Anal. Chem. 2003, 75, 3972-3978
Articles
Micellar Electrokinetic Capillary ChromatographyElectrochemical Detection for Analysis of Biogenic Amines in Drosophila melanogaster Paula J. Ream,† Steven W. Suljak,‡ Andrew G. Ewing,† and Kyung-An Han*,§
Department of Chemistry, Pennsylvania State University, 152 Davey Laboratory, University Park, Pennsylvania 16802, Department of Chemistry, University of Florida, P.O. Box 117200, Gainesville, Florida 32611, and Department of Biobehavorial Health, Pennsylvania State University, 315 Health & Human Development East, University Park, Pennsylvania 16802
Micellar electrokinetic chromatography coupled to amperometric electrochemical detection was used to investigate the chemical environment of the fruit fly, Drosophila melanogaster. Preliminary studies focused on the employment and optimization of the system to separate electroactive amine-containing molecules present in the head and body of male and female flies. Ultimately, biogenic amines significant to the fly including L-3,4dihydroxyphenylalanine, dopamine, tyramine, and serotonin were identified and their relative abundance quantified. Transgenic Drosophila with functionally ablated dopamine and serotonin neurons were analyzed to demonstrate the sensitivity of the technique. The separation method developed in this study should offer an advantage in elucidating the critical role that electroactive biogenic amines play in complex physiological processes correlated with Drosophila behavior. The fruit fly Drosophila melanogaster has been fundamental to the study of genetics. Exploitation of Drosophila is especially attractive because of the fly’s small size, relatively short life cycle, and ease of molecular and genetic manipulation. Moreover, information this tiny organism can provide extends far beyond Mendelian genetics. Numerous studies reveal remarkable similarities between Drosophila and mammals in their molecular and cellular components underlying various physiological processes ranging from basic cellular function to reproductive and developmental biology.1 The recent completion of the Drosophila genome project2 has heightened interest in this insect because it further facilitates genetic approaches and provides versatile utility in broad disciplines including computational and functional genomics. * Corresponding author. E-mail:
[email protected]. Phone (814) 865-8409. Fax (814) 863-7525. † Department of Chemistry, Pennsylvania State University. ‡ University of Florida. § Department of Biobehavorial Health, Pennsylvania State University. (1) The FlyBase Consortium. Nucleic Acids Res. 2003, 31, 172-175. http:// flybase.org/. (2) Adams, M. D.; Celniker, S. E.; Holt, R. A.; Evans, C. A.; et al. Science 2000, 287, 2185-2195.
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Drosophila also offers an invaluable resource for understanding neuronal processes and behaviors. The key molecules and signal transduction pathways involved in synaptic transmission, sensory processing, and higher-order brain functions are highly conserved between Drosophila and mammals.3-6 Neuromodulators such as biogenic amines contribute to a variety of physiological processes including responses to stress, emotion, sleep, learning, and memory.7 Multiple lines of evidence suggest the mechanisms of action for biogenic amines in mammals are analogous to those in Drosophila.8 For example, dopamine (DA), serotonin (5-HT), and histamine present in the mammalian central nervous system (CNS) and peripheral nervous system also function as neuromodulators in Drosophila to mediate diverse physiological processes.9,10 Other biogenic amines such as tyramine (TA) and octopamine (OA) serve as fly neuromodulators and likely represent invertebrate counterparts of mammalian norepinephrine (NE) and epinephrine (E).8,11,12 Thus, neurochemical research on mammalian systems can be expanded to the smaller and more controllable fly model. Studies of biogenic amines in flies have been accomplished primarily through two approaches: anatomical localization and molecular genetics of the enzymes involved in the biosynthetic pathways.12 In particular, immunocytochemistry and histochemistry have been employed to detect the catecholamines10,13 as well as OA and histamine in developing and adult CNS.10,14 These approaches have proven very useful in the identification of amines (3) Fernandez-Chacon, R.; Sudhof, T. C. Annu. Rev. Physiol. 1999, 61, 753-776. (4) Korsching, S. Curr. Opin. Neurobiol. 2002, 12, 387-392. (5) Panda, S.; Hogenesch, J. B.; Kay, S. A. Nature 2002, 417, 329-335. (6) Waddell, S.; Quinn, W. G. Trends Genet. 