Anal. Chem. 1999, 71, 28-33
Monitoring Cellular Release with Dynamic Channel Electrophoresis Yi-Ming Liu,† Tatiana Moroz, and Jonathan V. Sweedler*
Department of Chemistry and the Beckman Institute, University of Illinois, Urbana, Illinois 61801
A channel electrophoresis system consisting of a 50 µm by 75 mm by 25 mm separation channel has been adapted to follow stimulated release from individual and small groups of isolated neurons. The cells of interest are placed in a nanoperfusion chamber located near the exit of a sampling capillary. The capillary is scanned across the mouth of the channel so that compounds released from the cells are dynamically introduced into the separation channel. The position of the sampling capillary along the channel entrance yields temporal information, and electrophoresis in the channel length dimension provides the chemical data. NDA/CN- is placed in the inlet vial between the sampling capillary and channel so that primary amine-containing compounds released from the cell are derivatized prior to separation as they enter the channel. The performance of this method is evaluated, and the optimum NDA/CN- concentration and separation conditions for this on-line derivatization are presented, with detection limits for most underivatized amino acids of ∼500 nM at a particular time slice. The time-resolved electropherograms from single and a small group of cerebral ganglion neurons from Aplysia californica stimulated with KCl show multiple components released with different time courses. To understand cellular communication in even relatively simple systems of neurons, knowledge of the chemical, spatial, and temporal distribution of neurotransmitters and neuromodulatory compounds is important. The simultaneous time-resolved detection of multiple signaling molecules used by neurons is particularly difficult. At this time, no approach can completely provide such information. Various microscopic techniques with appropriate fluorescent probes and immunohistochemical techniques are commonly used to spatially localize specific compounds.1-3 In addition, there has been tremendous progress in using microseparations to provide snapshots of selected compounds in individual † Current address: Department of Chemistry, Jackson State University, Jackson MS 39217. (1) Haugland, R. P. Molecular Probes Handbook of Fluorescent Probes and Research Chemicals; Molecular Probes: Eugene, OR, 1996-8. (2) Kojima, H.; Nakatsubo, N.; Kikuchi, K.; Kawahara, S.; Kirino, Y.; Nagoshi, H.; Hirata, Y.; Nagano, T. Anal. Chem. 1998, 70, 2446-2453. (3) Bach, P., Baker, J., Eds. Histochemical and Immunohistochemical Techniques: Applications to Pharmacology and Toxicology; Chapman and Hall: New York, 1991.
28 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
neurons.4-9 Mass spectrometry is also useful for determining multiple compounds from single cells.10-13 As a small fraction of the material in a cell is released, even for relatively long release events,14,15 monitoring release is more difficult. The time-resolved detection of electroactive molecules released from individual cells using carbon fiber microelectrodes has been reported in several systems.16-20 However, many compounds are not electroactive and cannot be detected with this methodology. Tong and Yeung followed the release of epinephrine and norepinephrine from bovine adrenal chromaffin cells by the novel method of adhering the cell to the side of a CE capillary, stimulating them with a plug of acetylcholine and separating the released compounds in the separation capillary.21 Here we adapt a channel system to follow release from neurons with both the chemical specificity provided by a separation and the time resolution of multiple parallel assays. The approach is (4) Kennedy, R. T.; Oates, M. D.; Cooper, B. R.; Nickerson, B.; Jorgenson, J. W. Science 1990, 246, 57-63. (5) Lillard, S. J.; Yeung, E. S. Capillary electrophoresis for the analysis of single cells: laser-induced fluorescence detection. In Handbook of Capillary Electrophoresis, 2nd ed.; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1997; pp 523-544. (6) Swanek, F. D.; Ferris, S. S.; Ewing, A. G. Capillary electrophoresis for the analysis of single cells: electrochemical, mass spectrometric and radiochemical detection. In Handbook of Capillary Electrophoresis, 2nd ed.; Landers, J. P., Ed.; CRC Press: Boca Raton, FL, 1997; pp 496-522. (7) Jankowski, J. A.; Tracht, S.; Sweedler, J. V. Trends Anal. Chem. 1995, 14, 170-176. (8) Fuller, R. R.; Moroz, L. L.; Gillette, R.; Sweedler, J. V. Neuron 1998, 20, 173-181. (9) Ru ¨ chel, R. J. Chromatogr. 