Anal. Chem. 2005, 77, 7190-7194
Subcellular Analysis of D-Aspartate Hai Miao, Stanislav S. Rubakhin, and Jonathan V. Sweedler*
Department of Chemistry and the Beckman Institute, University of Illinois at UrbanasChampaign, Urbana, Illinois 61801
D-Aspartate (D-Asp)
is an especially intriguing molecule found within neurons of the central nervous system of animals ranging from mollusks to vertebrates. It has a large variety of roles ascribed to it, including an involvement in cell-to-cell signaling. To determine the D-Asp content in cells and in subcellular domains, a laboratoryassembled capillary electrophoresis system with laserinduced fluorescence (LIF) detection has been used. The system allows chiral separations with sufficient sensitivity and selectivity to measure the D-Asp content in specific subregions of a single neuron, including neuronal processes. The method uses microvial sampling, analyte derivatization with naphthalene-2,3-dicarboxaldehyde, cyclodextrin-mediated micellar electrokinetic capillary chromatography, and sheath flow cell-based LIF detection. Manipulating neuronal processes is difficult as they often disintegrate during the transfer to the sampling vial. We describe a glycerol treatment that stabilizes cell morphology during sample preparation, thereby alleviating the deleterious effects of the high-salt extracellular matrix on the electrophoretic separation. D-Asp percentages in processes from identified neurons from Aplysia californica differ significantly depending on the cell studied. Subcellular analysis reveals more compounds in the cell body than in the processes. In the nervous system, individual neurons directly communicate with hundreds of other cells using specialized extensions (in some cases, centimeters long) termed processes. Why study single processes? Processes are involved in transferring the chemical and electrical signals from one neuron to the next, as well as detecting, integrating, and conducting similar signals originating from other cells. The biochemical composition of processes can be indicative of chemical signals used by the neuron. Despite their significant length, processes have small volumes as they are typically just several micrometers in diameter and thus contain small amounts of analytes. It is difficult to investigate such structures individually with most analytical techniques. Investigation of the major nerves in an organism that contain hundreds of individual processes, glia, and other supporting cells provides important information, but often one must examine individual structures. Some neurotransmitters have multiple functions besides signaling and may be present in cells in significant amounts; thus, the presence of a compound in a process yields important clues as to function that are lost with
* To whom correspondence should be addressed. Telephone: 217-244-7359. Fax: 217-244-8068. e-mail:
[email protected].
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information just on its presence in a cell soma. Here, using D-aspartate (D-Asp) as a model, we report methods to detect neurotransmitters/neuromodulators at the subcellular level using capillary electrophoresis (CE)-laser-induced fluorescence (LIF). D-Asp is found in the central nervous system in a surprising variety of animals, including mammals,1-5 birds,6 reptiles,7 amphibians,8 and even mollusks.9,10 What are the physiological functions of D-Asp? D-Asp has been shown to be released from different tissues upon chemical or electrical stimulation.11-13 D-Asp modulates luteinizing hormone and growth hormone release in rat neuroendocrine tissues,14 the synthesis of testosterone in rat testes,15 and the activity of the aromatase enzyme in lizard.7 While these facts suggest a role relating to signaling, the function of D-Asp in intercellular signaling is still unclear. Recently, it was shown that free D-Asp is present in the nucleoli but not in either dendrites or axon terminals,16 and Bharathi et al. demonstrated that an aluminum D-Asp complex induces topological changes in supercoiled DNA,17 suggesting a function within the nucleus. Several approaches have been used to characterize the D-Asp content in biological tissues, including immunohistochemical (1) Aidulis, D.; Pegg, D. E.; Hunt, C. J.; Goffin, Y. A.; Vanderkelen, A.; Van Hoeck, B.; Santiago, T.; Ramos, T.; Gruys, E.; Voorhout, W. Cell Tissue Bank 2002, 3, 79-89. (2) Fisher, G. H.; Petrucelli, L.; Gardner, C.; Emory, C.; Frey, W. H., 2nd; Amaducci, L.; Sorbi, S.; Sorrentino, G.; Borghi, M.; D’Aniello, A. Mol. Chem. Neuropathol. 