Anal. Chem. 1996, 68, 3912-3916
Identification of Multiple Compartments of Dopamine in a Single Cell by CE with Scanning Electrochemical Detection Franklin D. Swanek, Guangyao Chen, and Andrew G. Ewing*
152 Davey Laboratory, Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania 16802
A method is described for the direct identification of dopamine from two separate vesicular compartments of a fully developed neuron in Planorbis corneus by capillary electrophoresis with scanning electrochemical detection. By manipulating the lyse time of the neuron after it is injected on the capillary, the two vesicular compartments are observed as two individually resolved peaks. Scanning electrochemical detection allows voltammetry to be obtained as the peaks elute in order to confirm their identity. Quantitation was accomplished through the use of calibration curves. The average amount of dopamine present was found to be ∼400 fmol. Dopamine concentrations were also determined and compared favorably with those of previous experiments on P. corneus. The extreme complexity of the nervous system has led many researchers to break it down into different regions and substructures for study. Extracellular fluid, whole cells, and subcellular regions such as cytoplasm and vesicles have been studied in this manner and are the subject of several recent reviews.1-6 To understand how these substructures work together to make up the nervous system, it is necessary to understand how cells communicate with one another. Communication in the nervous system occurs through the process of neurotransmission, where chemical messengers are released from one cell to elicit a response from a neighboring cell. These messengers are often stored in vesicles and are released through a process called exocytosis. The giant dopamine-containing neuron in the brain of the pond snail, Planorbis corneus, provides an excellent model for the study of neurotransmission in single cells. It is relatively large (75-200 µm in diameter), easily identified, and known to contain the neurotransmitter dopamine, and it has been shown to store dopamine in vesicles and release it through exocytosis.7-9 (1) Ewing, A. G.; Strein, T. M.; Lau, Y. Y. Acc. Chem. Res. 1992, 25, 440-447. (2) Wightman, R. M.; Finnegan, J. M.; Pihel, K. Trends Anal. Chem. 1995, 14, 154-158. (3) Huang, L.; Kennedy, R. T. Trends Anal. Chem. 1995, 14, 158-164. (4) Jankowski, J. A.; Tracht, S.; Sweedler, J. V. Trends Anal. Chem. 1995, 14, 170-176. (5) Gilman, S. D.; Ewing, A. G. J. Cap. Electrophor. 1995, 2, 1-13. (6) Swanek, F. D.; Ferris, S. S.; Ewing, A. G. In Handbook of Capillary Electrophoresis, 2nd ed.; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1996. (7) Berry, M. S.; Pentreath, V. W; Turner, J. D.; Cottrell, G. A. Brain Res. 1974, 76, 309-324. (8) Pentreath, V. W; Berry, M. S.; Cottrell, G. A. Cell Tissue Res. 1976, 151, 369-384. (9) Chen, G.; Gavin, P. F.; Luo, G.; Ewing, A. G. J. Neurosci. 1995, 15, 77477755.
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The existence of two vesicular compartments of neurotransmitter has long been hypothesized.10,11 This hypothesis states that two distinct types of neurotransmitter-containing vesicles exist within nerve cells. The first is described as a functional compartment, as it is available for immediate release via exocytosis upon stimulation. The second type is nonfunctional, as it is internalized within the cell and not available for immediate release. The second type might be a long-term storage compartment or possibly play a role in potentiation of response after long-term stimulation. To determine the validity of this model, a technique must be employed which can sample from the cellular environment and detect minute quantities present there. Microscale separation techniques such as capillary electrophoresis (CE) and opentubular liquid chromatography (OTLC) provide an excellent means to study the nervous system at the single-cell level.12-14 Using CE, Kristensen et al. reportedly observed two vesicular compartments of dopamine in the giant dopamine neuron of P. corneus.15 Capillary electrophoresis was utilized to inject an intact cell, lyse the plasma membrane on column, and separate the cellular components. A cation was detected with the same electrophoretic mobility as dopamine, as well as a neutral species and the anionic metabolites uric acid and dihydroxyphenylacetic acid (DOPAC). In addition to the identifiable species, a second cation was observed. When a longer lyse time was permitted, the second peak disappeared, while the first became larger. Upon incubation of the cell with reserpine, a vesicle depleting agent, only the first peak was observed. Incubation with amphetamine, which also displaces dopamine from the vesicles into the cytoplasm, has also been demonstrated to deplete the second peak, resulting in a single dopamine peak.16 This evidence strongly suggests that the two cations observed in these separations are, in fact, both dopamine. The second, unidentified peak was believed to be due to dopamine present in a second compartment that was observed when the cell was not completely lysed before beginning the separation. In support of the two-compartment model, the first dopamine peak represented the functional compartment which was immediately released when the cell began (10) Besson, M. J.; Cheramy, A.; Feltz, P.; Glowinski, J. Proc. Natl. Acad. Sci. U.S.A. 1969, 62, 741-748. (11) Shore, P. A. J. Pharm. Pharmacol. 