Fig. 3

Astrocyte VMAT2 deletion disrupts DA metabolism and leads a decrease in dopamine levels in the PFC. a HPLC quantification of total dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), norepinephrine (NE) and serotonin (5-HT) (ng per mg of protein) in the PFC and VTA of recombined aVMAT2cKO and control LoxTAM mice (P40). The error bars indicate the SEM. b Histograms show the average basal extracellular levels of DA calculated in the extracellular perfusates of the in vivo microdialysis in control LoxTAM and recombined aVMAT2cKO mice (P40). The microdialysis probes were placed in the PFC, and DA levels (pg/μl) were measured at baseline for 30 min. The error bars indicate the SEM. c Histograms show the average basal extracellular levels of DA calculated in the extracellular perfusates of the in vivo microdialysis of control LoxTAM (grey) and recombined aVMAT2cKO mice (green) at different time points after the first TAM injection. The microdialysis probes were placed in the PFC, and DA levels (pg/μl) were measured at baseline for 150 min. The error bars indicate the SEM. d Diagram shows the contribution of astrocytic VMAT2 to DA metabolism. e Histograms shows enzymatic activities of MAOA, MAOB and COMT measured in PFC brain homogenates of control LoxTAM and recombined aVMAT2cKO mice (P40). The error bars indicate the SEM. f Dopamine accumulation in wt-derived primary cultured astrocytes using [³H]-Dopamine. Curves represent time–dependent specific accumulation of [³H]-Dopamine (black curve, 3 mM DA and 150 nM of [³H]-Dopamine as a tracer) in cultured astrocytes incubated with reserpine (red curve) or reserpine plus deprenyl (blue curve). Note that in the presence of reserpine the accumulation of [³H] is the sum of [³H]-Dopamine and [³H]-DOPAC. The error bars indicate the SEM. g Histograms shows [³H]-Dopamine accumulation in primary cultured astrocytes derived from control LoxTAM and recombinant aVMAT2cKO mice in the presence or in the absence of deprenyl (1 μM). The error bars indicate the SEM. h Time-course effect of reserpine (1 μM) and deprenyl (1 μM) on the intracellular levels of DOPAC/DA ratio calculated in LoxTAM or aVMAT2cKO-derived primary cultured astrocyte. Cultured astrocytes were incubated with DA (3 mM) in presence or absence (black curve) of reserpine (red curve) and reserpine plus deprenyl (blue curve) for 10, 20, 40, 50 and 60 min. Note that in the presence of reserpine there is an increased accumulation of DOPAC. The error bars indicate the SEM. i Histograms show intracellular levels of DOPAC in cultured astrocytes derived from control LoxTAM and recombinant aVMAT2cKO mice. Cultured astrocytes were incubated with DA (3 mM) in presence or absence of deprenyl (1 μM) for 40 min. The error bars indicate the SEM. j Graph shows the average levels of DA calculated in extracellular perfusates of the in vivo microdialysis in control LoxTAM and recombined aVMAT2cKO mice (P40) treated with deprenyl (10 mg/kg, i.p.) or D22 (1,1-diethyl-2,2-cyanine iodide, 100 μM). The microdialysis probes were placed in the PFC, and DA levels (pg/μL) were measured at baseline for 30 min. The error bars indicate the SEM. k Histograms shows the average levels of DA calculated in extracellular perfusates of the in vivo microdialysis in the PFC of control LoxTAM and recombined aVMAT2cKO mice locally infected with astrocyte-targeted with lentiGFP or lentiVMAT2viruses, respectively (P40). Data expressed as fold percentages of baseline levels. The error bars indicate the SEM