This study provides evidence of the existence of presynaptic inhibitory sphingosine-1-phosphate receptor 1 (S1P1R) and facilitatory S1P3R in cortical nerve endings (synaptosomes) of healthy mice. The conclusion relies on the findings that (i) the S1P1R agonist CS-2100 (0.1-30 nM) inhibits the 12 mM KCl-evoked glutamate exocytosis (quantified as the release of [3H]D-aspartate) while the S1P3R allosteric agonist CYM-5541 potentiates it and (ii) these effects are inhibited by the S1P1R antagonist Ex 26 (30-300 nM) and the S1P3R antagonist TY-52156 (100-1000 nM), respectively. Confocal microscopy and western blot analysis confirmed the presence of S1P1R and S1P3R proteins in cortical glutamatergic synaptosomes, which were scarcely accessible to biotin in a biotinylation study. Then, we demonstrated that S1P1R and S1P3R densities and their release activity are amplified in cortical synaptosomes of mice suffering from experimental autoimmune encephalomyelitis (EAE), despite receptors maintain their preferential internal distribution. Receptor changes recover following chronic oral therapeutic FTY720 (0.03 mg/Kg/day). These results improve our knowledge of the role of presynaptic release-regulating S1P1Rs and S1P3Rs controlling glutamate transmission in the CNS also unravelling functional adaptations during EAE that recover following chronic FTY720. In a whole, these findings provide new information on the central neuroprotectant activities of FTY720.