Click here to close
Hello! We notice that you are using Internet Explorer, which is not supported by Xenbase and may cause the site to display incorrectly.
We suggest using a current version of Chrome,
FireFox, or Safari.
???displayArticle.abstract???
Metabolites are involved in a diverse range of intracellular processes, including a cell's response to a changing extracellular environment. Using single-cell capillary electrophoresis coupled to electrospray ionization mass spectrometry, we investigated how placing individual identified neurons in culture affects their metabolic profile. First, glycerol-based cell stabilization was evaluated using metacerebral neurons from Aplysia californica; the measurement error was reduced from ∼24% relative standard deviation to ∼6% for glycerol-stabilized cells compared to those isolated without glycerol stabilization. In order to determine the changes induced by culturing, 14 freshly isolated and 11 overnight-cultured neurons of two metabolically distinct cell types from A. californica, the B1 and B2 buccal neurons, were characterized. Of the more than 300 distinctive cell-related signals detected, 35 compounds were selected for their known biological roles and compared among each measured cell. Unsupervised multivariate and statistical analysis revealed robust metabolic differences between these two identified neuron types. We then compared the changes induced by overnight culturing; metabolite concentrations were distinct for 26 compounds in the cultured B1 cells. In contrast, culturing had less influence on the metabolic profile of the B2 neurons, with only five compounds changing significantly. As a result of these culturing-induced changes, the metabolic composition of the B1 neurons became indistinguishable from the cultured B2 cells. This observation suggests that the two cell types differentially regulate their in vivo or in vitro metabolomes in response to a changing environment.
Figure 1. Analyte extraction strategies for single isolated MCC neurons of
the A. californica CNS. (a) PCA score
plot of the CE-ESI-MS data revealed differences between sample extracts:
cells isolated in ASW and 33% glycerol-ASW solutions form separate
data clusters. Duplicate analytical measurements are included. (b)
The PCA loading plot helped to identify specific metabolic differences
between the cell extracts. Underlined numbers correspond to compounds
identified in Table 1. (c) The composition
of the cell-isolation solution had a pronounced effect on extraction
efficiency for many, but not all, metabolites. For example, when isolating
neurons in glycerol-ASW, the ion signal intensities did not appreciably
vary for glycine betaine, significantly increased for adenosine, and
decreased for ornithine. Bars correspond to individual cells measured
in technical duplicates. (d) Histograms show the cumulative measurement
error as RSD for 35 metabolites measured in duplicate. Gaussian curves
(solid lines) fitted on these data had a median and width of ∼24%
(RSD) and ∼16% for cells isolated in ASW, and ∼6% (RSD)
and ∼13% for those treated with 33% glycerol-ASW. The higher
analytical reproducibility offered by glycerol stabilization was beneficial
for assessing chemical changes upon neuron culturing. Key: MCC1–4 = freshly isolated and MCC5–8 = glycerol-stabilized MCC cell extracts.
Figure 2. Metabolic differentiation between freshly isolated and
cultured
neurons of A. californica. (a) PCA
score plots of the CE-ESI-MS data uncovered differences in metabolite
abundances between the freshly isolated B1 and B2 neurons. (b) In
contrast, these neurons possessed indistinguishable chemistries after
cell culture. Respective loading plots are shown in Figure S2. Metabolic differences were (c) clear between the
freshly isolated and cultured B1 cells and (d) minor between the freshly
isolated and cultured B2 neurons. These results indicate that despite
metabolic dissimilarities in the freshly isolated state, B1 and B2
neuron chemistries became similar upon culturing. Key: B11–7 = freshly isolated B1; B21–7 = freshly isolated B2; cB11–5 = cultured B1; and cB21–6 = cultured
B2 neuron extracts. Technical replicate measurements are included.
