Locomotor activity of DAT-KO and WT mice was measured in an Omnitech CCDigiscan. To assess effects of 3-MT in normal and TAAR1-KO mice, the animals were placed in the locomotor activity chamber and 30 min later various doses of 3-MT were administered i.c.v. over 4 minutes. After infusion, animals were put back into experimental chamber and behavior was monitored for 90 min after administration. 3-MT induced significant behavioral activation in DDD mice. Striatal tissue was collected from DDD mice treated with 36 µg of 3-MT 30 minutes after administration was analyzed for ERK activity. 3-MT caused a significant increase in the level of phosphorylated Erk2, thus indicating that certain receptor-mediated processes in striatal cells are affected by this treatment.

Mitochondria were isolated from whole brains of Sprague-Dawley rats. Mitochondria were suspended in 1 mL of incubation buffer containing 0.095MKCl, 0.075Mmannitol,0.025Msucrose, 0.005MKH2PO4, 0.020MTris-HCl, 0.001MEGTA, pH 7.4 (adjusted with KOH) at a final concentration of between 0.40 and 0.80 mg protein/mL. Malate and pyruvate were added to a final concentration of 10 mM. Oxygen consumption was carried out at 30°C in a closed chamber containing a Clark type oxygen electrode connected to a YSI model5300 oxygen monitor. Following a 10-minute incubation, resting conditions (no ADP) were monitored for 5 minutes, followed by the addition of ADP (0.25 mMfinal concentration) and measurement of active respiration. ADP depleted respiration was also monitored. Pre-incubation with the mitochondrial inhibitor 3-Methoxytyramine (3-MT) was carried out for15 min at 30°C prior to addition of ADP. Incubation with 3-MT was carried out in the dark for the first 10 minutes. IC50measurements represent the millimolar concentration of inhibitor required to inhibit 50% malate/pyruvate supported respiration compared with controls containing no inhibitor following a 15-min incubation at 30°C. 3-MT was found to inhibit brain mitochondrial respiration by MAO-dependent and -independent mechanisms. The IC50 of mitochondrial respiratory inhibition for 3-MT was determined to be 19.6 mM.