The efficacy of the serotonin 1A receptor (5-HTiA-R)-specific ligands in treating depression, anxiety, and many related symptoms has prompted studies into the signaling mechanisms of this neurotransmitter receptor. Its absence in the forebrain, specifically during early postnatal development of mice, results in elevated anxiety levels. The applicant's groups has shown that stimulation of this receptor in a hippocampal neuron-derived cell line causes activation of the mitogen activated protein kinase (MARK) isozymes Erk1/2, which in turn signal through protein kinase C alpha (PKCa) to cause inhibition of the proapoptotic protein caspase-3. Furthermore, in postnatal day-6 (P6) hippocampal slice cultures from mice, activation of Erk1/2 was dependent on an unidentified PKC isozyme, whereas at P15, PKCa was activated by Erk1/2 in 5-HTiA- R signaling. Since MAPK is known to promote division as well as protection and maturation of neuronal cells, preceding observations suggest that this curious switch in the 5-HTiA-R-dependent MAPK pathway could play an important role in the transition from the initial burst of proliferation to the later, post-mitotic stages of maturation of neurons in the brain. To test this hypothesis cultured hippocampal slices from 5-HT1A-R (+/+) and 5-HT1A-R (-/-) mice at P6-P20 will be treated with a 5-HT1A agonist. Immunohistochemistry and Western blotting will be used to study stimulation of key proteins, like PKCa, Erk1/2, and CREB, which could elicit age-dependent effects on division (measured by BrdU incorporation) and maturation (measured by MAP-2 and synaptophysin staining) of neuronal cells. Likely anti-apoptotic effects of the 5-HT1A-R-^Erk1/2-^PKCa pathway will be tested by using an anti-active caspase-3 antibody to record caspase-3 inhibition and deoxynucleotidyl transferase-mediated dUTP nick end labeling to monitor inhibition of DNA fragmentation. The novel Erk1/2-dependent PKCa stimulation suggests that Erk1/2 activate PKCa via direct phosphorylation at Threonine-638. To test this possibility activated Erk1/2 and pure PKCa will be used in an in vitro Erk1/2 assay. Finally, we will test if the observed switch in the hierarchy of PKC in the 5-HTiA- R-^Erkl/2 pathway between P6 and P15 was due to an age-dependent change in expression of PKC isozymes. This project will delineate a novel pathway that could play a key role in early brain development. Knowledge of this pathway will help in designing better therapies to combat developmental brain disorders.
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