Sigeng Chen; Geoffrey C. Owens; David B. Edelman

“ Although it has been suggested that D2 receptors, and not D1 receptors, are involved in the regulation of Akt signaling pathways in the striatum [8] , other studies employing cultured striatal neurons have shown that D1 receptors are also involved in the regulation of cAMP-dependent Akt signaling [23] .
Axonal movement of mitochondria and other cargoes requires an association between the organelle and specific motor proteins, which, in turn, mediate movement of cargoes on cytoskeletal elements in an ATP-dependent manner [1] . ”

“ We found that dopamine has a net inhibitory effect on mitochondrial movement in hippocampal neurons (Fig. 3A–C) . Subsequent experiments with different receptor-specific agonists and antagonists revealed that this observed inhibition likely reflects the net outcome of the opposing effects of different dopamine receptor subtypes within the same cells. Specifically, a D2R agonist inhibited mitochondrial movement (Fig. 4A–C) , whereas a D1R agonist promoted mitochondrial movement (Fig. 5A–C) . ”

“ 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 (Fig. 9A) . 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 5-HT. In an earlier study, we showed that the addition of 5-HT increases Akt phosphorylation [6] . ”

“ B. Western blot analysis shows that dopamine counteracts the effect of 5-HT on Akt activity in the presence of 5-HT. C. Prior to treatment with dopamine signals, hippocampal neurons were pretreated with 5-HT. Each mean speed value in the graph represents three repeat experiments, i.e., separately prepared cultures (n = 3, paired t-test; p<0.02; 103–130 mitochondria were tracked in calculating the average speeds of each set of mitochondria in a given treatment interval) . ”

“ Whole cell extracts from hippocampal neuronal cultures (DIV 17) were used in Western blots. In B and D, intensity values were normalized to total Akt or total GSK3β values. Given the importance of the cAMP pathway in dopamine signaling [21] , [22] , we decided to elevate cAMP levels directly in hippocampal neurons by treating cultures with 3-isobutyl-1-methylxanthine (IBMX, 100 μM) . In the striatum, D1R signaling is coupled to an increase in adenylyl cyclase activity [21] , [22] ; we therefore anticipated that IBMX treatment might cause an increase in mitochondrial movement. ”

“ A D1R agonist promotes mitochondrial transport, whereas a D1R antagonist inhibits mitochondrial movement D1 receptors are the second major class of dopamine receptors found in hippocampal neurons (Figs. 1, 2) . Since signals from D2R and D1R may have opposing effects [15] , [21] , we decided to investigate the possible effect of a D1R-specifc agonist on mitochondrial movement. Administration of the D1R agonist, SKF38393 (33 μM) , markedly stimulated mitochondrial movement, primarily anterogradely, toward the axon terminal (Fig. 5A, B; Movies S9 and S10) . ”

“ Plating densities of hippocampal neurons were 2800 cells/cm² and 1200 cell/cm² in conventional 35-mm dishes for protein assays and 35 mm glass-bottom dishes for imaging studies, respectively. Cells were harvested at 14 DIV for protein assays or used at 17–21 DIV in time-lapse imaging studies. On the fourth day after initial plating and every four days thereafter, Ara-C was added to control growth of glial populations. ”

“ Cultures were infected with the Flx1.8/MitoEYFP lentivirus [6] and used in live imaging experiments. Images were collected for 2 hours prior to the addition of dopamine (30 nM) , and then for 2 hours after exposure to dopamine (representative time-lapse movies are available in Supporting Information) . Following the same criteria used previously, patterns of mitochondrial motility during long-term observation were categorized into three distinct groups: a stationary population, an oscillatory population, and a directionally moving population [6] . ”

“ This result suggests that haloperidol blocked the effects of any endogenous dopamine in the cultures. In earlier studies, dopamine uptake by cultured rat astrocytes was demonstrated [19] , [20] , suggesting that astrocytes might be a possible source of endogenous dopamine in our culture system, in which we use glia-conditioned media. ”

“ Pie charts represent the initial and final proportions of each population category represented in the total pool of experiments. Speed plots represent the mean speeds of pooled directionally moving populations from all experiments, calculated at 15 minute intervals. ”

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