No abstract

By using matrix-assisted laser desorption͞ ionization time-of-f light MS, individual peptidergic neurons from Aplysia are assayed. A semiquantitative method is developed for comparing single-cell profiles by using spectral normalization, and peptides are localized to specific cells by mass spectrometric cell mapping. In addition to all previously identified products of the egg-laying hormone (ELH) gene, other peptides are formed from proteolytic hydrolysis of Leu-Leu residues within ELH and acidic peptide (AP). AP exhibits further processing to yield AP 1-20 and AP 9 -27 . These peptides appear to be colocalized in vesicles with ELH, transported to specific neuronal targets, and released in a Ca 2؉ -dependent manner. A differential peptide distribution is observed at a specific target cell, and a low-frequency variation of AP, [Thr 21 ]AP, is detected in a single animal.

One of the most widely used transgenic animal models in biology is Drosophila melanogaster, the fruit fly. Chemical information from this exceedingly small organism is usually accomplished by studying populations to attain sample volumes suitable for standard analysis methods. This paper describes a direct sampling technique capable of obtaining 50-300 nL of hemolymph from individual Drosophila larvae. Hemolymph sampling performed under mineral oil and in air at 30 s intervals up to 120 s after piercing larvae revealed that the effect of evaporation on amino acid concentrations is insignificant when the sample was collected within 60 s. Qualitative and quantitative amino acid analyses of obtained hemolymph were carried out in two optimized buffer conditions by capillary electrophoresis with laser-induced fluorescence detection after derivatizing with fluorescamine. Thirteen amino acids were identified from individual hemolymph samples of both wild-type (WT) control and the genderblind (gb) mutant larvae. The levels of glutamine, glutamate, and taurine in the gb hemolymph were significantly lower at 35%, 38%, and 57% of WT levels, respectively. The developed technique that samples only the hemolymph fluid is efficient and enables accurate organism-level chemical information while minimizing errors associated with possible sample contaminations, estimations, and effects of evaporation compared to the traditional hemolymph-sampling techniques.

Many neuroscientists assume that ambient extracellular glutamate concentrations in the nervous system are biologically negligible under nonpathological conditions. This assumption is false. Hundreds of studies over several decades suggest that ambient extracellular glutamate levels in the intact mammalian brain are ~0.5 to ~5 μM. This has important implications. Glutamate receptors are desensitized by glutamate concentrations significantly lower than needed for receptor activation; 0.5 to 5 μM of glutamate is high enough to cause constitutive desensitization of most glutamate receptors. Therefore, most glutamate receptors in vivo may be constitutively desensitized, and ambient extracellular glutamate and receptor desensitization may be potent but generally unrecognized regulators of synaptic transmission. Unfortunately, the mechanisms regulating ambient extracellular glutamate and glutamate receptor desensitization remain poorly understood and understudied.

Matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI/TOF‐MS) is a viable technique for the examination of biological environments. Clearly, sample preparation plays a pivotal role in the ability to obtain mass spectra from samples as complex as biological cells. The physiological salt concentrations associated with neurons from marine specimens interfere with MALDI analysis. A unique and simple rinsing procedure allows cellular clusters, individual neurons and connective tissues to be directly assayed for peptides with minimal sample handling. Isolated cells and tissues, including egg‐laying hormone‐releasing cells, from the central nervous systems of the model marine molluscs Aplysia californicaand Pleurobranchaea californicaare used to demonstrate the salt removal method. In addition to facilitating sample ionization, the MALDI matrix 2,5‐dihydroxybenzoic acid serves to (i) aid in microdissections by stabilizing cell membranes, (ii) deactivate endogenous proteolytic enzymes and (iii) reduce high salt concentrations in order to improve spectral quality. Representative MALDI mass spectra are presented which indicate the presence of several neuroactive peptides previously characterized by conventional biochemical methods. More than ten individual peptides can be detected in a single cell. In spite of the chemically complex sample, the mass spectra are surprisingly free of extraneous peaks. Furthermore, both mass resolution and mass accuracy are similar to those encountered with more common MALDI samples and protocols.