1976, 28, 855-857. (12) Kennedy, R. T.; Oates, M. D.; Cooper, B. R.; Nickerson, B.; Jorgenson, J. W. Science 1989, 246, 57-63. (13) Mesaros, J. M.; Ewing, A. G.; Gavin, P. F. Anal. Chem. 1994, 66, 527A537A. (14) Ewing, A. G. J. Neurosci. Methods 1993, 48, 215-224. (15) Kristensen, H. K.; Lau, Y. Y.; Ewing, A. G. J. Neurosci. Methods 1994, 51, 183-188. (16) Sulzer, D.; Chen, T.-K.; Lau, Y. Y.; Kristensen, H. K.; Rayport, S.; Ewing, A. G. J. Neurosci. 1995, 15, 4102-4108. S0003-2700(96)00570-7 CCC: $12.00
© 1996 American Chemical Society
to lyse, whereas the second represented the nonfunctional compartment which was internalized and more difficult to lyse. However, to further support this theory, direct identification of the peaks as they elute from the capillary is necessary. In the previously described experiment, amperometric detection was used to detect species as they eluted from the CE column. Here, a species is detected if it is oxidized at the potential of the electrode and results in a current response. The technique is highly sensitive, with the capability of detecting species at attomole to zeptomole (10-18 to 10-21) levels; however, no information is provided as to the identity of the species. Scanning electrochemical detection with capillary electrophoresis provides an excellent means to confirm the identity of the species detected. Scanning electrochemical detectors are not new to separations, as their application to LC and flow injection analysis systems is well documented.17-20 Recently, Ferris et al. demonstrated the ability of scanning electrochemical detection to increase resolving power of closely eluting solutes in capillary electrophoresis separations.21 The principle of scanning electrochemical detection is that the potential applied to the electrode is rapidly stepped over a range of voltages while the current is measured at each voltage. The current versus voltage trace at any given time during the separation yields a characteristic voltammogram for the analyte under study. Therefore, scanning electrochemical detection can be used as a means of identification, as the voltammetry of an analyte may be used to distinguish it from other compounds. This paper describes the application of scanning electrochemical detection to identify the peaks in the capillary electrophoretic separation of components in the giant dopamine neuron of P. corneus. Voltammetric information is presented that indicates dopamine is stored in two separate compartments within the cell. The effect of lyse time on the two compartments is examined. Finally, quantitation of the two compartments as well as wholecell dopamine content is shown. EXPERIMENTAL SECTION Capillary Electrophoresis System. The capillary electrophoresis system used for these experiments was similar to that described by Sloss and Ewing.22 Briefly, the apparatus consisted of a fused silica capillary with dimensions of 25-µm i.d., 150-µm o.d. (Polymicro Technologies, Phoenix, AZ), cut to a length of 80-85 cm and placed between two buffer reservoirs with high voltage applied at the injection end, while the reservoir containing the electrochemical detection system was held at ground potential. Separations were carried out at an applied voltage of 25-30 kV. Injections were performed electrokinetically, with injected volumes calculated on the basis of electroosmotic flow as measured by a neutral marker. Both the injection and detection ends of the capillary were specially treated prior to use. On the injection end, a microinjector was fashioned to facilitate the injection of a whole cell. On the detection end, a similar treatment was performed to allow end column detection. The procedures for both techniques are similar and have been described previously.15,22 Briefly, microinjectors (17) White, J. G.; St. Claire, R. L.; Jorgenson J. W. Anal. Chem. 1986, 58, 293298. (18) Kennedy, R. T.; St. Claire, R. L., III; White, J. G.; Jorgenson, J. W. Mikrochim. Acta 1987, 11, 37-45. (19) Kennedy, R. T.; Jorgenson, J. W. Anal. Chem. 1989, 61, 436-441. (20) Trojanek, A.; De Jong, H. Anal. Chim. Acta 1992, 11, 325-329. (21) Ferris, S. S.; Luo, G.; Ewing, A. G. J. Microcolumn Sep. 1994, 6, 263-268. (22) Sloss, S. E.; Ewing, A. G. Anal. Chem. 1993, 65, 577-581.