1994, 23, 115-124. (3) Hashimoto, A.; Kumashiro, S.; Nishikawa, T.; Oka, T.; Takahashi, K.; Mito, T.; Takashima, S.; Doi, N.; Mizutani, Y.; Yamazaki, T.; et al. J. Neurochem. 1993, 61, 348-351. (4) Sakai, K.; Homma, H.; Lee, J. A.; Fukushima, T.; Santa, T.; Tashiro, K.; Iwatsubo, T.; Imai, K. Brain Res. 1998, 808, 65-71. (5) Schell, M. J.; Cooper, O. B.; Snyder, S. H. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 2013-2018. (6) Neidle, A.; Dunlop, D. S. Life Sci. 1990, 46, 1517-1522. (7) Assisi, L.; Botte, V.; D’Aniello, A.; Di Fiore, M. M. Reproduction 2001, 121, 803-808. (8) Di Fiore, M. M.; Assisi, L.; Botte, V.; D’Aniello, A. J. Endocrinol. 1998, 157, 199-207. (9) Zhao, S.; Liu, Y. M. Biomed. Chromatogr. 2001, 15, 274-279. (10) Zhao, S.; Liu, Y. M. Cell Mol. Biol. 2001, 47, 1217-1222. (11) Nakatsuka, S.; Hayashi, M.; Muroyama, A.; Otsuka, M.; Kozaki, S.; Yamada, H.; Moriyama, Y. J. Biol. Chem. 2001, 276, 26589-26596. (12) Savage, D. D.; Galindo, R.; Queen, S. A.; Paxton, L. L.; Allan, A. M. Neurochem. Int. 2001, 38, 255-267. (13) Wolosker, H.; D’Aniello, A.; Snyder, S. H. Neuroscience 2000, 100, 183189. (14) D’Aniello, A.; Di Fiore, M. M.; Fisher, G. H.; Milone, A.; Seleni, A.; D’Aniello, S.; Perna, A. F.; Ingrosso, D. FASEB J. 2000, 14, 699-714. (15) D’Aniello, A.; Di Cosmo, A.; Di Cristo, C.; Annunziato, L.; Petrucelli, L.; Fisher, G. Life Sci. 1996, 59, 97-104. (16) Wang, H.; Wolosker, H.; Morris, J. F.; Pevsner, J.; Snyder, S. H.; Selkoe, D. J. Neuroscience 2002, 109, 1-4. (17) Bharathi; Jagannatha Rao, K. S.; Stein, R. J. Biol. Inorg. Chem. 2003, 8, 823-830. 10.1021/ac0511694 CCC: $30.25
© 2005 American Chemical Society Published on Web 10/07/2005
staining with anti-D-Asp antibody,18 HPLC separation after derivatizing D-Asp with a chiral fluorogen,14,19 colorimetric detection of D-Asp enzymatic oxidation products,20,21 and radionuclide labeling.22 Until now, effective methods to quantitate chiral amino acids, such as D-Asp in subcellular domains, have been lacking. Immunohistochemistry provides excellent spatial information. However, this approach suffers from difficulty in generating highly specific antibodies for such small and structurally similar antigens as chiral amino acids and problems with preserving the spatial localization of D-Asp, as this molecule is difficult to cross-link using typical fixatives. HPLC and colorimetric-based analyses are not well-suited to studying analytes in subcellullar samples. Radionuclide labeling provides the requisite sensitivity, although the labeling involves an extra step of tissue exposure to radiolabeled analytes12,23 and still often requires a separation step for distinguishing between D-Asp and its possible metabolites. Recently, CE with LIF detection has been applied to study chiral amino acids such as D-Ser and D-Asp in small-volume biological samples due to its low detection limit, small consumption of sample, relatively fast separation, high separation efficiency, and ease of chiral separation.24-26 In addition, CE with LIF detection has been adapted to other subcellular measurements, including measurement of the content of individual organelles within the cell.27-30 In this work, our effort is focused on the analysis of D-Asp in processes from individual neurons of the well-characterized Aplysia central nervous system (CNS). The existence of D-Asp in all five major ganglia of Aplysia CNS was recently demonstrated.9,31 We present a unique sampling protocol using glycerol to stabilize the neuron during isolation, which allows the measurement of the D-Asp in different subcellular regions. Low amounts of glycerol in artificial seawater (ASW) are compatible with the CE separation without causing peak distortion. We demonstrate the assay of D-Asp in the processes originating from single neurons, including individual processes and sections from a single process. The percentage of Asp in the D-form in the processes from different types of neurons varies significantly, while D-Asp percentages in processes from the same sensory neuron are similar. To the best of our knowledge, this is the first demonstration of chiral amino acid characterization with CE at the subcellular level. In Aplysia neurons, D-Asp is located outside the nucleus of the neuron, (18) Wang, H.; Wolosker, H.; Pevsner, J.; Snyder, S. H.; Selkoe, D. J. J. Endocrinol. 2000, 167, 247-252. (19) Aswad, D. W. Anal. Biochem. 