Figure 3. Morphological and statistically significant metabolic
changes upon
single-cell culturing. (a) In culture, B1 and B2 neurons typically
formed a network of neurites overnight, as demonstrated in the microscope
image of a representative cultured B2 cell. (b) Statistical analysis
of the data revealed that culturing imposed cell-type dependent variations
in neuron chemistries. For example, both neuron types in culture became
depleted in alanine. (c) Acetylcholine abundance decreased in the
B2 but not the B1 neurons. (d) In stark contrast, other compounds
such as glycine accumulated in the B2 neurons only. Bars correspond
to individual cells measured in technical duplicates. Key: square,
box, and whisker represent statistical median, standard error, and
confidence interval, respectively. NS labels statistically insignificant
variations, and asterisk (*) and two asterisks (**) mark p-values below 0.05 and 0.005, respectively. Scale = 50 μm.
Ainla,
A microfluidic pipette for single-cell pharmacology.
2010, Pubmed
Ainla,
A microfluidic pipette for single-cell pharmacology.
2010,
Pubmed Amantonico,
Single-cell MALDI-MS as an analytical tool for studying intrapopulation metabolic heterogeneity of unicellular organisms.
2010,
Pubmed Amantonico,
Analytical techniques for single-cell metabolomics: state of the art and trends.
2010,
Pubmed Blow,
Metabolomics: Biochemistry's new look.
2008,
Pubmed Borland,
Chemical analysis of single cells.
2008,
Pubmed Cecala,
Sampling techniques for single-cell electrophoresis.
2012,
Pubmed Church,
Peptidergic motoneurons in the buccal ganglia of Aplysia californica: immunocytochemical, morphological, and physiological characterizations.
1991,
Pubmed Coello,
Atmospheric pressure femtosecond laser imaging mass spectrometry.
2010,
Pubmed Fan,
Collection of peptides released from single neurons with particle-embedded monolithic capillaries followed by detection with matrix-assisted laser desorption/ionization mass spectrometry.
2011,
Pubmed Fuller,
Single neuron analysis by capillary electrophoresis with fluorescence spectroscopy.
1998,
Pubmed Gach,
Isolation and manipulation of living adherent cells by micromolded magnetic rafts.
2011,
Pubmed Gaiarsa,
Contribution of metabotropic GABA(B) receptors to neuronal network construction.
2011,
Pubmed Gao,
GABA release from mouse axonal growth cones.
2000,
Pubmed Greving,
Nanostructure-initiator mass spectrometry metabolite analysis and imaging.
2011,
Pubmed Hatcher,
5-HT and 5-HT-SO4, but not tryptophan or 5-HIAA levels in single feeding neurons track animal hunger state.
2008,
Pubmed Heinemann,
Single cell metabolomics.
2011,
Pubmed Huh,
From 3D cell culture to organs-on-chips.
2011,
Pubmed Kane,
Liver-specific functional studies in a microfluidic array of primary mammalian hepatocytes.
2006,
Pubmed Kreiner,
Localization of Aplysia neurosecretory peptides to multiple populations of dense core vesicles.
1986,
Pubmed Kress,
Cell stimulation with optically manipulated microsources.
2009,
Pubmed Kurczy,
Mass spectrometry imaging of mating Tetrahymena show that changes in cell morphology regulate lipid domain formation.
2010,
Pubmed Kwon,
Glutamate induces de novo growth of functional spines in developing cortex.
2011,
Pubmed Lapainis,
Capillary electrophoresis with electrospray ionization mass spectrometric detection for single-cell metabolomics.
2009,
Pubmed Lapainis,
Contributions of capillary electrophoresis to neuroscience.
2008,
Pubmed Lin,
Chemical analysis of single cells.
2011,
Pubmed Lloyd,
Release of neuropeptides during intracellular stimulation of single identified Aplysia neurons in culture.
1986,
Pubmed Lloyd,
Biochemical and immunocytological localization of molluscan small cardioactive peptides in the nervous system of Aplysia californica.
1985,
Pubmed Lyck,
Culture-induced changes in blood-brain barrier transcriptome: implications for amino-acid transporters in vivo.
2009,
Pubmed Maric,
GABA expression dominates neuronal lineage progression in the embryonic rat neocortex and facilitates neurite outgrowth via GABA(A) autoreceptor/Cl- channels.
