENT This work was supported in part by grants from the Office of Naval Research and the National Science Foundation. A.G.E. is a Camille and Henry Dreyfus Teacher Scholar. Received for review May 12, 1994. Accepted July 22, 1994." Abstract published in Aduance ACS Abstracts, August 15, 1994.
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