1984, 137, 405-409. (20) D’Aniello, A.; Di Fiore, M. M.; D’Aniello, G.; Colin, F. E.; Lewis, G.; Setchell, B. P. FEBS Lett. 1998, 436, 23-27. (21) D’Aniello, S.; Spinelli, P.; Ferrandino, G.; Peterson, K.; Tsesarskia, M.; Fisher, G.; D’Aniello, A. Biochem. J. 2004, 386, 331-340. (22) Granata, A. R.; Reis, D. J. Brain Res. 1983, 259, 77-93. (23) Muzzolini, A.; Bregola, G.; Bianchi, C.; Beani, L.; Simonato, M. Neurochem. Int. 1997, 31, 113-124. (24) Quan, Z.; Liu, Y. M. Electrophoresis 2003, 24, 1092-1096. (25) Wan, H.; Blomberg, L. G. J. Chromatogr., A 2000, 875, 43-88. (26) O’Brien, K. B.; Esguerra, M.; Klug, C. T.; Miller, R. F.; Bowser, M. T. Electrophoresis 2003, 24, 1227-1235. (27) Chen, Y.; Walsh, R. J.; Arriaga, E. A. Anal. Chem. 2005, 77, 2281-2287. (28) Chiu, D. T.; Lillard, S. J.; Scheller, R. H.; Zare, R. N.; Rodriguez-Cruz, S. E.; Williams, E. R.; Orwar, O.; Sandberg, M.; Lundqvist, J. A. Science 1998, 279, 1190-1193. (29) Fuller, K. M.; Arriaga, E. A. Curr. Opin. Biotechnol. 2003, 14, 35-41. (30) Gunasekera, N.; Musier-Forsyth, K.; Arriaga, E. Electrophoresis 2002, 23, 2110-2116. (31) Sweedler, J. V.; Miao, H.; Rubakhin, S. S. Program No. 497.4. 2004 Abstract Viewer and Itinerary Planner; Society for Neuroscience: Washington, DC, 2004. Online.
supporting the idea of the involvement of this molecule in cellto-cell signaling. EXPERIMENTAL SECTION Reagents and CE-LIF System. All solutions were prepared with ultrapure Milli-Q water (Milli-Q filtration system, Millipore, Bedford, MA) to minimize the presence of impurities. Five hundred milliliters of 50 mM borate buffer (pH 9.4) was made by dissolving 2.384 g of sodium borate (Na2B4O7‚10H2O; Sigma, St. Louis, MO) in 456 mL of ultrapure deionized water and adding ∼44 mL of 0.2 M NaOH. This buffer was used for sample preparation and as sheath flow buffer. The separation buffer was 20 mM β-cyclodextrin, 50 mM sodium dodecyl sulfate in 50 mM borate buffer (pH 9.4), and 15% methanol (V/V). All solutions were filtered using 0.2-µm filters (Gelman Sciences, Ann Arbor, MI) to remove particulates. The buffers were degassed by ultrasonication for 10 min to minimize the chance of bubble formation in the capillary and sheath flow cell during separation. Naphthalene-2,3dicarboxaldehyde (NDA) and KCN were used to derivatize primary amines. The 10 mM NDA was dissolved in methanol and 20 mM KCN in Milli-Q water. Both NDA and KCN were carefully filtered before derivatization. NDA and KCN were stored in a refrigerator, and fresh solutions were made weekly. Glycerol (minimum purity of 99%) was from Sigma. L-Cysteic acid (L-Cya), dissolved in 50 mM borate buffer (pH 9.4), was used as internal standard and made fresh for all experiments. L-Cya was used as the internal standard as it did not comigrate with any of the 22 amino acids we studied in control experiments and its signal intensity showed higher reproducibility for these samples than L-norleucine (data not shown), another compound commonly used as internal standard. Analyte separation and detection were performed using the previously described CE-LIF system;32 in this work, the excitation was shifted to 457.9 nm by changing the laser, focusing lens, and optics as follows: an argon ion laser (Melles Griot, Carlsbad, CA) provided the 457.9-nm wavelength line and a Mitutoyo infinitycorrected long working distance objective (Catalog No. H46144, Edmund, Barrington, NJ) was used to focus the beam to a postcolumn sheath flow assembly. The fluorescence was collected by an all-reflective microscope objective and a focusing lens (fl 8.0 cm). A 488-nm notch filter, a 475-nm long-pass filter (03FCG465/ GG475, Melles Griot, Irvine, CA), and a 550-nm short-pass filter (03SWP408, Melles Griot) were used to select the fluorescence signal. The microinjector, high-voltage power supply, sheath flow assembly, and photomultiplier tube were the same as previously described.32 The analyte separation occurred in the 65-75-cmlong, 50-µm-i.d./360-µm-o.d. uncoated fused-silica capillary (Polymicro Technologies, Phoenix, AZ). Animals and Dissection. Aplysia californica (200-300 g) were obtained from Charles Hollahan (Santa Barbara, CA) and kept in an aquarium containing continuously circulating, aerated, and filtered ASW at 14-15 °C until used. Animals were anesthetized by injection of isotonic MgCl2 (∼30 to ∼50% of body weight) into the body cavity. The CNS was dissected and placed in ASW containing (in mM) 460 NaCl, 10 KCl, 10 CaCl2, 22 MgCl2, 6 MgSO4, and 10 HEPES, pH 7.8 or in ASW-antibiotic solution: (32) Miao, H.; Rubakhin, S. S.; Sweedler, J. V. Anal. Bioanal. Chem. 2003, 377, 1007-1013.