1977, 32, 451-468. (10) Li, K. W.; Hoek, R. M.; Smith, F.; Jime´nez, C. R.; van der Schors, R. C.; van Veelen, P. A.; Chen, S.; van der Greef, J.; Parish, D. C.; Benjamin, P. R.; Geraerts, W. P. M. J. Biol. Chem. 1994, 269, 30288-30292. (11) Valaskovic, G. A.; Kellcher, N. L.; McLafferty, F. W. Science 1996, 273, 1199-1202. (12) Garden, R. W.; Shippy, S. A.; Li, L.; Moroz, T. P.; Sweedler, J. V. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3972-3977. (13) Hofstadler, S. A.; Swanek, F. D.; Gale, D. G.; Ewing, A. G.; Smith, R. D. Anal. Chem. 1995, 67, 1477-1480. (14) Newcomb R.; Scheller, R. H. Brain Res. 1990, 521, 229-237. (15) Conn, P. J.; Kaczmarek, L. K. Mol. Neruobiol. 1989, 3, 237-273. (16) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J., Near, J. A.; Diliberto, E. J.; Viveros, O. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10754-10758. (17) Leszczyszyn, D. J., Jankowski, J. A.; Viveros, O. H.; Diliberto, E. J.; Jr.; Near, J. A.; Wightman, R. M. J. Biol. Chem. 1990, 265, 14736-14737. (18) Alvarez de Toledo, G.; Ferna´ndez-Chaco´n, R.; Ferna´ndez, J. M. Nature 1993, 363, 554-558. (19) Chen, G.; Gavin, P. F.; Luo, G.; Ewing, A. G. J. Neurosci. 1995, 15, 77477755. (20) Bruns, D.; Jahn, R. Nature 1995, 337, 62-65. (21) Tong, W.; Yeung, E. S. J. Neurosci. Methods 1997, 76, 193-201. 10.1021/ac9808977 CCC: $18.00
© 1998 American Chemical Society Published on Web 12/01/1998
and channel electrophoresis. We demonstrate the performance of this system using a series of neurons isolated from the CNS of the neuronal model system Aplysia californica and stimulating release from the cells using KCl. EXPERIMENTAL SECTION
Figure 1. Schematic diagram emphasizing the sampling device, placement of the neuron, and the pressurized sample inlet to precisely control the flow rate of media past the cell. The output of the sampling capillary is derivatized with NDA as it enters the channel, and then the individual components are separated. The time axis is generated by the precise movement of the sampling capillary across the entrance to the channel.
based on scanning a sampling capillary across a thin electrophoresis channel as described by Mesaros et al.22 Such systems have been used for proof of concept studies with derivatized amino acids,22,23 to follow reaction kinetics, 24,25 DNA separations,26,27 and multidimensional separations28 using both fluorescent22-28 and electrochemical detection.29 Analysis of the compounds released from a neuron is aided by high collection efficiency. Unlike approaches consisting of a capillary located near the cell of interest, we place the neuron into a nanoperfusion chamber constructed in the sampling capillary. All the releasate is collected by continuously flowing cell media past the cell in the capillary. As shown in Figure 1, by scanning the outlet end of the sampling capillary across the entrance to the separation channel, a time axis is generated. The time resolution in channel electrophoresis is limited by diffusional spreading which occurs while the analyte is in the sampling capillary, during the injection, and in the channel during electrophoresis.25,26 By placing the neuron against a frit located near the outlet of the sampling capillary, a higher time resolution is obtained as diffusional spreading is limited to the injection process (22) Mesaros, J. M.; Lou, G.; Roeraade, J.; Ewing, A. G. Anal. Chem. 1993, 65, 3313-3319. (23) Mesaros, J. M.; Ewing, A. G.J. Microcolumn Sep. 1994, 6, 483-494. (24) Liu, Y. M.; Sweedler, J. V. J. Am. Chem. Soc. 1995, 117, 8871-8872. (25) Liu, Y. M.; Sweedler, J. V. Anal. Chem. 1996, 68, 2471-2476. (26) Bullard, K. M.; Beyer Hietpas, P.; Ewing, A. G. Electrophoresis 1998, 19, 71-75. (27) Beyer Hietpas, P.; Bullard, K. M.; Gutman, A.; Ewing, A. G. Anal. Chem. 1997, 69, 2292-2298. (28) Liu, Y. M.; Sweedler, J. V. Anal. Chem. 1996, 68, 3928-3933. (29) Gavin, P. F.; Ewing, A. G. Anal. Chem. 1997, 69, 3838-3845.
