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An in vitro model of human neocortical development using pluripotent stem cells: cocaine-induced cytoarchitectural alterations.

Kindberg AA, Bendriem RM, Spivak CE, Chen J, Handreck A, Lupica CR, Liu J, Freed WJ, Lee CT - Dis Model Mech (2014)

Bottom Line: This model was used to examine the effects of cocaine during neocorticogenesis.These cocaine-induced changes were inhibited by the cytochrome P450 inhibitor cimetidine.This in vitro model enables mechanistic studies of neocorticogenesis, and can be used to examine the mechanisms through which cocaine alters the development of the human neocortex.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Research Branch, Intramural Research Program (IRP), National Institute on Drug Abuse, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Baltimore, MD 21244, USA.

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Cocaine induced ROS generation in neocortical progenitors and premature neuronal differentiation. (A) ROS formation in neocortical progenitors, at day 19, treated with 3 μM cocaine, for 30 minutes, determined by DCFH-DA; n=7. (B,C) Expression of BrdU and TUJ1 (B) by immunocytochemistry in the presence or absence of cocaine and/or cimetidine, at day 27. Percentages of BrdU+ and TUJ1+ cells of total BrdU+ cells are shown in C. Scale bars: 100 μm; n=5. *P<0.05, **P<0.01. Error bars, s.e.m. Cell line: H9.
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f4-0071397: Cocaine induced ROS generation in neocortical progenitors and premature neuronal differentiation. (A) ROS formation in neocortical progenitors, at day 19, treated with 3 μM cocaine, for 30 minutes, determined by DCFH-DA; n=7. (B,C) Expression of BrdU and TUJ1 (B) by immunocytochemistry in the presence or absence of cocaine and/or cimetidine, at day 27. Percentages of BrdU+ and TUJ1+ cells of total BrdU+ cells are shown in C. Scale bars: 100 μm; n=5. *P<0.05, **P<0.01. Error bars, s.e.m. Cell line: H9.

Mentions: To study how cocaine affects neocorticogenesis and the mechanisms involved in cytochrome-P450-mediated N-oxidative metabolism of cocaine in humans, we first examined whether cocaine induces ROS formation in NE cells derived from hPSCs on day 19. As shown in Fig. 4A, a pharmacologically relevant dose of cocaine, 3 μM (Kourrich et al., 2013), caused a significant increase in ROS 30 minutes after cocaine treatment. Pretreatment of NE cells, 30 minutes prior to cocaine exposure, with the cytochrome P450 inhibitor cimetidine, which blocks the N-oxidative metabolism of cocaine, inhibited cocaine-induced ROS formation (Fig. 4A). We previously reported that cocaine concentrations in the fetal rat brain decrease rapidly less than 1 hour after cocaine injection (Lee et al., 2008). Therefore, in order to create a physiologically meaningful drug treatment schedule in vitro, hPSC-derived neocortical cells were treated with 3 μM cocaine for 1 hour at days 20, 22 and 24, the beginning of neocortical neurogenesis, during which the NE cells are actively cycling.


An in vitro model of human neocortical development using pluripotent stem cells: cocaine-induced cytoarchitectural alterations.

Kindberg AA, Bendriem RM, Spivak CE, Chen J, Handreck A, Lupica CR, Liu J, Freed WJ, Lee CT - Dis Model Mech (2014)

Cocaine induced ROS generation in neocortical progenitors and premature neuronal differentiation. (A) ROS formation in neocortical progenitors, at day 19, treated with 3 μM cocaine, for 30 minutes, determined by DCFH-DA; n=7. (B,C) Expression of BrdU and TUJ1 (B) by immunocytochemistry in the presence or absence of cocaine and/or cimetidine, at day 27. Percentages of BrdU+ and TUJ1+ cells of total BrdU+ cells are shown in C. Scale bars: 100 μm; n=5. *P<0.05, **P<0.01. Error bars, s.e.m. Cell line: H9.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4257008&req=5

f4-0071397: Cocaine induced ROS generation in neocortical progenitors and premature neuronal differentiation. (A) ROS formation in neocortical progenitors, at day 19, treated with 3 μM cocaine, for 30 minutes, determined by DCFH-DA; n=7. (B,C) Expression of BrdU and TUJ1 (B) by immunocytochemistry in the presence or absence of cocaine and/or cimetidine, at day 27. Percentages of BrdU+ and TUJ1+ cells of total BrdU+ cells are shown in C. Scale bars: 100 μm; n=5. *P<0.05, **P<0.01. Error bars, s.e.m. Cell line: H9.
Mentions: To study how cocaine affects neocorticogenesis and the mechanisms involved in cytochrome-P450-mediated N-oxidative metabolism of cocaine in humans, we first examined whether cocaine induces ROS formation in NE cells derived from hPSCs on day 19. As shown in Fig. 4A, a pharmacologically relevant dose of cocaine, 3 μM (Kourrich et al., 2013), caused a significant increase in ROS 30 minutes after cocaine treatment. Pretreatment of NE cells, 30 minutes prior to cocaine exposure, with the cytochrome P450 inhibitor cimetidine, which blocks the N-oxidative metabolism of cocaine, inhibited cocaine-induced ROS formation (Fig. 4A). We previously reported that cocaine concentrations in the fetal rat brain decrease rapidly less than 1 hour after cocaine injection (Lee et al., 2008). Therefore, in order to create a physiologically meaningful drug treatment schedule in vitro, hPSC-derived neocortical cells were treated with 3 μM cocaine for 1 hour at days 20, 22 and 24, the beginning of neocortical neurogenesis, during which the NE cells are actively cycling.

Bottom Line: This model was used to examine the effects of cocaine during neocorticogenesis.These cocaine-induced changes were inhibited by the cytochrome P450 inhibitor cimetidine.This in vitro model enables mechanistic studies of neocorticogenesis, and can be used to examine the mechanisms through which cocaine alters the development of the human neocortex.

View Article: PubMed Central - PubMed

Affiliation: Cellular Neurobiology Research Branch, Intramural Research Program (IRP), National Institute on Drug Abuse, National Institutes of Health (NIH), Department of Health and Human Services (DHHS), Baltimore, MD 21244, USA.

Show MeSH
Related in: MedlinePlus