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ASW supplemented with 100 units/mL penicillin G, 100 µg/mL streptomycin, and 100 µg/mL gentamicin, pH 7.8. In some cases, to improve isolation of cellular clusters and individual neurons, the ganglion sheath was enzymatically treated by incubating the CNS or its regions in 1% protease (Type IX: Bacterial; Sigma) ASW-antibiotic solution at 36 °C for 1-2 h depending on animal size and season. Next, the ganglia were washed in fresh ASW. Using sharp needles made from 0.38-µm-diameter tungsten wire (WPI, Sarasota, FL), the ganglia were pinned dorsal side up to a silicone elastomer (Sylgard, Dow Corning, Midland, MI) layer in a plastic chamber containing 3-4 mL of ASW-antibiotic media. The ganglia, individual neurons, and their processes were dissected mechanically using dissection scissors. In some experiments, 30% glycerol-ASW mixture was substituted for ASW. Extracellular solution was removed after 1-5-min exposure of the cell culture to the glycerol solution using a Pasteur pipet. Cells and processes were isolated using sharp tungsten needles or glass pipets attached to a micromanipulator. These samples were stored at -80 °C in a freezer and usually analyzed within 1 day after dissection. Sample Derivatization. The samples (cell bodies or neuronal processes) were put in the center of the dome caps of 0.2-mL Thermowell tubes (Corning Inc., Corning, NY). For the F- and C-cluster neurons, 10 µL of 1.7 × 10 µM L-Cya solution, 5 µL of 20 mM KCN, and 5 µL of 10 mM NDA were added. For single sensory neuron soma and process samples, 0.5 µL of 10 µM L-Cya solution, 0.5 µL of 20 mM KCN, 0.5 µL of 10 mM NDA, and 1 µL of 50 mM borate buffer were added. For each R2 neuron process fragment, 10 µL of 3.3 × 10 µM L-Cya solution, 5 µL of 20 mM KCN, and 5 µL of 10 mM NDA were added. All solutions were carefully mixed with pipets and left for 30 min at room temperature for the reaction to complete in closed tubes. Usually, the total volume after mixing and the 30-min reaction was less than the sum of individual volumes added because of evaporation. Approximately 400 nL was transferred to a 400-nL metal nanovial. The nanovial was positioned into the microinjection box, and sample was immediately analyzed to minimize liquid evaporation. The sample was loaded into the capillary using electrokinetic injection (5 kV for 3 s) with an injection current of ∼5 µA. The separation voltage was 21 kV with an associated current of ∼25 µA. Morphology. The preparations were viewed and photographed using a Zeiss Axiovert inverted microscope equipped with a CCD camera and software package (Carl Zeiss, Oberkochen, Germany). RESULTS AND DISCUSSION Protocol Optimization. To maintain the viability of tissues throughout the experiments, they are placed in ASW after isolation. However, due to the high ionic strength of ASW, its use as a sample buffer causes electropherogram peak distortion and an unstable baseline during CE separation.32 If the tissue samples are relatively large, such as entire neuronal ganglia, neuronal clusters, nerves, connectives, or even the larger identified single neurons, the presence of ASW does not cause a problem; the amount of material is large enough that the samples can be significantly diluted with borate buffer to decrease their ionic strength prior to injection into a CE column. The excellent detection limit of the CE-LIF system (∼5.0 × 10-10 M), and the relatively high concentration of the D-Asp (near millimolar) in 7192 Analytical Chemistry, Vol. 77, No. 22, November 15, 2005
Figure 1. Electropherograms showing the D-Asp content of the Cand F-cluster top layer neurons sampled with and without glycerol treatment. The electropherogram is scaled so several peaks are truncated to allow better visibility of the D- and L-Asp peaks. Peak identities: D-Asp, D-aspartate; L-Asp, L-aspartate; I.S., internal standard, L-cysteic acid.