Chemicals. Amino acids and 3-(cyclohexylamino)propanesulfonic acid (CAPS) are purchased from Sigma (St. Louis, MO). NDA, OPA, and fluorescamine were from Molecular Probes (Eugene, OR). All chemicals are of analytical grade or better. Milli-Q (Millipore, Bedford, MA) water was used throughout. Channel Electrophoresis. The channel electrophoresis system, spatially resolved fluorescence detection, and data acquisition have been described previously.24,25 Briefly, two microscope slides (75 mm long, 25 mm wide, and 1 mm thick) are glued together using the epoxy/glass bead spacer method.22-25 Electrophoresis is along the 75-mm dimension, with the width (25 mm) acting as a time axis by scanning the sampling capillary across the mouth of the channel. Detection takes place near the channel outlet and involves illuminating a strip across the channel width. Fluorescence emission is detected with a custom filter fluorometer/ multichannel CCD detector so that the fluorescence intensity at any point along the illuminated zone can be quantitated. Flow Control. To maintain the viability of the neurons, there must be a continuous and steady slow flow of media (at the correct ionic strength, temperature, and pH) past the cells at all times. To introduce pharmacological agents, the ability to inject materials at the sampling capillary inlet and then restore flow is also important. We have assembled a ∼50-mL gastight container out of acrylic that contains two solution vials and an O-ring fitted lid. To generate the desired flow rate through the sampling capillary, a precise volume of air can be injected using a syringe through a syringe port/septum, generating the slight pressure needed for a particular constant flow rate (dependent on the amount of air injected and the sampling capillary diameter and length). This system allows for precise and stable flow rate control; as one example, for a 1-mL air injection, we obtain a 10.8 ( 0.2 nL/s flow rate over 1 h. Nanoperfusion Chamber. Injection into the channel involves scanning a sample introduction capillary across the entrance of the separation channel. In addition, a portion of the sample introduction capillary now acts as the nanoperfusion chamber to hold the cell(s). The device for stimulating the cellular sample and introducing the releasate into the separation channel is depicted in Figure 1. A sintered glass frit is made at one end of a 25-mm-long cylindrical capillary (100-µm i.d.), and the frit is polished smooth to prevent damaging the cells placed against it. The end of the capillary is inserted into a 5 mm long piece of poly(vinyl) tubing (250 mm i.d.) connected to a 350-mm-long 100µm-i.d. capillary, with media flow provided by connecting the end of the capillary to the pressurized box for flow control. While the inlet of the 25-mm-long capillary contains the cell, the outlet of the capillary is scanned across the channel entrance. A. californica (50-150 g) are obtained from the Aplysia Research Facility (Miami, FL) and anesthetized with MgCl2. The cerebral ganglion is dissected and transferred to a dish containing artificial seawater. Under a microscope, the cells of interest are isolated and introduced into the tubing and placed onto the frit. Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
29
While several identifiable large cells (up to 500-µm diameter) are available, we use smaller (typically 30-100-µm diameter and unidentified) cerebral cells for these experiments. Next, the poly(vinyl) tubing is connected to the 350-mm-long 100-µm-i.d. capillary that was placed in a reagent solution vial. A flow of artificial seawater or seawater with pharmacological reagents is generated in the capillary via the pressurized delivery system. After passing around the cell, the solution containing cellular releasate is deposited along the entrance of the channel system in a timedependent manner. On-Line Derivatization. The electrophoresis running buffer is 20 mM CAPS aqueous solution (pH 9.6-10.0) containing 10% (v/v) methanol. On the sample introduction side of the channel, NDA and KCN are added so that analytes exiting the sample introduction capillary are derivatized prior to introduction into the separation channel (see Figure 1). To optimize fluorogenic reaction conditions, a commercial fluorometer is used (Hitachi F-4010, Hitachi Ltd., Tokyo, Japan). For the derivatization optimization studies, hydrodynamic injection with a 20-cm height displacement for 5 s is used. RESULTS AND DISCUSSION Precolumn Tagging of Cell Releasates. The overall goal of these experiments is to detect the neurotransmitters and neurohormones released from neurons. However, as most transmitters are not fluorescent, derivatization is needed to monitor the release from cells. Precolumn derivatization is difficult as the cell needs to be maintained in a medium containing high salts (seawater) without fluorogenic tagging reagents; the NDA or other reagents likely will affect cellular physiology and so cannot make contact with the cell directly. We have chosen to add the reagents into the buffer reservoir on the sample entrance side of the channel. Thus, the analytes are tagged at the interface between the outlet of the cylindrical capillary containing the cell and the entrance of the rectangular separation channel. In essence, this is similar to a gap reactor developed by Albin et al.30 and refined by Gilman et al. 31 and uses the natural “gap” between the sampling capillary and channel. The