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RESEARCH PROFILES Subcellular measurements of D-amino acids
JONATHAN V. SWEEDLER AND STANISLAV S. RUBAKHIN
and his team have applied their tech“What’s important here is the subcelThe amino acid D-aspartate (D-Asp) has nique to an analysis of the distribution been identified from the central nervous lular sampling,” says Sweedler. The CE/ of D-Asp in individual neurons of known LIF method is similar to existing protosystem of every animal thus far investicols, he says, and maintains the character- identity. Whereas earlier studies in mamgated, from humans to octopuses. The istic low detection limit (~5.0 10–10 M), malian neurons showed a D-Asp distrimolecule is released from various tissues under electrical or chemical stimulation, picoliter-range sample consumption, and bution restricted to the cell nucleus, and an aluminum–D-Asp complex was fast, efficient chiral separation. “But we’ve Sweedler’s team found high ratios of worked out methods to pull out very recently shown to cause topological D-Asp to L-Asp in both cell body and changes in the structure of supercell process samples of several coiled DNA. But despite its apparAplysia neurons, including ratios ent ubiquity and proposed involveof ~70% and ~75%, respectively, ment in cell–cell signaling and in for one bipolar pleural ganglion processes occurring in the cell nusensory neuron. Overall, Sweedler cleus, this enantiomer’s biological and his colleagues found that DProcesses Cell body role remains mysterious. Asp ratios varied widely between Until now, an effective assay for different identified neurons but subcellular determination of D-Asp that ratios were similar throughout different morphological areas and other neurochemicals has been of single neurons. lacking. The use of antibodies for “Now we know what cells to quantitative analysis of D-amino A sensory neuron, freshly isolated from Aplysia. Careful look at, and we can start to ask acids in biological tissues is difficult. sample preparation and handling allow for biochemical more questions,” says Sweedler. “Is Similarities in molecular shape and analysis of distinct cellular regions, including individual mass, along with the presence of D-Asp released? What does it do in neuronal processes. both enantiomers, make their analypostsynaptic cells?” In a way, he sis by MS problematic. By combining an says, these are “retro experiments,” parsmall pieces of the cell and analyze optimized CE/laser-induced fluorescence those,” says Sweedler. “Every time the alleling the early physiological work on (LIF) detection system with a glycerol neurotransmitters such as serotonin, sample touches a new wall [or] a pipette sample preparation method, Jonathan V. “testing particular neurons and asking tip, there are losses, so we had to miniSweedler, Stanislav S. Rubakhin, and Hai mize manipulations drastically.” what does D-Asp do.” Miao have filled that gap in analytical Attempting to dissect and manipulate Although the analytical system and measurement ability. individual neuronal processes—delicate approach are optimized for derivatizaIn the November 15 issue of Analyt- structures in the 20-µm size range— tion and measurement of D-Asp, they ical Chemistry (pp 7190–7194), the recan be “like isolating a piece of tissue are also suitable for analysis of D-glutasearchers, based at the University of Illi- paper,” says Sweedler. “It falls apart mate with minimal changes, Sweedler nois at Urbana–Champaign, describe a very easily.” Traditional fixatives, like says, and can be more broadly adapted new method for measuring the ratio of formaldehyde, cross-link with memto study a wide range of amino acids. branes and cellular constituents, interThe technique can also be combined D- and L-Asp in individual cell bodies fering with electrophoresis. But the with other methods to extend the range and cellular processes that have been of compounds and processes analyzed. mechanically isolated from neurons. The glycerol treatment stabilizes cell membranes sufficiently to allow for mechaniAlready, Sweedler and his co-workers introduction of glycerol into the extrahave gone on to show that when radiocellular physiological media during sam- cal dissection of cell processes without significant sample loss. “It turns out labeled L-Asp is supplied externally to ple preparation allows for improved stathat this was an old technique that has bilization of cellular morphology and Aplysia neurons, the cells convert it to not been used much recently,” says keeps the cell hydrated even during D-Asp in a predominantly one-way conSweedler. “We weren’t aware of it at sample manipulations in air. Especially version. Now, they’re developing protofor very small samples such as individual the time, but there is a large literature.” cols to sample neurotransmitter release Working with the marine slug nerve terminals, the use of glycerol induring physiological stimulation of indistead of a high ionic strength extracellu- Aplysia californica, a well-studied neuvidual neurons. “The next step,” rological model organism in which lar solution decreases peak distortion Sweedler says, “is to adapt the techcomplex behavior is controlled by a rel- nique to study dynamic processes.” a and improves baseline stability during atively simple nervous system, Sweedler CE, without excessive sample dilution. —Thomas Hayden 450 A
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