some regions of the CNS, allow the dilution of tissue samples with buffer while maintaining an adequate S/N ratio in the resulting electropherograms. However, such a strategy cannot be applied to small samples, such as neuronal processes, because addition of sufficient buffer to reduce the ionic strength of these samples can result in the dilution of analyte below the detection limit of the CE system. Can one stabilize the samples to aid in the required manipulations by substituting the ASW with a lower ionic strength solution? There have been some reports showing that glycerol is a good additive to maintain both cell function and cell morphology.1,33,34 In a recent report from our group,35 a 30-50% glycerol-ASW mixture was substituted for ASW during sample preparation. In comparison to ASW, this solution provided excellent signal quality, without analyte spreading, for MALDI-MS profiling of the spatial distribution of neuropeptides within single neurons. MALDI-MS images demonstrate that there is little peptide redistribution during the glycerol treatment. Here we show that glycerol treatment of single neurons is compatible with our CE separation and does not change the D-Asp and L-aspartate (L-Asp) presence in the neurons we studied. Typical electropherograms of samples treated with or without glycerol are shown in Figure 1. No observable difference is found between C- and F-cluster top neurons treated either with glycerol or analyzed without glycerol treatment. D-Asp in Processes from Individual Neurons. We analyzed the D-Asp content in the processes of a range of individual neurons using glycerol treatment during sample preparation. Aplysia neurons range in size from several hundred micrometers to less (33) Hoving, S.; Gerrits, B.; Voshol, H.; Muller, D.; Roberts, R. C.; van Oostrum, J. Proteomics 2002, 2, 127-134. (34) Pegg, D. E. Semin. Reprod. Med. 2002, 20, 5-13. (35) Rubakhin, S. S.; Greenough, W. T.; Sweedler, J. V. Anal. Chem. 2003, 75, 5374-5380.
Figure 3. Processes of the same neuron have similar biochemical profiles, including D-/L-aspartate ratio. Shown are electropherograms of single processes. Process 1 (A), process 2 (B), and soma (C) are from a single pleural sensory neuron. Peak identities: Glu, glutamate; D-Asp, D-aspartate; L-Asp, L-aspartate; Leu, leucine; I.S., internal standard, L-cysteic acid; *, unidentified peak.
was reported only in the nucleus compartment of soma.16 As shown below, in other neurons, less than 10% of the Asp is in the D-form. D-Asp in Individual Processes from a Single Neuron. For larger identified neurons, individual processes from a single neuron have been analyzed in order to answer questions about whether D-Asp has a heterogeneous distribution in different processes originating from the same neuron soma. Figure 3 shows the electropherograms of two single processes (Figure 3A and B) and the corresponding cell body (Figure 3C). As in the above-reported experiments, more compounds are detected in the cell soma than in the processes (Figure 3C). The electropherograms of different processes appear similar. Interestingly, unlike the heterogeneous distribution of D-Asp in nerves, clusters, and identified neurons, the D-Asp percentages in individual processes from the same neuron are similar. R2 is an identified giant cholinergic neuron (Figure 4) localized on the right dorsolateral margin of the abdominal ganglion of Aplysia; it has a major process that extends all the way to the pleural ganglion and other distal regions.36-38 Figure 4C is a photomicrograph of an R2 neuronal soma separated from its neurite. The neurite was further divided into two sections, and each section was derivatized and analyzed separately. Panels A and B of Figure 4 present the electropherograms of each section of the neurite. In addition to D-Asp, glutamate (Glu) was also detected. As seen from the figure, D-Asp amounts and percentages were similar in different regions of the neurite while the amount of Glu was different; the Glu amount in section 2 (Figure 4B) was ∼5 times higher than in section 1 (Figure 4A). Assuming the D-Asp
Figure 2. Detection of D-Asp in the processes of sensory neurons. (A) Image of an isolated sensory neuron acquired using variable relief contrast. Electropherograms of D-Asp in (B) the processes and (C) the soma from a single pleural sensory neuron. Peak identities: Glu, glutamate, with a comigrating compound; D-Asp, D-aspartate; L-Asp, L-aspartate; Leu, leucine; Phe, phenylalanine; I.S., internal standard, L-cysteic acid; *, unidentified peaks. The y-axis scale has been chosen to allow visualization of the D- and L-Asp peaks, so that several other peaks are off scale.