reaction time is quite short and so conditions need to be carefully optimized. This form of the gap reactor is different from previous reactors as it results in precolumn instead of postcolumn derivatization. Since the time that the analytes stay in the gap is short, the tagging reaction must proceed quickly. Three possible fluorogenic tagging reagents, NDA, OPA, and fluorescamine, have been studied for this purpose. Fluorescamine reacts very fast.32,33 However, fluorescamine hydrolyzes quickly (forming a white precipitate), which makes longer release experiments problematic as fluorescamine will not stay reactive throughout the experiment. One can minimize hydrolysis using organic solvents in the inlet of the separation channel. However, when organic solvent (for example, 90% formamide) is used as the running buffer, the sample is not effectively injected into the channel system for separation (30) Albin, M.; Weinberger, R.; Sapp, E. Anal. Chem. 1991, 63, 642-648. (31) Gilman, S. D.; Pietron, J. J.; Ewing, A. G. J. Microcolumn Sep. 1994, 6, 373-384. (32) Shippy, S. A.; Jankowski, J. A.; Sweedler, J. V. Anal. Chim. Acta 1995, 307, 163-171. (33) Udenfriend, S.; Stein, S.; Bohlen, P.; Dairman, W.; Leimgruber, Weigele, M. Science 1972, 178, 871-874.
30
Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
and detection. OPA reactions are fast enough to be practical. However, the excitation maximum for the CBI derivatives is ∼350 nm, which is not ideal for the Ar/Kr ion mixed gas laser because of power limitations in this wavelength range and because of the high fluorescence background from the glass microscope slides used to manufacture our channels. Hence, we have chosen to use NDA as the fluorogenic reagent. To optimize reaction times and reagent concentrations, we monitored the reaction of various amino acids using a spectrofluorometer. Under optimal conditions, we observe significant product formation with Arg, Glu, and the dipeptide Pro-Phe within several seconds. The excitation maximum for NDA is 445 nm, which is close to the wavelength of a laser line from the Ag/Kr ion laser (457 nm). Moreover, NDA is relatively stable in aqueous solution, making multiple experiments from a single neuron with the same set of fluorogenic reagent practical. The NDA-containing running buffer is effective for ∼2 h, which is sufficient for all release experiments. The concentration of NDA is an important consideration in our particular application. If the concentration is not high enough, poor derivatization efficiency and substantial differences in derivatization yields are observed for the amino acids tested, i.e., Arg, Gly, and Glu. This effect is exacerbated by the short reaction time allowed in our arrangement. For example, the reaction between Arg and NDA is slower than the reaction between NDA and either Gly or Glu. For a mixture of these three, the derivatization yield for Arg is poor when the NDA concentration is low. This problem is reduced by increasing the concentration of NDA higher than the total amino acid concentration. The concentration of NDA, however, cannot be too high because of the limited solubility of NDA in aqueous solutions and the increase in fluorescence baseline which is NDA-dependent. Figure 2 shows a comparison of the derivatization results at two different NDA concentrations. Based on the data similar to that presented in Figure 2, for all further experiments, the concentration of tagging reagents is 2 mM for NDA and 1 mM for CN-. Electrolyte composition determines the background fluorescence signal, which should be minimized. Using a spectrofluorometer, several buffer solutions have been studied; NDA and CNare added into cuvettes containing borate buffer (10 mM Na3BO3 at pH 8.8) or CAPS buffer (25 mM CAPS at pH 9.6) with fluorescence excitation spectra measured over 1 h. While no significant difference in the background signal is found, CAPS running buffer is more effective for separations of CBI-amino acid derivatives and is used for further experiments. In addition, we add 10% (v/v) methanol to increase the solubility of NDA, reduce µeo and the channel current, and improve separation efficiency. Under optimized conditions, detection limits for underivatized amino acids injected from the sampling capillary (and subsequently derivatized and detected) are ∼5 × 10-7 M. Because of reaction time limitations in our precolumn system and the slow speed of NDA/peptide derivatizations, the suitability of the online derivatization scheme has been tested for peptide derivatization. Using a mixture containing five peptides, Gly-Tyr, Val-TyrVal, Tyr-Gly-Gly-Phe-Leu, Tyr-Gly-Gly-Phe-Met, and Asp-Arg-ValTyr-Ile-His-Pro-Phe, no electrophoretic peaks are detected even with 1 µg/mL peptide concentrations. While NDA can react with peptides, we find that the reaction for peptides is much slower than those for amino acids and so no product is observed within
Figure 2. Injection of Arg, Gly, and Glu into the sampling capillary, and derivatization as the analytes enter the separation channel for two different NDA/KCN concentrations. In (A), [NDA] is 0.5 mM and the amino acids are at 50 µg/mL, and in (B), [NDA] is 2.0 mM and the amino acids are at 1 µg/mL. (C) Electropherogram showing the entire channel output for a single injection of 11 µg/mL Arg, Gly, and Glu injected into a 40% T, 5% C polyacrylamide gel showing the higher separation efficiency possible with off-line derivatization and gel-filled channels.