2001, 17, 719-726. (7) Bergquist, J.; Sciubisz, A.; Kaczor, A.; Silberring, J. J. Neurosci. Methods 2002, 113, 1-13. (8) Mercer, A. R. In Arthropod Brain: Its Evolution, Brain, Structure, and Functions; Gupta, A. P., Ed.; Wiley-Interscience: New York, 1987; pp 399414. (9) Blenau, W.; Baumann, A. Arch. Insect Biochem. Physiol. 2001, 48, 13-38. (10) Monastirioti, M. Microsc. Res. Technol. 1999, 45, 106-121. (11) Watson, D. G.; Zhou, P.; Midgley, J. M.; Milligan, C. D.; Kaiser, K. J. Pharm. Biomed. Anal. 1993, 11, 1145-1149. (12) Restifo, L. L.; White, K. Adv. Insect Physiol. 1990, 22, 115-219. (13) Budnik, V.; White, K. J. Comput. Neurol. 1988, 268, 400-413. (14) Han, K. A.; Millar, N. S.; Davis, R. L. J. Neurosci. 1998, 18, 3650-3658. 10.1021/ac034219i CCC: $25.00
© 2003 American Chemical Society Published on Web 06/26/2003
and their receptors as well as their localization in the nervous system. However, there is no technique that generally provides quantitative information. In recent years, separation-based methods have been added as tools to confirm or measure biogenic amine content. High-performance liquid chromatography analysis with electrochemical detection (HPLC-EC) has been applied to the study of DA levels in early development15 and aging,16 as well as in genetic variants.17-19 These experiments have been conducted to substantiate biogenic amine presence in the fly, but quantitation was not the focal point of these studies. Therefore, they provide limited information on amine levels. Independent studies of four fly strains using gas chromatography coupled with mass spectrometry (GC/MS) have revealed the presence and quantity of several biogenic amines in Drosophila head homogenates.11 However, this approach requires difficult preparative procedures, including derivatization and volatilization of the fly sample, and complex equipment. The research presented here was directed to alleviate these problems by employing an alternate approach, micellar electrokinetic capillary chromatography with amperometric electrochemical detection (MEKC-EC). MEKC is ideally suited for the analysis of a complex mixture of monoamines. The addition of a surfactant to the electrophoresis buffer resolves analytes with similar charges and electrophoretic mobilities such as the catecholamines while the use of a narrowdiameter capillary facilitates sampling of extremely small volumes making it ideal for small systems such as Drosophila.20-22 In addition, this technique yields enhanced selectivity and is relatively simple with rapid analysis times. The use of amperometry provides sensitive detection for the monoamines while selectively excluding interferences from nonelectroactive species. Moreover, MEKCEC does not require complex sample preparation and easily distinguishes subtle changes in analytes. Thus, the coupling of MEKC-EC creates a powerful analysis tool capable of quantitation as well as identification. This paper describes the first application of capillary electrophoresis in general and specifically MEKC-EC for the analysis of biogenic amines in Drosophila homogenates. Drosophila is one of the most widely studied model systems for genetic, physiological, and behavioral processes, yet functional quantitative tools have not been extensively developed by analytical chemists. The emphasis of this work has been to optimize the analytical system by adjusting separation parameters and sampling methodologies. To this end, we have achieved reproducible separations of relevant biogenic amines present in the fly head and body. Preliminary quantitative data revealed the distinct content of biogenic amines in the Drosophila head and the sensitivity of MEKC-EC to detect changes in monoamine quantities in transgenic animals. This study suggests the potential of MEKC-EC as a tool for monitoring biogenic amines involved in complex physiological and behavioral processes in Drosophila. (15) Neckameyer, W. S. Dev. Biol. 1996, 176, 209-219. (16) Neckameyer, W. S.; Woodrome, S.; Holt, B.; Mayer, A. Neurobiol. Aging 2000, 21, 145-152. (17) Ramadan, H.; Alawi, A. A.; Alawi, M. A. Cell Biol. Int. 1993, 17, 765-771. (18) Borycz, J.; Vohra, M.; Tokarczyk, G.; Meinertzhagen, I. A. J. Neurosci. Methods 2000, 101, 141-148. (19) Monastirioti, M.; Linn, C. E., Jr.; White, K. J. Neurosci. 1996, 16, 3900-3911. (20) Wallingford, R. A.; Ewing, A. G. Anal. Chem. 1988, 60, 258-263. (21) Wallingford, R. A.; Ewing, A. G. J. Chromatogr. 1988, 441, 299-309. (22) Wallingford, R. A.; Curry, P. D.; Ewing, A. G. J. Microcolumn Sep. 1989, 1, 23-27.
