This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. We sought an in vitro primate model for studying serotonin neurons. Using the rhesus embryonic stem cell (ESC) line 366.4, a protocol as developed which generates a high percentage of serotonin neurons as determined by immunocytochemistry for tryptophan hydroxylase and the serotonin reuptake transporter. In addition, the ESC-derived neurons express estrogen receptor beta (ERbeta) and progesterone receptors (PR) in a manner similar to serotonin neurons in the macaque brain. We further optimized yield and obtained functional characteristics of the derived serotonin neurons. Sequential treatments of ESC 366.4 during expansion stage with fibroblast growth factor 4 and sonic hedgehog markedly increased the yield of serotonin neurons. These serotonin neurons propagated action potentials and expressed GABA receptors. Also for the first time we demonstrate that these ESC-derived serotonin neurons exhibit functional high affinity transporter sites, as well as high-affinity serotonin (5HT1A) binding sites, which are essential targets of common psychoactive drugs. Finally, to test the generality of this method, we utilized another rhesus ESC line, ORMES-22, which efficiently differentiated into serotonin neurons. Together these findings demonstrate the feasibility of our protocol to direct different primate neuronal ESC lines to serotonin neurons with physiological characteristics, which makes them a useful in vitro model system.
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