title: Monitoring mouse serotonin transporter internalization in stem cell-derived serotonergic neurons by confocal laser scanning microscopy.
abstract: In the central nervous system serotonergic neurotransmission is terminated by the rapid removal of serotonin (5-hydroxytryptamine, 5HT) out of the extra-cellular space back into the presynaptic neuron. This task is fulfilled by a specific serotonin transporter (SERT) protein which controls the concentration of extra-cellular 5HT. Consequently, one mechanism to regulate the efficacy of serotonergic neurotransmission is via modulation of the density of SERT molecules on the cell membrane. In this regard it has been shown, that chronic activation of the p38 mitogen-activated protein kinase (p38 MAPK) leads to enhanced SERT surface expression whereas activation of protein kinase C (PKC) reduces SERT surface expression. In addition, it has been reported that exposure to selective serotonin re-uptake inhibitors (SSRIs) leads to a down-regulation of SERT expression in vivo and in vitro in different cellular systems. Here, we have studied interactions between kinase- and SSRI-induced SERT internalization in mouse stem cell-derived serotonergic neurons expressing the native SERT allele in its natural surroundings. Therefore we established a method to quantify the amount of cell surface-expressed SERT molecules on individual cells by antibody detection combined with confocal laser scanning microscopy. Using this methodology we could show that activation of PKC, inhibition of the p38 MAPK as well as exposure to the SSRI citalopram each induced a significant reduction of cell surface-expressed SERT over time. Combinations of PKC activation, p38 MAPK inhibition and SSRI exposure led to a more pronounced down-regulation of SERT surface expression depending on the time of drug exposure.
authors.affiliation: Central Institute of Mental Health, Mannheim, Germany.
authors.forename: Thorsten
authors.initials: T
authors.lastname: Lau
authors.forename: Sandra
authors.initials: S
authors.lastname: Horschitz
authors.forename: Dusan
authors.initials: D
authors.lastname: Bartsch
authors.forename: Patrick
authors.initials: P
authors.lastname: Schloss
mesh_terms: D000818:Animals; D002454:Cell Differentiation / Q000502:physiology; D002460:Cell Line; D019070:Cell Lineage / Q000502:physiology; D015283:Citalopram / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004705:Endocytosis / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal; D055495:Neurogenesis / Q000502:physiology; D009474:Neurons / Q000166:cytology / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D012701:Serotonin / Q000378:metabolism*; D050486:Serotonin Plasma Membrane Transport Proteins / Q000378:metabolism*; D017367:Selective Serotonin Reuptake Inhibitors / Q000494:pharmacology; D013234:Stem Cells / Q000166:cytology / Q000378:metabolism*; D009435:Synaptic Transmission / Q000187:drug effects / Q000502:physiology; D048051:p38 Mitogen-Activated Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D015283:Citalopram; D004791:Enzyme Inhibitors; D011493:Protein Kinase C; D012701:Serotonin; D050486:Serotonin Plasma Membrane Transport Proteins; D017367:Selective Serotonin Reuptake Inhibitors; D048051:p38 Mitogen-Activated Protein Kinases; C493231:Slc6a4 protein, mouse