the time constraints of our precolumn reactor. One potential way to reduce this limitation is to use faster reacting fluorogenic reagents such as fluorescamine and modify the sample introduction method to use a dual-capillary design; one would be the sampling capillary with the cell and the other would deliver fresh reagent to the channel inlet. For on-column derivatization, the separation efficiencies are lower than previous free-solution channel separations. Prior work using FITC-labeled amino acids obtained separation efficiencies of 5000-10000,24,25 while we obtain ∼500. The reduced efficiency is due, to a large extent, to the on-line derivatization process and the reaction between the amino acids and NDA. Figure 2c is a channel electropherogram of the same mixture of three amino acids (each at 11 µg/mL) prederivatized with NDA and injected into a polyacrylamide (40% T, 5% C) gel-filled channel,28 which results in greatly improved separation efficiencies. We find that some electroosmotic flow is required for the on-line derivatization to work reproducibly and the gel slowly becomes fluorescent from contact with the NDA solution so that the gel-filled channels cannot be used at this time with the on-line derivatization. Simulated Release Experiment. Before using a cell, a simulated cell release experiment is performed. Four plugs of a mixture solution containing Arg and Gly (each at 5 µM) are introduced into the sampling capillary from a reagent vial. These amino acids are carried to the entrance of the channel by the flow of artificial seawater, where they are derivatized as they enter the separation channel. As can be seen in Figure 3a, these analyte spots are well separated and easily detected. Diffusion of the
analytes in the sampling capillary and channel is evident as these electrophoretic peaks are Gaussian, resulting in reduced time resolution. However, the analytes are injected at the far end of the sample introduction capillary (more than 350 mm from the channel end). For cell work, the cell is positioned 25 mm from the entrance of the separation channel. Therefore, diffusion of the components after being released is reduced. Release from Cerebral Ganglion Neurons. Figure 3b shows a two-dimensional electropherogram obtained from an experiment with several (∼5) neurons isolated from the A. californica cerebral ganglion and placed on the frit of the capillary. At all times after the cells are positioned in the sample capillary, the seawater perfusion is maintained to keep the cells alive. To stimulate cell release, a short (∼100-nL) plug of artificial seawater containing 50 mM elevated KCl is introduced into the flow. KCl is a secretogogue for Aplysia. Figure 3b shows the resulting twodimensional electropherogram after the cells are stimulated with KCl. Two compounds are observed with different electrophoretic migration times, one of which is released continuously for more than 5 min after the stimulation. To verify we are observing physiological release, we repeat the experiment but first inject a plug of isotonic MgCl2 before the KCl plug. Mg2+ interferes with the Ca2+ channels and so blocks neurotransmitter release.34 As expected, the observed release is greatly reduced. To further verify this effect, we changed the perfusion media to fill the separation capillary with the MgCl2 solution and observed virtually no detectable release. In control experiments (not shown), we demonstrated reproducible release when performing two serial KCl injections. The reproducibility of release and the ability of a plug of Mg2+ preceding the K+ plug to greatly reduce release indicate we are observing physiologically relevant release and not cell lysis or permeation. Our efforts to identify the chemical nature of the released material are in progress. Single Neuron Release. An experiment involving a single neuron has also been performed to demonstrate the ability to follow release from a single cell. For this experiment, a plug of L-arginine (Arg) and L-aspartate (Asp) (each at 5 µM) is injected 60 s after the KCl injection. This standard injection serves three purposes: (1) to verify the performance of the system including derivatization, sample introduction into the separation channel, and detection; (2) to provide analyte peaks to calibrate the electrophoretic performance of the channel system, making it possible to compare the elution times of the analytes with those of standard components in the same run; (3) to calibrate the time delay after analytes are