Article Figures & Data
Figures
- Figure 1.
d-Serine distribution in the rat cerebral cortex. A, Confocal microscopy analysis of immunostainings of d-serine versus astroglial [glutamine synthase (GS)] or neuronal (β3-tubulin) markers in the rat cerebral cortex. Arrowheads, d-Serine-containing neurons; arrows, d-serine-positive astrocytes; Control, staining with a nuclear marker, DAPI (blue), and an anti-d-serine antibody previously preabsorbed with 500 μm liquid-phase d-serine glutaraldehyde conjugate. Scale bars, 30 μm. B, Representative electron micrographs of d-serine preembedding immunohistochemistry using the peroxidase method showing that d-serine immunoreactivity is mainly distributed in astrocytic processes (arrows) at the level of asymmetric synapses in the rat cerebral cortex. C, Representative low-magnification (top) and high-magnification (bottom) electron micrographs showing that no peroxidase staining is detected with a preabsorbed anti-d-serine antibody. Ast, Astrocyte; D, dendrite; S, dendritic spine; B, axonal bouton. Scale bars, 500 nm.
- Figure 2.
Characterization of GVs, immunoisolated from cultured astrocytes using beads coated with anti-Sb2 monoclonal antibodies. A, Electron micrographs of the bead fraction, showing vesicles bound to the surface of the beads. Ctl, Immunobeads coated with mouse IgG that were processed in parallel; no bound vesicles are detectable. Arrows, Bead-bound vesicles. Scale bars, 100 nm. B, Size distribution of the immunoisolated vesicles. A total of 300 vesicles was measured, with the numbers falling into the respective categories indicated in the histogram. The black curve shows a Gaussian fit of the size distribution. C, Astroglial LSS and bead-bound material (IP) were immunoblotted for Sb2. Equal proportion of samples was loaded showing that a major fraction of Sb2-positive organelles was recovered in the bead fraction. Light and heavy chains correspond to light and heavy chains of mouse control (lane 2) and monoclonal anti-Sb2 antibodies (lane 3). D, E, Immunoblots of the fractions shown in C for various marker proteins specific for SVs (D) and subcellular compartments (E). Sph, Synaptophysin; Stg 1, synaptotagmin 1.
- Figure 3.
Amino acid content of immunoisolated GVs and SVs. A, Astroglial LSS, Sb2-containing vesicles immunoisolated at 21°C and 0°C with or without RgDAAO treatment were analyzed by CE. Electropherograms are adjusted to the same scale to facilitate the comparison between traces. Arrows point to the position of l-serine (lS), d-serine (dS), GABA + unknown substance (γ*), glycine (G), and l-glutamate (E). B, Quantification of amino acid content of GVs and SVs isolated at 0°C. All values were corrected for nonspecific adsorption as determined by isolating vesicles at 21°C. The figure shows mean ± SEM values of four independent experiments, normalized to Sb2 as a vesicle marker.
- Figure 4.
Quantitation of vesicle amino acid content using CE–LIF and CE–MS. To validate quantitation via CE–LIF chiral separations as shown in Figure 3A, two additional measurements using non-chiral separation conditions were performed using CE–LIF and CE–MS detection to validate the quantitative measures and peak assignments. A, Astroglial Sb2-containing vesicles immunoisolated at 21°C and 0°C were analyzed using a non-chiral CE–LIF separation. Arrows point to the serine (S), GABA (γ), glycine (G), and glutamate (E) peaks. B, The same samples were analyzed by CE–MS. Electropherograms were adjusted to the same scale to facilitate comparison between approaches. C, A table shows representative quantification results of serine using these three different methods.
- Figure 5.
Preembedding immunogold staining for d-serine at glia–neuron interfaces in rat cerebral cortex. A, Low-magnification shows that immunogold-positive particles (arrows) are detected in astrocytic processes surrounding asymmetric synapses. High magnifications reveal that d-serine is associated with vesicle-like structures located in astrocytic processes facing the asymmetric synapses. Please note that the insets are rotated compared with the low-magnification image. B, Representative low-magnification (top) and high-magnification (bottom) electron micrographs showing that no immunogold-positive particle is detected with a preabsorbed anti-d-serine antibody. Ast, Astrocyte; D, dendrite; S, dendritic spine; B, axonal bouton. Scale bars, 500 nm.
- Figure 6.
Characterization of immunoisolated SVs. A, Cortex homogenate (Input) and bead-bound material (IP) were immunoblotted for Sb2, V-ATPase (subunit a1), synaptophysin, synaptotagmin 1, and for the endosomal marker EEA1. Equal proportion of samples was loaded to allow direct comparison. Light and heavy chains correspond to light and heavy chains of antibodies. Ctl, Control. B, Cortex homogenate, Sb2-containing SVs immunoisolated at 21°C and 0°C were analyzed by CE–LIF. Electropherograms are adjusted to the same scale to facilitate the comparison between traces. Arrowheads point to the position of l-serine (lS), d-serine (dS), GABA (γ), glycine (G), and l-glutamate (E). Quantification of amino acid content in SVs is presented in Figure 3B for comparison with GVs.
