Nemes P et al. (2012), Single-cell metabolomics: changes in the metabo...

XB-ART-51830

ACS Chem Neurosci 2012 Oct 17;310:782-92.

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Single-cell metabolomics: changes in the metabolome of freshly isolated and cultured neurons.

Nemes P , Knolhoff AM , Rubakhin SS , Sweedler JV .

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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.

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Species referenced: Xenopus
Genes referenced: gnl3

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	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.

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