injected into the sampling capillary (containing the neuron) until they appear at the channel entrance. Figure 3c shows the time-resolved electropherogram from the cell release. A compound is observed that has an elution time that matches the electrophoretic mobility of Asp standard injected in the same run within several percent. No other tested amino acid (including Glu) matches this peak; further confirmation of the identity of this compound is needed as our relatively poor separation efficiency makes definitive assignments difficult. Asp is a known neurotransmitter in mammals,35,36 and Asp has also (34) Hille, B. Ionic Channels of Excitable Membranes, 2nd ed.; Sinauer Associates: Sunderland, MA, 1992; Chapter 4. (35) Lo´pez-Colome´, A. M.; Somohano, F. Brain Res. 1984, 298, 159-162. (36) Yuzaki, M.; Forrest, D.; Curran, T.; Connor, J. A. Science 1996, 273, 112114.
Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
31
Figure 3. False color two-dimensional channel electropherograms, where red indicates the highest fluorescence intensity and blue the lowest. (A) Simulated cell release experiment with four replicate injections of 5 µM Arg and Gly. The injection is at the inlet end of the sampling capillary so that greater lateral (time-based) spreading occurs than would occur for the cell located near the exit of the sampling capillary. (B) Series of electropherograms showing release from the same group of cerebral ganglion neurons for different injected solutions, with the injection protocol shown schematically over each electropherogram. While release is observed for KCl stimulated release, the amount released is dramatically reduced with a short injection of MgCl2 prior to the KCl, and virtually no release is observed while the neuron is bathed in isotonic MgCl2. (C) An electropherogram from stimulating a single cerebral ganglion cell with elevated KCl in seawater. An Arg and Asp solution is injected 60 s after the KCl injection as internal standards. The vertical line in (C) is caused by a bubble at a particular position on the channel during the experiment.
been reported to be a likely transmitter in Aplysia.37-39 The putative Asp band is released almost immediately after the neuron is stimulated by KCl and is released for 10 ( 3 s. Two other compounds are detected in the releasate with distinct migration times and time courses, both of which serve to identify them as separate components. The release of one component lasts 60 ( 6 s, longer than expected for classical amino acid transmitters, although certainly possible for some neuromodulators. These compounds have not been identified yet and further work involves characterizing these compounds. (37) Yarowsky, P. J.; Carpenter, D. O. Science 1976, 192, 807-809. (38) Zeman, G. H.; Carpenter, D. O. Comp. Biochem. Physiol. 1975, 52C, 2326. (39) McCreery, M. J.; Carpenter, D. O. Cell. Mol. Neurobiol. 1984, 4, 91-95.
32 Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
Conclusions. Channel electrophoresis coupled with the capillary-based nanoperfusion chamber described here allows high collection efficiency of released compounds and allows the release of neurotransmitters to be measured from individual cells as a function of time. Large differences in the temporal release of different species is observed for single stimulation events, and most cells appear to have multiple compounds with derivatizable amino groups released with different time courses. Future experiments will verify the performance of this system with neurons with known transmitter complements and will work on improving the separation efficiency and the use of standards to improve our ability to identify the neuronal releasates. The ability to precisely control the contact time between a cell and various
pharmacological reagents and follow the effects of such agents on release allows the physiological effects of many important pharmaceuticals to be studied in a way not possible with other techniques.
Dreyfus Foundation are gratefully acknowledged. Aplysia californica are partially provided by the NCRR National Resource for Aplysia at the University of Miami under National Institutes of Health Grant RR10294.
ACKNOWLEDGMENT The assistance of Scott Shippy in initial cell isolation and injections is appreciated. The support of NSF (Grant CHE-9622663), the National Institute of Health (Grant NS31609), and the
Received for review August 12, 1998. Accepted October 23, 1998. AC9808977
Analytical Chemistry, Vol. 71, No. 1, January 1, 1999
33