EXPERIMENTAL SECTION Reagents. All amine standards, L-3,4-dihydroxyphenyl alanine (L-DOPA) and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES) were purchased from Sigma (St. Louis, MO). Sodium dodecyl sulfate (SDS) and a 48% aqueous solution of hydrofluoric acid were obtained from Aldrich (Milwaukee, WI). All chemicals were used as received. Separations were performed in 10 mM TES buffer containing 30 mM SDS and 2% 1-propanol, adjusted to pH 7.1. All standards were prepared as 10 mM stock solutions in 0.1 M perchloric acid and were diluted to the desired concentration with additional 0.1 M perchloric acid. Drosophila Strains. Canton-S flies, a wild-type Drosophila strain maintained in the laboratory, were used to obtain head and body homogenates. Transgenic flies with functionally ablated dopa decarboxylase (Ddc) neurons were also utilized. They were generated by crossing flies carrying pDdc-GAL4 that express the transcription factor GAL4 in Ddc-expressing cells23,24 with flies containing pUAS-tetanus toxin light chain (TNT). In these animals, TNT expression was induced by GAL4 in Ddc neurons to inhibit synaptic transmission.25 Flies were cultured on standard cornmeal/ agar medium and collected between 2 and 4 days after emerging from pupal cages. Homogenate Preparation. Flies were prepared by quickfreezing in a liquid nitrogen or dry ice/ethanol bath. The heads and bodies were separated by vortexing and gentle sifting through mesh minisieves (Scienceware, Pittsburgh, PA), then homogenized in 1 µL of 0.1 M perchloric acid per head and 2 µL per body, placed in an Eppendorf centrifuge with a fixed angle rotor (Brinkman Instruments, Westbury, NY), and spun at 13 000 rpm for 5 min at 4 °C. For a subset of samples as described in the text, the supernatant was subsequently centrifuged (13 000 rpm, 4 °C) through Amicon Ultrafree-MC centrifuge filters (Fisher Scientific, Pittsburgh, PA). Dihydroxybenzylamine (DHBA, 5-100 µM) was added as the internal standard to a subset of samples as indicated. All resulting filtrates were dispensed into 1-µL aliquots and frozen until use. Instrumentation and Analysis. The CE system with endcolumn amperometric detection utilized in this study was built in-house and has been described previously.20,26 Briefly, 45-50 cm of fused-silica capillary with an outer diameter of 148 µm and inner diameter (i.d.) of 16 µm (Polymicro Technologies, Phoenix, AZ) was used for the separations. Amperometric EC detection was carried out with a two-electrode format in which a 5-µm carbon fiber microelectrode, coated with Nafion for enhanced selectivity,27 was held between +0.70 and +0.75 V versus Ag/AgCl reference electrode (World Precision Instruments, Sarasota, FL). Faradaic currents were amplified with a Keithley model 427 current amplifier (Cleveland, OH) and collected with a LabView 5.1 interface (National Instruments, Austin, TX). Microsoft Excel (Redmond, WA) was used to generate the electropherograms. Data analysis was performed using both GC Workmate running under LabView (23) Li, H.; Chaney, S.; Roberts, I. J.; Forte, M.; Hirsh, J. Curr. Biol. 2000, 10, 211-214. (24) Heimbeck, G.; Bugnon, V.; Gendre, N.; Keller, A.; Stocker, R. F. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 15336-15341. (25) Keller, A.; Sweeney, S. T.; Zars, T.; O’Kane, C. J.; Heisenberg, M. J. Neurobiol. 2002, 50, 221-233. (26) Sloss, S.; Ewing, A. G. Anal. Chem. 1993, 65, 577-581. (27) Brazell, M. P.; Kasser, R. J.; Renner, K. J.; Feng, J.; Moghaddam, B.; Adams, R. N. J. Neurosci. Methods 1987, 22, 167-172.
