Fig 2: Environmental enrichment improves OL numbers and myelination in the mPFC of APO-SUS rats.a Schematic representation of experimental paradigm. APO-SUS and APO-UNSUS rats were housed in standard housing (SH) or in environmental enrichment (EE) conditions starting at P21, and sacrificed at P90 for immunohistochemical (IHC) or transmission electron microscopy (TEM) analyses. b Number of OLIG2+ lineage cells; *p = 0.045, ^p = 0.018 in two-tailed independent samples t-test, c number of OLIG2+NG2+ OPCs; ^p = 0.013 in two-tailed independent samples t-test, d number of OLIG2+GPR17+BCAS1+ early-myelinating OLs; *p = 0.012 in two-tailed independent samples t-test, and e number of OLIG2+CC1+ mature OLs per mm2 in the IL subregion of the mPFC of APO-SUS and APO-UNSUS rats in standard housing and environmental enrichment conditions (n = 3–6, specified in the graphs). f TEM analysis and quantification in the IL of APO-SUS (n = 4) and APO-UNSUS (n = 3) rats in standard housing and environmental enrichment conditions; *p = 0.021 in two-tailed independent samples t-test. g Average myelinated axon G-ratio in IL of APO-SUS (n = 4) and APO-UNSUS (n = 3) rats in standard housing and environmental enrichment conditions. h Axon caliber cumulative distributions of myelinated axons in IL of APO-SUS and APO-UNSUS rats in standard housing and environmental enrichment conditions; *p = 0.012; 0.2–0.4 ^p = 0.041; 0.8–1.0 ^p = 0.047; 1.0–1.2 ^p = 0.031 in two-tailed independent samples t-test. Scale bars 20 µm in immunofluorescence images and 1 µm in electron micrographs. Error bars represent standard error of the mean. Source data are provided as a Source data file.

Fig 3: Montelukast modulates microglia in old rats and neuroinflammatory gene expression.(a) Iba1+ immunostaining in the DG of the different age- and treatment groups (white arrowheads point toward representative Iba1+ cells). Control stainings, in which the primary goat Iba1 antibody was omitted, excluded unspecific labelling of the secondary antibody. (b) Total numbers of Iba1+ cells, and (c) percentages of proliferating microglia (Iba1+/PCNA+) in the DG of young (4 months) and old (20 months) rats after a 6-week treatment with montelukast (10 mg kg−1 daily p.o.). (d) Soma sizes of Iba1+ cells in the DG of young and old montelukast- and vehicle-treated rats. (e) Representative images of CD68+ immunostaining in Iba1+ microglia (arrowheads) of the different experimental groups. (f) Percentage of Iba+ cells that contain CD68+ particles. (g) Average size of CD68+ particles in Iba1+ cells of in the DG. (h) Immunoreactivity for CysLTR1 and for GPR17 in BV-2 cells. (i) mRNA expression of NOS2, CCL2, TNF, TGF-beta1 and of ARG1 in BV-2 cells 24 h after treatment with 100 nM LTD4 and/or with 15 μM montelukast, and with vehicle control. N per group in (b–d,f,g): 10 (young vehicle), 10 (young montelukast), 7 (old vehicle), 7 (old montelukast). Every tenth section (400 μm interval) of one brain hemisphere (b,c) or four sections (d,f,g) per animal were analysed. In (i), 3–5 biological replicates were performed in technical duplicates. Data are shown as mean±s.d. *P<0.05, **P<0.01, ***P<0.001. Significant differences between the old age groups and the young vehicle group were indicated by # (#=P<0.05; ###=P<0.001). One-way ANOVA (b–d,f,g) and Two-way ANOVA (i) with Bonferroni post hoc tests were performed. Scale bars, 50 μm (a); 100 μm (e). yg veh, young vehicle; yg mtk, young montelukast; old veh, old vehicle; old mtk, old montelukast; DG, dentate gyrus.

Fig 4: Survival and maturation of OLs are modified by A2BAR activation in SCZ mice. (A) Immunohistochemical images of NG2+ OPC staining in the cerebral cortex (scale bar, 50 µm) The arrows and insert box indicate the NG2+ cells; the inset boxes are at x8 magnification. (B) MK-801 treatment markedly reduced the number of NG2+ OPCs in the cerebral cortex. Administration of BAY 60-6583 in the SCZ mice notably increased the number of NG2+ OPCs in the cerebral cortex. (C) Representative immunoblots of the NG2 protein levels. (D) MK-801-treated mice showed a significant reduction in NG2, while BAY 60-6583 administration increased NG2 protein levels in the cerebral cortex. (E) Representative immunoblots of GPR17 protein expression levels. (F) GPR17 protein expression levels were significantly increased in MK-801-treated mice and were restored in MK-801 + BAY-treated mice. (G) The number of CC-1+/Olig2+ cells was reduced in the MK-801 group, while a significant increase was observed in the MK-801 + BAY group. (H) Immunofluorescence staining was performed for CC-1/Olig2 in the cerebral cortex (scale bar, 50 µm). Data are presented as the mean ± SEM. *P<0.05. OLs, oligodendrocytes; A2BAR, A2B adenosine receptor; SCZ, schizophrenia; NG2, chondroitin sulfate glycoprotein 4; OPCs, OL precursor cells; MK-801, dizocilpine maleate; GPR17, G protein-coupled receptor 17; CC-1, anti-adenomatous polyposis coli clone CC-1; Olig2, OL transcription factor 2; BAY 60-6583, A2BAR agonist; PSB 603; A2BAR antagonist.

Fig 5: GPR17 and CysLTR1, target receptors of montelukast, are expressed within the dentate gyrus of old rats.(a–f) GPR17 expression pattern in the DG of old (20 months) rats. GPR17 immunoreactivity was localised to cells of the oligodendroglial lineage (Olig2+), a), to some DCX+ neuronal progenitors (c), to a subset of NeuN+ granular neurons (d) and to a fraction of Iba1+ microglia (f). GPR17 expression was neither observed in Sox2+ neural stem cells (b) nor in GFAP+ astrocytes (e). (g–l) CysLTR1 immunoreactivity in the DG of 20-month old rats was localised to a few Sox2+ cells (h), to cells of the oligodendroglial lineage (Olig2+, g), to GFAP+ astrocytes (k), and to some Iba1+ microglia (l). CysLTR1 expression was not observed in DCX+ neuronal progenitors (i) or in NeuN+ granular neurons (j). Scale bars, 50 μm (a–l). Arrowheads indicate receptor co-localisation. DG, dentate gyrus.