were prepared by removing approximately 5 mm of the polyimide polymer coating from one end of a capillary to expose the fused silica. The tip was then etched in 40% aqueous hydrofluoric acid (HF) for 15 min while purging the capillary with 80 psi of helium. This etches the outside of the capillary to approximately 100 µm and the inside to a funnel-like structure of about 75 µm at the tip. After etching, the tip was neutralized in sodium bicarbonate and washed with doubly distilled water. For the detection end of the capillary, the procedure was repeated, except that the capillary was etched for a shorter time, creating an inner diameter of approximately 40 µm. Scanning Electrochemical Detection. Detection was performed in the scanning electrochemical mode with a two-electrode cell. The working electrode consisted of a 5-µm-diameter carbon fiber (Amoco Performance Products, Greenville, SC) with an exposed length of 200-500 µm protruding from a drawn glass capillary. The electrode was inserted into the etched end of the electrophoresis capillary. The potential of the working electrode was referenced to a Ag/AgCl electrode (World Precision Instruments, Sarasota, FL). A 486DX33 personal computer (Gateway 2000, N. Sioux City, SD) was interfaced to a 12-bit Lab Master interface (Scientific Solutions, Solon, OH) and used for waveform generation and data acquisition. Electrode scanning and data acquisition were controlled by software written in-house. The D/A converter was connected to the reference electrode through a potentiostat (EI400 Ensman Instrumentation, Bloomington, IN) and was used to control the electrochemical cell potential. The potential at the electrodes was scanned in a staircase fashion from -0.2 to +1.0 V at a rate of 1 V/s in 6-mV steps, as depicted in Figure 1. One complete scan required 1.2 s, with a 0.2-s delay between scans. The potentiostat was used to amplify the current at the working electrode before connection to the A/D converter. A gain of 2 × 108 V/A and a filter cutoff frequency of 10 Hz were employed. After acquisition, data could be viewed in three formats: current versus potential (voltammogram), current versus time at any chosen potential (electropherogram), or current versus potential and time (threedimensional surface plot). Software-controlled options for data treatment included background subtraction in the voltammetric mode. Three-dimensional surface plots were obtained by importing the data into Transform (Fortner, Inc., Sterling, VA). Reagents. Dopamine (DA), catechol (CAT), and 2-(N-morpholino)ethanesulfonic acid (MES) were obtained from Sigma and were used as received. The electrophoresis buffer was 25 mM MES, adjusted to pH 5.65 with NaOH. Calibration standards were prepared as 10 mM stock solutions in perchloric acid and diluted to the desired concentration in electrophoresis buffer. HF was obtained as a 40% aqueous solution from Aldrich. Cell Preparation and Microinjection of Whole Cells. Planorbis corneus (Nasco, Fort Atkinson, WI) were dissected as described by Chien et al.,23 except all the membranes and connective tissues on the left pedal ganglion were removed so that the whole dopamine cell was exposed. The etched tip of the capillary was placed in contact with the giant dopamine cell (bathed with Ringer’s solution), using a micromanipulator (E. Leitz, Inc., Rockway, NJ) with the aid of a stereomicroscope (Carl Zeiss, Inc., Thornwood, NY). Cell sizes were measured with the aid of a calibrated eyepiece accurate to (1 µm. A platinum wire (23) Chien, J. B.; Wallingford, R. A.; Ewing, A. G. J. Neurochem. 1990, 54, 633638.
Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
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Figure 1. (A) Schematic depiction of the potential waveform used in scanning electrochemical detection. Potential is stepped from 0.0 to 1.2 V at a rate of 1 V/s, with a 0.2-s delay between subsequent scans. (B) Representation of a voltammogram obtained by monitoring current versus voltage over the course of one scan. (C) Electropherogram obtained by monitoring current at one electrode potential over the course of many scans.
was placed in the Ringer’s solution to serve as the electrophoresis anode, and an injection potential of 10 kV was applied for ∼10 s to transport the whole cell into the etched capillary tip. After injection, the etched end of the separation capillary was removed from the Ringer’s solution and placed into a buffer reservoir. Cell lysis was accomplished by injecting a plug of the separation buffer over the cell and allowing it to incubate. The effect of lyse time was studied with times ranging from 1 to 10 min. After the cell was lysed, a potential of 25 kV was applied to carry out an electrophoretic separation. RESULTS AND DISCUSSION An electropherogram of the total cellular contents of a single dopamine neuron from P. corneus after a 1-min lyse time is shown in Figure 2A. Scanning electrochemical detection has been employed in this separation; however, for clarity, the data are plotted simply as the current versus time at an electrode potential of 0.75 V. Figure 2B shows the post-cell standard injection of dopamine and catechol. Three peaks are readily detected in the cell separation: one at a time corresponding to the electrophoretic mobility of dopamine (A), another for a cation eluting slightly later than dopamine (B), and a third due to uncharged electroactive species in the cellular buffer (C). DOPAC and uric acid are present at low levels and are often observed in cell separations, but they are not present here. This same peak sequence has been observed in previous experiments involving the whole-cell sampling of P. corneus.15,24 In these experiments, the identity of the (24) Olefirowicz, T. M.; Ewing, A. G. Chimia 1991, 45, 106-108.
3914 Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
Figure 2. (A) Electrophoretic separation of the components of a single dopamine neuron after injection and lysing in the capillary tip for 1 min with buffer. (B) Separation of a standard solution containing 10-5 M DA and catechol. Conditions: capillary length, 85 cm; buffer, 25 mM MES (pH ) 5.65); injection, 5 s at 5 kV (0.68 nL); separation potential, 25 kV. The electropherograms were obtained by extracting the current versus time data at 0.75 V versus Ag/AgCl from the total scanning data that were collected.
second cation (B) has been assigned to dopamine. This behavior may be explained by a model which states that dopamine is stored in two separate compartments within the cell. The first compartment (peak A) is composed of dopamine stored in functional vesicles that are released due to the injection procedure in addition to a 1-min lysing of the cell. Also present in peak A is cytoplasmic dopamine. However, previous studies have shown that cytoplasmic dopamine makes up only 2% of the total stores under resting conditions.23 Peak B has been hypothesized to be made up of dopamine that is stored in vesicles deeper within the cell in a nonfunctional compartment that is not immediately available for stimulated release. This dopamine must wait for running buffer to penetrate and lyse the interior structure of the cell before it is free to begin migration. The time required for this to occur represents the time difference between peaks A and B in the separation. In addition, this process may add additional width to peak B, as the lysing occurs over a finite time during the separation. The electropherogram shown in Figure 3 is another separation of the contents of a giant dopamine neuron. In this case, the cell was allowed to lyse on the column for a period of 10 min rather than 1 min. In the resulting electropherogram, only one peak is observed due to cationic species. This peak migrates with an electrophoretic mobility similar to that of the peak A in Figure 2A, hence the typical mobility of dopamine (note the separation
Figure 3. Electrophoretic separation of the components of a single dopamine neuron after lysing in the capillary tip for 10 min. Conditions are as described in Figure 2, except the capillary length, which was 90 cm. The rate of electroosmotic flow differs from that of Figure 2.
Figure 5. Three-dimensional electropherogram of the electrophoretic separation of the contents of a single dopamine neuron shown in Figure 2A.
Figure 4. Three-dimensional electropherogram of the separation of dopamine and catechol shown in Figure 2B. Electrode potential is scanned from 0.0 to 1.2 V at a rate of 1 V/s.
is on a different capillary and both peaks are shifted to longer times). The reason for this occurrence is most likely due to the facts that the cell now has sufficient time to lyse all the membranes and that all the dopamine contained in internalized vesicles begins migrating at the same time and is, therefore, detected as a single peak. The indirect evidence presented thus far is fairly convincing; however, direct identification of the two compartments as they elute is required in order to confirm this hypothesis. Figure 4 illustrates a three-dimensional surface plot of the data obtained through scanning electrochemical detection of a standard mixture of dopamine and catechol. The two peaks are clearly resolved along the time axis. In addition, if the current is monitored along the potential axis at a time corresponding to the elution of a peak, the voltammogram for that peak may be extracted, and voltammograms for dopamine and catechol are significantly different. Thus, scanning electrochemical detection provides direct qualitative information. In Figure 5, scanning electrochemical detection has been applied to a cell separation. While Figure 2A displays the electrophoretic data from a single electrode potential, Figure 5 represents the total data over all electrode potentials from 0.0 to 1.0 V. The three peaks detected correspond to the two dopamine peaks and neutral species observed in the separation shown in Figure 2A.