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Table 1. Optimization of Separation Conditionsa parameters
conditions attempted
effect on resolution
effect on peak shifting
field strength
capillary length applied potential
improved with increased field strength
no effect
buffer/pH
SDS TES/SDS (pH 7) vs phosphate/SDS (pH 2)
increased with optimized SDS concn decreased with lower pH
no effect increased at low pH
additives
CTAB Z1 methyl reagent ethylene glycol
minimal to no enhancement
increased, inconsistent
pre/posttreatment
HCl NaOH methanol
decreased
increased, inconsistent
homogenization conditions
TES/SDS (pH 7) HClO4
no effect; poor detection no effect; improved detection
no effect decreased with HClO4
a Summary of the applied separation parameters and optimization of the biogenic standards and homogenates using a 16-µm-i.d. capillary. The effects of each parameter on peak resolution and shifting are presented.
and PeakFit Separation and Analysis Software (SPSS Inc., Chicago, IL) to estimate individual peak areas in all electropherograms. Procedures. Capillaries were filled with separation buffer using a stainless steel reservoir with applied He pressure (100 psi). Buffer solutions were filtered with a 0.2-µm nylon filter (Alltech, Deerfield, IL) prior to use. Injections were performed electrokinetically at 5 kV for 5 s extracting ∼1 nL of homogenate from the thawed homogenate aliquot. To enhance microelectrode placement, the capillary inner diameter was enlarged via HF etching as previously described.26 Approximately 2 mm of the polyimide coating was removed from the capillary to expose the fused silica. The exposed portion of the capillary was placed in HF for 15 min, after which the same segment of capillary was placed in a sodium bicarbonate solution to neutralize the acid and was washed with water. Safety Considerations. An in-house-built safety interlock box was utilized to protect the user from high voltage. Since HF could cause severe burns, it was used with extreme care. HF was neutralized with sodium bicarbonate before disposal. RESULTS AND DISCUSSION Characterization of the Method. The use of MEKC-EC represents a new approach to the study of biogenic amines in the important Drosophila system. To demonstrate the utility of MEKCEC and optimize separation parameters, a mixture of pure compounds has been used as references or standards. The electroactive biogenic amines L-DOPA, NE, OA, E, DA, TA, and 5-HT were chosen for their potential role in Drosophila and mammalian neurotransmission and thus probable presence in in vivo samples. Prior to the analysis of in vivo samples, several separation parameters were explored and summarized in Table 1. They include various field strengths, capillary treatments, and buffer additives. Separation of the standards was easily accomplished at high potentials with good resolution and peak shapes. Use of organic additives such as hexadecyltrimethylammonium bromide (CTAB) and ethylene glycol did not improve resolution of the standards; however, the use of 2% propanol enhanced peak shapes and, thus, was used in all the separations presented. While buffer pH was explored, the goal for this research is to stay within the realm of physiological pH; thus, neutral TES buffer was chosen. 3974
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Limited reproducibility of migration times between runs is often a problem in CE, which could raise questions regarding peak identification. Capillary pre- and posttreatments are often used to correct for this problem; however, they did not improve separation reproducibility or resolution in our work (see Table 1). Therefore, we addressed this issue by using the same capillary and electrode for both the reference and homogenate samples. Figure 1A illustrates three successive separations of the reference mixture. Because there is limited charge attraction to the negative SDS micelles, the zwitterionic analyte L-DOPA elutes first followed by cationic NE, E, OA, DA, TA, and 5-HT in the order of increasing hydrophobicity. Cationic DHBA was used as an internal standard for in vivo samples, eluting between OA and DA in the