- Figure 7.
Features of vesicular d-serine uptake into astrocytic immunopurified vesicles. A, ATP-dependent uptake of d-[3H]serine and l-[3H]glutamate. The values represent FCCP-sensitive uptake. FCCP (40 μm) was added together with MgATP (4 mm). Control experiments are performed by replacing MgATP by ATPγS (4 mm), by decreasing temperature to 0°C, or by inhibiting V-ATPase with bafilomycin A1 (1 μm). B, d-Serine (7.5 mm)-mediated acidification of GVs is dependent on chloride (the figure shows representative traces from 3–6 independent measurements). To maintain ionic strength and osmolarity, chloride was exchanged for gluconate (potassium salts). Arrows correspond to addition of the tested compound [10 mm ATP and 50 mm (NH4)2SO4, final concentrations in this and the following figures]. At the end of the reaction, (NH4)2SO4 was added to dissipate ΔpH. C, Overlay of photometric traces showing acidification of GVs induced by various d-serine concentrations in the presence of 4 mm chloride (representative of 3–5 independent experiments). No change in fluorescence under any condition was observed when control (Ctl) beads (mouse IgG) were used. Scale bars, 2 arbitrary units per 1 min. D, Dependence of the initial rate of acidification on d-serine and l-glutamate concentration. E, F, Initial rate of acidification induced by d-serine (7.5 mm) or l-glutamate (5 mm) and d-[3H]serine uptake were measured in the presence of different chloride concentrations. G, Influence of both components of the proton electrochemical potential ΔμH+ on FCCP-sensitive ATP-dependent d-[3H]serine uptake. Additions: 5 μm nigericin (Nig), 20 μm valinomycin (Val). Error bars represent mean ± SEM; n = 3–6 independent experiments.
- Figure 8.
SVs transport l-glutamate but not d-serine. A, Photometric trace showing the absence of d-serine-induced acidification of SVs, whereas l-glutamate can acidify these organelles (10 mm each, n = 3). Scale bar, 2 arbitrary units per 1 min. B, ATP-dependent uptake of d-[3H]serine and l-[3H]glutamate by isolated SVs. The values represent FCCP-sensitive uptake. N.D., Not detectable. Values represent mean ± SEM of two independent experiments performed in triplicate. C, Western blot analysis of cortex homogenate (Input) and control (Ctl) or Sb2 immunoisolates (IP) for vGlut1 and vGlut2 and for SR.
- Figure 9.
Vesicular synergy between d-serine and l-glutamate transport activities in GVs. A, Partial photometric traces showing the acidification induced by 5 mm d-serine (top) and 5 mm l-glutamate (bottom) in the absence (black) or presence (gray) of 5 mm l-glutamate and 5 mm d-serine, respectively. The experiments were performed as in Figure 7, i.e., initiated with 10 mm ATP and ended with 50 mm (NH4)2SO4. Scale bar, 2 arbitrary units per 1 min. B, Histogram showing the reciprocal effect of each amino acid (d-serine or l-glutamate) on the acidification rate and uptake of the other one. Amino-acid-induced acidification was measured as in A. FCCP-sensitive ATP-dependent uptakes of l-[3H]glutamate or d-[3H]serine were measured in the presence of 10 mm d-serine or l-glutamate, respectively. Ctl, Control. Error bars represent mean ± SEM; n = 3 for acidification, 4 in duplicate for radioactive assay. *p < 0.05, **p < 0.01, Student's t test.
- Figure 10.
d-Serine uptake by immunoisolated GVs is coupled to endogenous SR activity. A, Photometric trace showing acidification induced by 10 mm l-serine (representative trace from 4 independent experiments). Scale bar, 2 arbitrary units per 1 min. B, Colocalization of SR (green) with Sb2 (red) in cultured astrocytes. Scale bars: top (low magnification), 10 μm; bottom (high magnification), 5 μm. The presence of SR on GVs is confirmed by immunoblotting of immunoisolated GV fractions. Ctl, Control. C, D, Inhibition of SR by HOAsp (400 μm, 10 min) abolishes l-serine-induced acidification without affecting d-serine-induced acidification (10 mm each, representative traces from 4–5 independent experiments). Scale bars, 2 arbitrary units per 1 min. ns, Not significant, *p < 0.05, **p < 0.01, Student's t test.
- Figure 11.
VSERT operates as a putative d-serine/H+ antiporter with a chloride leak or a coupled chloride cotransport. Although d-serine is a neutral amino acid, chloride acts as a counterion for V-ATPase and increases the pH gradient while reducing the membrane potential. Alternatively, chloride could modulate d-serine transport by regulatory binding to the transporter. SR is anchored to the membrane of GVs in which it converts l-serine into d-serine, allowing a local and efficient concentration of the gliotransmitter to be transported. However, preexisting d-serine is also taken up in GVs without any preference over a newly synthesized one.
