03 μm2, n = 27; p > 0.05; t test; Figure 1E). To explore the effects of mTOR inhibition and macroautophagy deficiency on the size of dopamine axonal profiles,

we injected pairs of DAT Cre and Atg7 DAT Cre mice with rapamycin (2 mg/kg) or vehicle (DMSO) 36 and 12 hr prior to perfusion. Rapamycin decreased the area of TH+ striatal axon profiles by 32% in RG7420 chemical structure DAT Cre mice but had no effect on DA terminals of the DAT Cre Atg7 mutant line (Figure 1F) (interaction between rapamycin and genotype, F = 6.72; p < 0.01; two-way analysis of variance [ANOVA]). We used cyclic voltammetry to directly measure evoked dopamine release and reuptake in the striatum. The peak amplitude of the signal is dependent on both neurosecretion and reuptake through DAT, whereas the half-life (t1/2) is a function of DAT activity (Schmitz et al., 2001). The amplitude of the dopamine signal evoked by a single pulse of electrical stimulation in Atg7 DAT Cre mice was 54% greater than in DAT Cre controls (n = 9 and n = 7, respectively; 4.0 ± 0.3 and 2.6 ± 0.2 nM, respectively; p < 0.005; t test; selleck inhibitor Figures 2A and 2B). As DAT Cre and Atg7 DAT Cre mice express a single functional copy of DAT, the signal duration in both genotypes was longer than in wild-type mice (mean t1/2: ∼490 ms) (Schmitz et al., 2001), but the mean t1/2 of DAT signals from DAT Cre and Atg7 DAT Cre slices was not different (Figure S2A; mean t1/2: 637 ± 51 and 662 ±

23 ms, respectively; p > 0.05; t test), which indicates that reuptake kinetics

are similar and that the increased peak amplitude in the Atg7-deficient line was due to greater dopamine release rather than decreased reuptake. To measure the rate of presynaptic recovery, we stimulated dopamine release with pairs of pulses separated by intervals that ranged from 1 to 60 s (Schmitz et al., 2002). Atg7 DAT Cre mice exhibited faster recovery (p < 0.05; repeated-measures Bay 11-7085 ANOVA; Figure 2D), suggesting that basal macroautophagy can restrict synaptic transmission. We then examined effects of rapamycin on evoked dopamine release. Striatal slices were bisected, and one striatum was exposed to rapamycin (3 μM, >5.5 hr)and the other to vehicle. Rapamycin decreased dopamine release evoked by a single electrical stimulus by 25% ± 3% in DAT Cre slices (n = 7) and by 6% ± 6% in Atg7 DAT Cre slices (n = 9; p < 0.05; two-way ANOVA; Newman-Keuls posttest; Figures 2E and 2F). Rapamycin did not significantly alter the t1/2 of the signals from DAT Cre (control: 718 ± 29 ms; Rapa: 675 ± 22 ms) or Atg7 DAT Cre (control: 753 ± 23 ms; Rapa: 743 ± 32 ms) mice (Figure S2A; p > 0.05; two-way ANOVA). The data indicate that the bulk of rapamycin’s inhibition of evoked dopamine release is mediated by macroautophagy. To confirm that these effects were not limited to DAT Cre mutants, we repeated the recordings in slices from wild-type mice and observed a similar rapamycin-induced reduction in dopamine secretion (Figure S2B).