ALC proneurogenic effects on adult hippocampal neural progenitors involved activation of the NF-κB pathway and acetylation of p65. (a) The 24 h treatment with ALC (0.01–1 mM) promoted neuronal differentiation from adult hippocampal NPCs by significantly increasing the percentage of MAP-2⁺ cells as compared with vehicle (veh). (b, c) ALC (300 μM) significantly reduced the percentage of GFAP⁺ astrocytes (b) and NG2⁺ oligodendrocyte precursors (c) derived from adult NPCs. (d, e) The percentage of apoptotic cells (d) and the amount of released LDH activity (e) were not significantly different in ALC- vs vehicle-treated cells. (f) The 2- and 72-h incubation of proliferating neural progenitors with ALC 300 μM did not affect BrdU incorporation as compared with vehicle-treated cells. BrdU (10 μM) was added to culture medium during the last 2 h of incubation. Data are expressed as %BrdU⁺ cells over total viable cells. (g) NF-κB inhibitors JSH-23 (3 μM ), SC-514 (3 μM), and SN-50 (10 μg/ml) counteracted the proneurogenic effects of 300 μM ALC. The inactive peptide SN-50M (10 μg/ml) had no effect. Data are mean±SEM of n=2 experiments in triplicate for (g) and n=3 experiments in triplicate for all the others. (h) The 0.3 and 1 mM L-carnitine (LC) produced no effect on MAP-2⁺ cells when compared with similar concentrations of ALC. (i) The 2-h treatment with ALC (300 μM), but not LC (300 μM) or vehicle, resulted in NF-κB p65 Lys310 acetylation in extracts of adult NPCs. Expression levels of total p65 and β-actin were also evaluated as internal controls. Data are mean±SD of n=3 experiments in triplicates. *P<0.05; ***P<0.001 vs vehicle-treated cells, ^§§§P<0.001 vs ALC-treated cells. Data in (a, d, f, g, and h) were analyzed by one-way ANOVA followed by Tukey's post hoc test, whereas data in (b, c, and e) were analyzed by Student's t-test.

Group II mGlu receptors, and in particular the mGlu2 subtype, are involved in ALC proneurogenic effects. (a) The mGlu2/3 agonist LY379268 increased the percentage of MAP-2⁺ cells in adult NPC cultures in a concentration-dependent manner. (b) The preferential mGlu2/3 antagonist LY341495 counteracted the effects of ALC (300 μM) on MAP-2⁺ cells. (c) The selective mGlu2 PAM LY487379 increased the percentage of MAP-2⁺ cells generated from adult NPCs. (a–c) Data are mean±SD of n=3 experiments in triplicates, analyzed by one-way ANOVA followed by Tukey's post hoc test. *P<0.05; **P<0.01; ***P<0.001 vs vehicle-treated cells and ^§§§P<0.001 vs ALC-treated cells. (d–i) Representative fluorescence microscopy images of MAP-2 immunolabeling (green) in cells treated for 24 h with vehicle (d), 300 μM ALC (e), 10 μM LY379268 (f), 1 μM LY487379 (g), 7.5 nM LY341495+300 μM ALC (h), and 7.5 nM LY341495 (i). Nuclei were counterstained with TOPRO (blue). Scale bar: 47.62 μm. (j) Immunoblot analysis and quantification of mGlu2 and mGlu3 protein levels in adult hippocampal NPCs treated for 24 h with vehicle, 3 μM JSH-23, 300 μM ALC in the presence of vehicle, or 3 μM JSH-23. Compared with vehicle, ALC treatment increased mGlu2 protein levels and JSH-23 counteracted these effects. No change in mGlu3 protein levels was observed in the presence of any treatment condition. Data represent the mean±SD of n=3 experiments from different cell preparations, normalized by α-tubulin expressions and analyzed by two-way ANOVA followed by Bonferroni post hoc test. *P<0.05 vs vehicle-treated cells; ^§§P<0.01 vs ALC-treated cells.

Chronic ALC treatment reverted UCMS-induced depressive-like behavior and upregulated mGlu2 protein levels in the hippocampi of stressed mice. (a) Schematic representation of the experimental procedure. (b, c) Sucrose preference (SP) in (b) stressed (n=11) and (c) unstressed (n=11) mice. Mice subjected to 8-week UCMS significantly reduced SP compared with their (b) baseline values (P<0.001) and with (c) unstressed mice (P<0.001). (b) Chronic ALC treatment (100 mg/kg, once a day, s.c.) reversed UCMS-induced anhedonia, with ALC-treated stressed mice (n=6) displaying SP values significantly higher than vehicle-treated stressed mice (n=5; P<0.001). (c) ALC (n=6) or vehicle treatment (n=5) had no effect on unstressed mice. (d, e) Behavioral analysis in the TST (d) and FST (e) following UCMS and after 21 days of saline/ALC treatment. After 8 weeks of stress, UCMS mice significantly increased time (in seconds) spent in immobility in both TST (d) and FST (e) as when compared with unstressed, control (ctrl) mice. ALC was able to revert stress-induced increase of immobility time in both TST (d) and FST (e), with ALC-treated UCMS mice immobility being significantly different from the vehicle-treated UCMS mice in both TST and FST. No significant difference was observed between vehicle- and ALC-treated unstressed mice in both TST (d) and FST (e). Data are mean±SD (*P<0.05, **P<0.01, ***P<0.001, ^§P<0.05, ^§§§P<0.001). (f–h) Representative experiments of immunoblot analysis of mGlu2 (f), mGlu3 (g), and PAR-1 (h) levels in hippocampi of vehicle-treated unstressed, ALC-treated unstressed, vehicle-treated UCMS, and ALC-treated UCMS mice. Densitometric values are mean±SD of mGlu2/α-tubulin, mGlu3/α-tubulin, and PAR-1/α-tubulin ratios from three independent experiments (n=4 mice/group). Data were analyzed by two-way ANOVA followed by Bonferroni post hoc test. **P<0.01 vs vehicle-treated stressed mice.

Chronic ALC treatment increased hippocampal neurogenesis in vivo. (a–d) Representative confocal microscopy images of immunofluorescent labeling for BrdU (blue)/NeuN (red)/GFAP (green) of hippocampal sections in control (Ctrl; a, b) and stressed (UCMS; c, d) mice subcutaneously injected with vehicle (a, c) or 100 mg/kg ALC (b, d) for 21 days. White arrowheads indicate BrdU⁺/NeuN⁺/GFAP⁻ cells within the dentate gyrus. Scale bar: 75 μm. (e) Quantitative analysis of BrdU⁺/NeuN⁺/GFAP⁻ cells in stressed and unstressed mice. The number of newly born hippocampal neurons was significantly increased in the granular cell layer (GCL) of ALC-treated stressed and unstressed mice compared with vehicle-treated stressed and unstressed mice, respectively. Data are expressed as mean±SD of n=7 mice/group, and were analyzed by two-way ANOVA followed by Bonferroni post hoc test. *P<0.05 vs vehicle-treated unstressed mice; ^##P<0.01 vs vehicle-treated stressed mice. (f) Proliferation rate in the dentate gyrus of mice treated with 100 mg/kg ALC or vehicle for 21 days. The number of BrdU⁺ cells in the subgranular zone (SGZ) and GCL was not significantly different in vehicle- and ALC-treated mice killed 2 h after BrdU administration. Data are expressed as mean±SD of n=7 mice/group.