separation. All biogenic amines applied are clearly resolved in 25 min for all three runs. However, an increase in migration time is observed for all analytes with greater changes taking place for DA, TA, and 5-HT in the consecutive runs. This occurrence could be the result of adsorption effects amplified by the long residence time of the analytes in the capillary and their interaction with the micellar phase. The result has been examined by determining the standard deviation for five consecutive separations. Although the largest variation is observed for 5-HT (peak 8, 2.8% standard deviation), standard deviations are less than 3% for all compounds (0.65% for L-DOPA, 1.6% for NE, 1.8% for E, 2.0% for OA, 2.2% for DHBA, 2.3% for DA, and 2.6% for TA). Thus, identification of the peaks should be straightforward as long as consistent changes in migration times are maintained. In addition, changes were observed at the detector. The coulometric efficiency of detection appears to decay in detector response over time, yielding a decrease in observed peak height over consecutive runs (Figure 1). This response is likely due to fouling of the electrode and is more pronounced after exposure to the biological sample. It should be noted that the peak heights for L-DOPA, NE, E, DA, and 5-HT are larger than those of OA and TA. This is apparently due to more efficient oxidation of the catecholamines (L-DOPA, NE, E, DA) and indolamine (5-HT) relative to the monophenolamines (OA, TA). This is consistent with different electron-transfer mechanisms for the monophenolamines (where n ) 1 electron transferred) versus the catecholamines and indolamine (where n ) 2 electrons transferred).
Figure 1. Overlaid electropherograms of three repetitive standard (A) and Drosophila homogenate (B) separations. A single separation of the 100 µM standards is shown in the upper right with the following identification: L-DOPA (1), NE (2), E (3), OA (4), DA (6), TA (7), and 5-HT (8). DHBA (5) is also used at lower concentration (10 µM). Separations were performed in a 16-µm-i.d. capillary with separation potentials of 563 (A) and 514 V/cm (B). The working electrode was held at +750 mV vs a Ag/AgCl reference electrode.
The initial analysis of unfiltered homogenate samples revealed irregular variation in elution times presumably due to frequent clogging of the capillary and rapid fouling of the electrode. These results were likely attributed to macromolecules present in biological samples. We subsequently found that filtering of homogenates through 5-µm PVDF membranes minimizes these problems and yields greater reproducibility in separation profiles. The use of perchloric acid during fly homogenization also aids in decreasing peak shift and biogenic amine oxidation (Table 1). Figure 1B illustrates the separation of male Drosophila head homogenates sequentially applied three times. The increase in migration time of the individual peaks on the second and third runs is consistent between reference and homogenate samples. The standard deviation of the mean for migration time is less than 2% for all biogenic amines when five separations were carried out on a homogenate sample. Therefore, it appears feasible to identify and quantitate peaks representing biogenic amines of interest by comparing electropherograms of standards and homogenates separated under the same conditions. Identification and Quantitation of Biogenic Amines. A primary application of MEKC-EC for Drosophila samples is to determine biogenic amines responsible for various physiological processes. The peaks representing electroactive species separated
