The efficacy of the serotonin 1A receptor (5-HT1A-R)-specific ligands in treating depression, anxiety, and many related symptoms has prompted studies into the physiological significance and signaling mechanisms of this neurotransmitter receptor. Such studies have shown that the absence of this receptor in the forebrain, specifically during early postnatal development of mice, results in elevated anxiety levels. Studies in the applicant's laboratory have shown that stimulation of this receptor in a hippocampal neuron-derived cell line causes protection of these cells against apoptosis. The mechanism responsible for this protection involves stimulation of the mitogen activated protein kinase (MAPK) pathway, which in turn signals through protein kinase C alpha (PKCc0 to cause inhibition of the proapoptotic protein caspase-3. Since the normal emotional state is a product of appropriate neuronal connections among the various regions of the fore brain, such as hippocampus, amygdala, and prefrontal cortex, preceding observations suggest that the 5-HT1A-R plays an important role in division and maturation of neurons in these brain regions. The current proposal will test this hypothesis by using both cultured hippocampal slices as well as 5-HT1A-R (+/+) and 5-HT1A-R (-/-) mice. Cultured hippocampal slices from mice at post-natal days 5, 10, 15, and 20 will be treated with the 5- HT1A agonist 8-hydroxy-2 (di-n-propylamino) tetralin (8-OH-DPAT) and immunohistochemical techniques used to study stimulation of key proteins, like PKC(x, MAPK and CREB, that promote neuronal division as well as maturation and survival. An important objective of performing such signaling studies will be to elucidate the profile of age-dependent regulation of 5-HT1A-R signaling and its effect on division (measured by BrdU incorporation) and maturation (measured by MAP-2 and synaptophysin staining) of neuronal cells. Regulation of apoptosis will be tested by using an anti-active caspase-3 antibody (to record activation of caspase-3) and deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) (to monitor apoptosis-associated DNA fragmentation). In parallel, the activation of MAPK, CREB, PKCcq and downstream transcription factors that relay the MAPK signals (like Elk-l), as well as the inhibition of caspase-3 will be measured by Western blot analysis using lysates of the hippocampal slices. RNA prepared from the treated slices will be reverse transcribed to prepare cDNA probes for microarray analysis to test for the regulation of gene expression, especially of the cytoskeleton-associated protein Arc and the immediate early proteins like Fos and Jun. Similar studies will be carried out by injecting 8-OH-DPAT into 5-HT1A-R (+/+) and 5-HT1A-R (-/-) mouse pups (d5, dl0, d15, d20) and preparing cryosections of brains to monitor activation of MAPK, CREB, PKCcq and also the effect of such 5-HT1A-R signaling on division and maturation of neuronal cells in the hippocampus and associated areas. Results obtained from 5-HT1A-R (+/+) and 5-HT_A-R (-/-) mouse pups will help delineate the specific effects of 5-HTtA-R signaling and shed new light on the mechanistic role of the 5-HT1A-R in brain development. ? ?
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