than 20 µm. Figure 2A is a microphotograph of a bipolar pleural ganglion sensory neuron. The diameter of the cell soma was ∼30 µm (a volume of 5 pL). The soma was carefully separated from its processes, and each was manually placed in vials for analysis (Figure 2B and C). We report the D-Asp as a percentage of total Asp in a sample, D-Asp/(D-Asp + L-Asp), as the ratio is independent of the volume of tissue used. A high percentage of D-Asp was found in both samples, with ∼70% of Asp in the D-form in the soma and ∼75% in the processes. In addition, the amounts of many amino acids, such as D-Asp, L-Asp, Leu, and Phe, are higher in the soma than in the processes investigated in these experiments, with the levels in some cases correlating to the larger internal volume of the soma compared to the volume of the analyzed processes. Larger numbers of compounds are detected in the cell soma (peaks marked with asterisks in Figure 2C) compared to the processes. This may indicate a lower biochemical complexity in the processes or that these compounds are below the detection limits of the detector. Like the cerebral ganglion F- and C-clusters, which consist mostly of multiple neuron soma, the D-Asp percentage in specific neurites can be high (above 70%).31 These results demonstrate an important difference between D-Asp-containing Aplysia and mammalian neurons; in mammalian neurons, the
(36) Ambron, R. T.; Kremzner, L. T. Proc. Natl. Acad. Sci. U.S.A. 1982, 79, 3442-3446. (37) Ambron, R. T.; Rayport, S. G.; Babiarz, J. J. Neurosci. 1988, 8, 722-731. (38) Yurchenko, O. P.; Grigoriev, N. G.; Turpaev, T. M.; Konjevic, D.; Rakic, L. Comp. Biochem. Physiol. C 1987, 87, 389-391.
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heterogeneous distribution in the same neurite. The soma of the R2 neuron had a relatively low percentage of D-Asp, usually below 10% of the total Asp. Similar results were obtained with another giant neuron, the serotonergic MCC, located in the cerebral ganglia of Aplysia. A broad range of D-Asp amounts was measured in cellular and subcellular domains. Determination of whether the presence of D-Asp in low amounts is the general case in giant motorneurons requires further investigation. CONCLUSIONS In this work, we demonstrate an approach for the quantitative investigation of the biochemical composition of subcellular regions of single neurons. Specifically, the colocalization of L- and D-Asp is observed using chiral CE-LIF separations with an optimized sampling procedure that preserves sample integrity and amino acid content in mechanically separated, individual subcellular structures. This approach can be applied to a large variety of biochemical and physiological experiments where the spatial and temporal changes in these small molecules is to be investigated. High-intensity signals obtained from segments of processes of Aplysia neurons suggest that the approach can be adopted for analysis of even the smaller processes of vertebrate neurons. Our results further demonstrate that D-Asp is present in the processes of individual neurons. In different identified neurons, the percentage of the Asp in the D-form varies considerably. However, morphologically distinct regions of the same neuron exhibited similar ratios of D-Asp, despite differences in the amounts observed. These results unequivocally demonstrate that D-Asp is not restricted to the nucleus of Aplysia neurons. In future research, we will use neurons with high D-Asp content to investigate possible mechanisms of uptake, synthesis, and transport, as well as the functional role of this putative intercellular signaling molecule. Figure 4. Different regions of the same process with similar D-Asp percentages. Shown are typical electropherograms from different regions of the same process of the giant identified neuron, R2: (A) part 1; (B) part 2. (C) Image of an R2 neuron and its main neurite acquired using variable relief contrast. Peak identities: Tau, taurine; Glu, glutamate, with a comigrating compound; D-Asp, D-aspartate; L-Asp, L-aspartate; I.S., internal standard, L-cysteic acid. The y-axis scale has been chosen to allow visualization of the D- and L-Asp peaks, so that the Tau peak is off scale in (B).
lengths/volumes of the two fragments were equal, Glu had a
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ACKNOWLEDGMENT This work is sponsored by the National Science Foundation and the National Institutes of Health through grants NIH DK070285 and NSF CHE-0400768.
Received for review June 30, 2005. Accepted September 8, 2005. AC0511694