from Drosophila homogenates have been evaluated by comparison with standards separated before and after the homogenate separation and by analysis of the homogenate samples spiked with known concentrations of a specific biogenic amine. The monoamine DHBA has been utilized as an internal standard for the homogenates. Figure 2 shows a representative electropherogram of the crude homogenate made from wild-type Canton S male heads. Several distinct peaks are consistently noted, and four have been identified as L-DOPA, DA, TA, and 5-HT as indicated by numbers corresponding to those in Figure 1A. L-DOPA, the synthetic precursor to the other amines, is abundant, although poorly resolved from the surrounding peaks (Figure 2B). When metabolites of 5-HT and the catecholamines including 5-hydroxyindoleacetic acid (5-HIAA), 3,4dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA) are added to the standards, they coelute at ∼6 min, prior to NE. It is probable that the poorly separated peaks at 6 min in the homogenate represent some of these metabolites. The remaining three biogenic amines are shown over a narrower range in Figure 2C, which reveals DA and TA in lower abundance than 5-HT. One of the most significant advantages of the MEKC-EC method is the quantitative information that can be collected on each analyte. Analyses of various concentrations of standards reveal the detection limit of the system to be as low as 4 amol for L-DOPA and 20 amol for E, with the remaining analytes falling in between. It is expected that biogenic amines in Drosophila are present at or above this attomole range. Calibration plots for L-DOPA and E are linear for concentrations from 250 nM to 5 µM with correlation coefficients of 0.929 and 0.997, respectively. Table 2 displays the representative quantities of four monoamines separated from homogenates of three independent preparations of Canton-S male heads. For each preparation, two to three different homogenate aliquots were analyzed and their average value per head is reported. The molar contents for each monoamine in the homogenates were calculated based on the known concentration of the corresponding monoamine in the standard mixture that were separated prior to or immediately after the homogenate. The relative abundance of the four monoamines has been replicated in independent preparations with L-DOPA being the highest (747 ( 59.8 fmol/head) and DA the lowest (15.9 ( 2.9 fmol/head). Although the range of biogenic amine content found in this study is comparable with that obtained using different separation methods,11,15,16,19 differences have been noted in the relative contents of DA and 5-HT. For example, GC/MS separations described by Watson et al. appeared to show a higher content for DA (722 fmol) than 5-HT (22.7 fmol) per fly head.11 GC/MS requires extensive sample processing involving volatilization of analytes while MEKC-EC utilizes simple centrifugation steps and aqueous analytes. The different extraction procedures may contribute to the discrepancies in the detected values. In addition, these levels could represent differences in environmental conditions (e.g., food, temperature, humidity, crowding) associated with fly culture. Comparison of Male and Female Drosophila Heads. Previous work using whole fly homogenates has reported DA levels to be sexually dimorphic.16 Homogenates of female and male heads were analyzed by MEKC-EC to investigate whether the dimorphic nature of DA is localized to the head versus the body of Drosophila. The overall separation profiles of female heads (data Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
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Figure 2. (A) MEKC-EC separation of a male head homogenate. (B) Enlargement of the first 6 min of the separation highlights L-DOPA (1). (C) Enlargement of the latter half of the electropherogram emphasizes peaks for DA (6), TA (7), and 5-HT (8). The numbers correspond to those in Figure 1A. The asterisk marks an unknown peak consistently observed in the head homogenate. Field strength for the separation was 491 V/cm. The working electrode was held at +750 mV vs Ag/AgCl.
Table 2. Quantitation of Four Biogenic Amines in Drosophila Head Homogenatesa fly culture
L-DOPA
(fmol)
DA (fmol)
TA (fmol)
5-HT (fmol)
1 2 3
629 791 822
18.4 10.1 19.2
38.8 10.9 26.0
288 374 420
av ( SEM
747 ( 59.8
15.9 ( 2.9
25.2 ( 8.1
361 ( 38.7
a Wild-type Drosophila male head homogenates from three independent fly cultures. Each value represents the average of triplicate (trial 1) or duplicate (trials 2 and 3) separations of the same homogenate preparation. The average and standard error of the mean show the variation among fly culture populations.