Figure 6. Background-subtracted voltammograms obtained from peaks A and B in Figure 5. Shown is the voltammogram from 0.0 to 0.6 V versus Ag/AgCl.
Figure 6 illustrates how the voltammetry that is obtained aids in identification of the peaks in a separation. The voltammograms of the peaks A and B from Figure 5 have been extracted and plotted together. Shown is the forward wave of a linear sweep voltammogram from 0.0 to 0.6 V versus a Ag/AgCl electrode. The resulting voltammograms are almost identical. This strongly suggests that both peaks detected from the P. corneus neuron are, indeed, dopamine. Another possible interpretation of the voltammetry could be that one of the peaks is a molecule similar to dopamine, such as epinephrine or norepinephrine. However, previous studies confirm that neither of these compounds is Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
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Table 1. Quantitation of Dopamine from Whole Dopamine Neurons by Capillary Electrophoresis lyse 1 min
peak 1 peak 2 total ratio of peak 2/1 a
(in fmol) (in µM) (in fmol) (in µM) (in fmol) (in µM)
lyse 10 min
x
SD
x
SD
174 301 287 564 461 865 1.6
114 143 114 289 92 278 0.64
344 601 nda nda 344 601 nda
162 187 nda nda 162 187 nda
Not detected.
present in this cell.25-27 This interpretation would also be inconsistent with the behavior observed at different lyse times. Although scanning electrochemical detection has been used as a means of qualitative identification, quantitative information is not sacrificed. The current response in scanning electrochemical detection has been shown to be linear over the concentration range of 10-3-10-5 M.21 Therefore, the current response from a single electrode potential or the averaged response over a range of potentials as measured from a standard of known concentration may be compared with the response from a cell in order to determine the amount of dopamine in each vesicular compartment or the entire cell. The results of this study are summarized in Table 1. The total amount of dopamine contained within a cell may be obtained by adding the amounts detected for peaks 1 and 2 for cells separated after a 1-min lyse time. This yields a result of 461 ( 92 fmol. In comparison, the amount detected in the single dopamine peak resulting from a 10-min lyse time is 344 ( (25) Powell, B.; Cotrell, B. A. J. Neurochem. 1974, 22, 605-606. (26) Osborne, N. N.; Priggemeier, E.; Neuhoff, V. Brain Res. 1975, 90, 261271. (27) Berry, M. S.; Penreath, V. W. In Biochemistry in Characterized Neurons; Osborne, N., Ed.; Permagon: Oxford, 1978.
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Analytical Chemistry, Vol. 68, No. 22, November 15, 1996
162 fmol. The two values are not different within the experimental uncertainty. This indicates that the two peaks observed after a short lyse time are made up of the same amount of dopamine as the single peak observed after long lyse times. This is not clear from previous experiments, where the results had a broader spread of data.15 The values reported here for the whole-cell dopamine concentration of dopamine in P. corneus do, however, agree with those previously reported, where considerably smaller cells were used (25-40 µm).15 To make the comparison, concentrations were calculated from the estimated cell volumes as determined from the measured radius while assuming a spherical geometry for the cell. CONCLUSIONS Scanning electrochemical detection has been used to identify the peaks from an electrophoretic separation of components of the giant dopamine neuron of P. corneus. The method demonstrates the utility of scanning electrochemical detection for both qualitative and quantitative identification in cellular analyses. Femtomole levels of dopamine have been quantitatively detected from single cells. Confirmation that two peaks, both with voltammetry identical to dopamine, are observed strongly supports the hypothesis that dopamine is stored in different vesicular compartments within the cell. In future experiments, it may be possible to see what other cell lines follow the two-compartment model and the implications this has on neurotransmission in these systems. ACKNOWLEDGMENT This research was supported, in part, by grants from the National Science Foundation and Office of Naval Research. Received for review June 11, 1996. Accepted August 30, 1996.X AC960570A X
Abstract published in Advance ACS Abstracts, October 15, 1996.