2001,
Pubmed Mellors,
Integrated microfluidic device for automated single cell analysis using electrophoretic separation and electrospray ionization mass spectrometry.
2010,
Pubmed Miyamoto,
Binomial analysis of quantal transmitter release at glycerol treated frog neuromuscular junctions.
1975,
Pubmed Monroe,
Vitamin E imaging and localization in the neuronal membrane.
2005,
Pubmed Nemes,
Laser ablation electrospray ionization for atmospheric pressure, in vivo, and imaging mass spectrometry.
2007,
Pubmed Nemes,
Metabolic differentiation of neuronal phenotypes by single-cell capillary electrophoresis-electrospray ionization-mass spectrometry.
2011,
Pubmed Nemes,
Spraying mode effect on droplet formation and ion chemistry in electrosprays.
2007,
Pubmed Neupert,
Single-cell peptidomics of drosophila melanogaster neurons identified by Gal4-driven fluorescence.
2007,
Pubmed Niepel,
Non-genetic cell-to-cell variability and the consequences for pharmacology.
2009,
Pubmed Northen,
Clathrate nanostructures for mass spectrometry.
2007,
Pubmed Pegg,
Long-term preservation of cells and tissues: a review.
1976,
Pubmed Romanova,
Engineering the morphology and electrophysiological parameters of cultured neurons by microfluidic surface patterning.
2004,
Pubmed Rubakhin,
Quantitative measurements of cell-cell signaling peptides with single-cell MALDI MS.
2008,
Pubmed Rubakhin,
Profiling metabolites and peptides in single cells.
2011,
Pubmed
,
Xenbase Rubakhin,
Spatial profiling with MALDI MS: distribution of neuropeptides within single neurons.
2003,
Pubmed Salehi-Reyhani,
A first step towards practical single cell proteomics: a microfluidic antibody capture chip with TIRF detection.
2011,
Pubmed Santama,
Neural network controlling feeding in Lymnaea stagnalis: immunocytochemical localization of myomodulin, small cardioactive peptide, buccalin, and FMRFamide-related peptides.
1994,
Pubmed Schoenherr,
CE-microreactor-CE-MS/MS for protein analysis.
2007,
Pubmed Scott,
Neuromuscular organization of the buccal system in Aplysia californica.
1991,
Pubmed Sernagor,
GABAergic control of neurite outgrowth and remodeling during development and adult neurogenesis: general rules and differences in diverse systems.
2010,
Pubmed Shrestha,
In situ cell-by-cell imaging and analysis of small cell populations by mass spectrometry.
2011,
Pubmed Silva,
Molecular and cellular cognitive studies of the role of synaptic plasticity in memory.
2003,
Pubmed Snijder,
Origins of regulated cell-to-cell variability.
2011,
Pubmed Stuart,
Serotonin catabolism depends upon location of release: characterization of sulfated and gamma-glutamylated serotonin metabolites in Aplysia californica.
2003,
Pubmed Svatoš,
Single-cell metabolomics comes of age: new developments in mass spectrometry profiling and imaging.
2011,
Pubmed Tapia,
Early expression of glycine and GABA(A) receptors in developing spinal cord neurons. Effects on neurite outgrowth.
2001,
Pubmed Tsuyama,
Mass spectrometry for cellular and tissue analyses in a very small region.
2011,
Pubmed Turrigiano,
Homeostatic plasticity in the developing nervous system.
2004,
Pubmed Urban,
High-density micro-arrays for mass spectrometry.
2010,
Pubmed Yang,
Detection of characteristic distributions of phospholipid head groups and fatty acids on neurite surface by time-of-flight secondary ion mass spectrometry.
2010,
Pubmed Yaqoob,
Influence of cell culture conditions on diet-induced changes in lymphocyte fatty acid composition.
1995,
Pubmed Zimmerman,
MALDI mass spectrometry imaging of neuronal cell cultures.
2011,
Pubmed