not shown) is indistinguishable from that of male heads, with the latter shown in Figure 2A. Quantitation of individual peaks carried out on three independently prepared samples has detected no difference in the basal monoamine content between the male and female heads. It is still conceivable that the level of certain biogenic amines could be different in male and female heads under certain physiological conditions or with different strains of flies. Comparison of Biogenic Amines in the Drosophila Head and Body. Analysis of biogenic amines in Drosophila has focused previously on either the head or the entire fly.11,15,16,19 The information obtained from these studies has indiscriminately been referred to as CNS biogenic amines. To explore whether the head and body contain different biogenic amines, the body represented by the thorax and abdomen has been subjected to MEKC-EC analysis (Figure 3). The four biogenic amines L-DOPA, DA, TA, and 5-HT, which are prevalent in the Drosophila head (Figure 2), are present in the body. However, an apparent difference is noted in their relative abundance. As shown in Figure 3C, the MEKC3976 Analytical Chemistry, Vol. 75, No. 16, August 15, 2003
EC separation of the body homogenate exhibits a larger peak for DA (30.2 fmol/body) when compared to Figure 2, whereas the L-DOPA (814 fmol/body) peak remains relatively the same (Figure 3B). The higher abundance of DA in the body may reflect its distinct function in the cuticle. Drosophila use DA not only as a chemical messenger in the CNS but also as a key component in sclerotization. DA is N-acetylated and cross-linked with proteins during this process to form the insect cuticle.16,28 Indeed, depletion of DA by genetic or pharmacological manipulation causes severe cuticle phenotypes.15 On the other hand, the 5-HT peak largely coelutes with the neighboring peak marked with an asterisk (Figure 3C) and is slightly broader and smaller than that in the head homogenate (Figure 2C). The most dramatic difference between the head and body homogenates is found in the level of TA observed. The quantity of TA is 26.3 fmol/head (Figure 2C) whereas the peak for TA is barely detectable in the body (Figure 3C). In addition, the unknown peak marked with an asterisk in Figure 2A and Figure 3A is greatly reduced in the body compared to the head samples. As a smaller proportion of the CNS is present in the body of Drosophila, it is not surprising to recover the reduced level of 5-HT and TA that are mostly if not exclusively present in the CNS. Likewise, the lower level of the unknown substance in the body implicates its function in the nervous system. Taken together the data presented in this study justify localized analysis of biogenic amines in the head, in lieu of the whole fly as adopted by several published studies, to monitor subtle changes in DA and 5-HT associated with CNS function. MEKC-EC Analysis of Transgenic Flies. To address whether MEKC-EC is capable of identifying changes in biogenic amine content due to variable physiological conditions, a Drosophila genetic variant was analyzed. The transgenic flies have been (28) Wright, T. R. F. Adv. Genet. 1987, 24, 127-222.
Figure 3. (A) Electropherogram obtained from a male body homogenate. The internal standard DHBA (5) is shown in this electropherogram at a concentration of 10 µM. (B) Enlargement of the first 5 min. (C) Enlargement of the latter half of (A). Peaks were identified with the same numbers as in Figure 1A: L-DOPA (1), DA (6), TA (7), and 5-HT (8). The asterisk marking an unknown peak appears to represent the same unknown peak marked in the head homogenate (Figure 2A). The field strength for the separation was 542 V/cm. The working electrode was held at +750 mV vs Ag/AgCl.
Figure 4. Comparison of female Canton-S and transgenic DdcTNT head homogenates. The Canton-S homogenate contained the monoamines L-DOPA (1), DA (6), TA (7), and 5-HT (8). The transgenic Ddc-TNT homogenate contained L-DOPA (1), TA (7), and 5-HT (8). A single separation of the transgenic Ddc-TNT homogenate is shown in the upper right, highlighting TA (7) and 5-HT (8). The internal standard DHBA (5) is shown in these electropherograms at a concentration of 100 µM for the wild-type and 7.5 µM in the DdcTNT flies. The diamond marks an additional peak unaffected by the toxin. Field strength for the separations was 525 V/cm. The working electrode was held at +750 mV vs Ag/AgCl.
