b, by 75% and 30%, respectively. Given the importance of the cAMP pathway in Ridaforolimus dopamine signaling, we decided to elevate cAMP levels directly in hippocampal neurons by treating cultures with 3-isobutyl-1methylxanthine. In the striatum, D1R signaling is coupled to an increase in adenylyl cyclase activity; we therefore anticipated that IBMX treatment might cause an increase in mitochondrial movement. However, as shown in 5-HT and dopamine exert opposing effects in the regulation of mitochondrial movement We have shown previously that exogenous 5-HT promotes mitochondrial trafficking in cultured hippocampal neurons. In the present study, we found that dopamine exerts a net inhibitory effect on mitochondrial movement in the same model system. It is therefore possible that the two neurotransmitters have opposing effects on mitochondrial movement, given that the AktGSK3b signaling pathway is affected by both signals. To explore this possibility, we performed experiments in which 5-HT and dopamine receptor agonists and antagonists were added sequentially in different combinations to the same culture and their cumulative effects on mitochondrial movements were observed over time using time-lapse fluorescence microscopy and by Western blot analysis to detect activated Akt levels under the same conditions. The results of these experiments showed that inhibition of mitochondrial movement by dopamine, a D2R agonist, or a D1R antagonist was not reversed by subsequent addition of 5-HT. Moreover, the increased speed of moving mitochondria induced by treatment with either a D2R antagonist or a D1R agonist was not further enhanced by subsequent treatment with 5-HT. Western blot analysis of cultures treated with the same drug regimen showed that Akt phosphorylation 
remained below control levels in the presence of dopamine, bromocriptine, or SCH23390 even after addition of 5HT. In an earlier study, we showed that the addition of 5-HT increases Akt phosphorylation. In the present study, Akt phosphorylation remained elevated in the presence of either SKF38393 or haloperidol and 5-HT. In contrast to these observations, when hippocampal neurons were pretreated with 5HT, the subsequent addition of a dopamine D2R agonist led to a The Akt-GSK3b signaling pathway is involved in dopamine-mediated regulation of mitochondrial movement Previously, using inhibitors of Akt or GSK3b and phosphospecific antibodies in a Western blot analysis, we showed that the Akt-GSK3b signaling pathway is explicitly involved in the modulation of mitochondrial movement in response to stimulation by 5-HT. We employed the same method here to determine if the Akt-GSK3b signaling cascade might also modulate the effects of dopamine on mitochondrial movement. Western blot analysis showed that the Akt-GSK3b signaling pathway is strongly implicated in dopamine signaling in hippocampal neurons treated with different dopamine agonists and antagonists. When neurons were treated with dopamine alone, levels of activated Akt decreased by 50%. In agreement with our imaging data, the effect of dopamine administration on the phosphorylation of Akt was evident after 15 minutes. Treatment with the D2R agonist, bromocriptine, or the D1R antagonist, SCH23390 also decreased levels of pAkt by more than 75% and 50%, respectively. As well, after treatment with bromocriptine or SCH23390, levels of inactivated Dopamine and Mitochondria 7 Dopamine and 15647369 target=_blank”>9874164 Mitochondria marked inhibitory effect on mitochondrial movement.
