Involvement of Sphingosine-1-Phosphate in Glutamate Secretion in Hippocampal Neurons

Inhibition of depolarization-evoked glutamate secretion by DMS. (A) Neurons cultured in 6-cm dishes were washed three times with phosphate-buffered saline. Neurons were pretreated without (vehicle) or with 10 μM DMS or 50 μM HACPT for 30 min and analyzed for glutamate secretion 1 min after treatment with either buffer or buffer containing 50 mM KCl. (B) Schematic representation of the protocol for the measurement of neurotransmitter secretion using the FM4-64 dye method. (C) Rat hippocampal neurons were treated without (vehicle; control) or with 10 μM DMS for 30 min. Neurons were washed and labeled with FM dye, which was incorporated into active presynaptic vesicles. The fluorescence of the dye at each punctum of interest was sequentially monitored after depolarization induced by 50 mM KCl. (D) Control or DMS-treated neurons were labeled with FM dye as in panel C, and the fluorescence of the dye was monitored after treatment with 100 μM glutamate. The arrow indicates the addition of glutamate. Data are means ± standard errors of the means of three independent experiments carried out in triplicate. For F(t), fluorescence at the selected region at time t, see text.

Involvement of SK1 in depolarization-evoked secretion. (A) Rat hippocampal neurons were transfected either with control or rSK1-siRNA together with expression vectors encoding rSK1, hSK1, or hSK1G82D. Two days after transfection neurons were analyzed for SK1 expression by immunoblot analysis. Endogenous SK1 mRNA and protein were quantitated in neurons transfected with either control or rSK1-siRNA by real-time quantitative PCR (B) or by immunoblot analysis using anti-SK1 antibody (C). Neurons transfected with either control or rSK1-siRNA together with vectors encoding hSK1, hSK1G82D, or an empty vector (mock) were prelabeled with FM4-64 and treated with 50 mM KCl (D) or 100 μM glutamate (E), and the fluorescence of the dye at each punctum of interest was sequentially monitored. The arrow indicates the addition of glutamate. Data are means ± standard errors of the means of three independent experiments carried out in triplicate (D and E). For F(t), fluorescence at the selected region at time t, see text.

Translocation of SK1 during depolarization. Hippocampal neurons were transiently transfected with expression vectors encoding GFP-SK1, free GFP, or SynPhy-GFP as indicated. Two days after transfection living cells were subjected to FRAP analysis using confocal laser scanning microscopy. After depolarization induced either by 50 mM KCl (A) or 100 μM glutamate (B), axonal puncta expressing GFP-fused proteins were photobleached. Subsequently, images were collected at the indicated time points. For the graphs the fluorescence recovery immediately after photobleaching (lowest fluorescence intensity, 0 s) at each punctum of interest was measured and is given as percent fluorescence recovery based on the initial value before bleaching. Data are means ± standard errors of the means of three independent experiments carried out in triplicate.

Depolarization-induced S1P receptor activation demonstrated by FRET analysis. (A) The strategy to detect S1P₁ interaction with β-arrestin (βArr) after S1P₁ activation using FRET is depicted. (B) Hippocampal neurons transfected with expression plasmids encoding S1P₁-CFP and YFP-β-arrestin were treated either with depolarization stimuli (50 mM KCl or 100 μM glutamate) or with 10 nM S1P and were analyzed for FRET in living cells. A representative emission ratio of the two fluorophores (excited at 458 nm) from five independent experiments is shown. Arrows indicate the addition of either control (buffer vehicle) or agonists. (C) Hippocampal neurons cotransfected with expression plasmids encoding S1P₁-CFP and YFP-β-arrestin were treated without (buffer) or with 50 mM KCl, 100 μM glutamate, or 10 nM S1P and were analyzed for FRET in living cells. Emission detected from an increase in donor fluorescence after acceptor photobleaching of puncta of interest was measured and expressed as FRET efficiency. Note that depolarization induced either by KCl or glutamate as well as S1P treatment caused a significant increase in FRET efficiencies (n = 50; a representative experiment of four independent experiments is shown; P < 0.01, Student's paired t test). (D) Neurons cotransfected with S1P₁-CFP and YFP-β-arrestin plasmids were untreated or treated with 10 μM DMS for 30 min before agonist stimulation. In some experiments neurons were transfected with control or rSK1-siRNAs together with plasmids encoding the fluorophore-conjugated proteins. Neurons were stimulated without (buffer) or with 100 μM glutamate, 50 mM KCl, or 10 nM S1P and fixed and measured for FRET efficiency after photobleaching of puncta or dendrite areas of interest. Data are means ± standard errors of the means of three independent experiments carried out in triplicate.

Functional roles of exogenous S1P as both an inducer and an enhancer of secretion. (A) Primary rat hippocampal neurons were prelabeled with FM4-64. FM dye-labeled cells were stimulated with various concentrations of either S1P or DHS1P for 6 s, and the changes in fluorescence intensity were monitored. At all points vehicle (methanol) concentrations were kept constant. (B) Neurons washed three times with PBS were stimulated with various concentrations of S1P for 6 s. Secreted glutamate was measured by an enzymatic fluorometric assay. (C) Neurons were treated without (vehicle) or with 10 μM DMS for 30 min and labeled with FM dye. Neurons were then stimulated with various combinations of 1 pM S1P and 100 μM glutamate for the indicated time, and the changes in fluorescence intensity were monitored. The arrow indicates the addition of agonists. (D) FM dye-prelabeled neurons were treated with various concentrations of glutamate without or with 1 pM S1P for 6 s, and the changes in fluorescence intensity were monitored. (E) Individual S1P receptor mRNAs were quantitated from primary rat hippocampal neurons by real-time quantitative reverse transcription PCR. Values of mRNA amounts were normalized to GAPDH expression. (F and G) Hippocampal neurons were transfected with control or S1P₁ or S1P₃ siRNA. Two days after transfection S1P₁ mRNA (F) or S1P₃ mRNA (G) levels were quantitated by real-time quantitative reverse transcription PCR. (H) Hippocampal neurons transfected with various combinations of control or S1P₁ and S1P₃ siRNAs were prelabeled with FM4-64. Neurons were stimulated with 10 nM S1P, and the changes in fluorescence intensity were monitored. The arrow indicates the addition of S1P. Data are the means ± standard errors of the means of five independent experiments carried out three (A to D and H) or six (E) times. For F(t), fluorescence at the selected region at time t, see text.