specifically bred to lack functional Ddc neurons throughout development by targeting the expression of the bacterial toxin TNT in cells containing Ddc, a synthetic enzyme for DA and 5-HT. TNT blocks synaptic transmission thus causing Ddc neurons in the transgenic animals to be functionally inactivated.25 The electropherograms shown in Figure 4 represent the head homogenate of wild-type females (top trace) compared to that of DdcTNT transgenic females (bottom trace). A lower concentration of internal standard has been employed in the transgenic fly to aid examination of the smaller amounts of monoamine. The DA peak is not observed in the Ddc-TNT flies whereas the other peaks are still present to varying levels. The amount of 5-HT is clearly diminished in the Ddc-TNT homogenate compared to the control (11.2 vs 145 fmol/head). Because neurons appear to survive in
the absence of prolonged synaptic function,24 the small amount of 5-HT present in the Ddc-TNT flies could be due to inefficient synaptic inhibition of Ddc cells by TNT. This is consistent with the overall lower levels of L-DOPA and putative metabolites including HVA, 5-HIAA, and DOPAC eluted around 6 min. The level of TA in the Ddc-TNT homogenate, however, remains relatively unchanged (21.2 fmol/head for control, 25.2 fmol/head for Ddc-TNT) as expected. The TA in the fly brain is present in neuronal populations that are distinct from Ddc neurons and is synthesized by different enzymes.29 Interestingly, a peak with unknown identity, marked with a diamond in Figure 4, also remains unaffected by the toxin. The sustained TA and unknown peak support that not all electroactive species in the fly have changed as a result of functional inhibition of the Ddc neurons. Other unidentified peaks have also been affected in the Ddc-TNT flies. These peaks are smaller compared to those in the normal head homogenate. It is yet to be determined whether this was due to direct or indirect effects of the absence of fully functional 5-HT and DA neuronal activities. Nevertheless, reductions of L-DOPA, 5-HT, and metabolites, and the complete loss of quantifiable DA, illustrate a detectable difference in biogenic amines in the transgenic fly. This study clearly demonstrates the relatively specific effect of TNT on the Ddc neurons in the fly and the sensitivity of MEKC-EC to discern it. CONCLUSIONS The studies presented here represent a new approach to the analysis of monoamines present in Drosophila samples. The small volumes handled by capillary electrophoresis are ideally suited for analysis of the small samples needed to compare biogenic amines in Drosophila head and body. Capillary electrophoresis in the micellar mode coupled to amperometric detection provides quantitative clues to the Drosophila nervous system, notably the (29) Livingstone, M. S.; Tempel, B. L. Nature 1983, 303, 67-70.
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differences in TA and 5-HT levels in the head versus the body. Drosophila is a key model organism used by biologists and behavioralists to study the neurochemical basis of numerous physiological conditions such as stress, aging, drug addiction, learning, and memory. The capability of MEKC-EC to distinguish and probe differences in the chemical environment of Drosophila and its genetic variants, as demonstrated in our studies on the Ddc-TNT transgenic flies, opens new opportunities to quantitatively examine this system. ACKNOWLEDGMENT This work was supported, in part, by grants from the National Institutes of Health (K.-A.H., A.G.E) and the National Science Foundation (A.G.E.). S.W.S. acknowledges the support of a National Science Foundation Graduate Fellowship. The authors
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thank Dr. Jay Hirsh at the University of Virginia and Dr. Cahir O’Kane at the University of Cambridge in the U.K. for providing the Ddc-GAL4 and UAS-TNT transgenic flies, respectively. Tracy Paxon and Elizabeth Smith Roddy are also gratefully acknowledged for their assistance with fly homogenization and data analysis, respectively. Note Added after ASAP Posting. The paper was posted on the Web (6/26/03) before all corrections had been made. The final version was posted on 6/30/03.
Received for review March 4, 2003. Accepted May 7, 2003. AC034219I
