No abstract

Expression of the brain-gut peptide cholecystokinin (CCK) in the developing olfactory-gonadotropin-releasing hormone-1 (GnRH-1) neuroendocrine systems was characterized, and the function of CCK in these systems was analyzed both in vivo and in vitro. We present novel data demonstrating that CCK transcript and protein are expressed in sensory cells in the developing olfactory epithelium and vomeronasal organ, with both ligand and receptors (CCK-1R and CCK-2R) found on olfactory axons throughout prenatal development. In addition, migrating GnRH-1 neurons in nasal regions express CCK-1R but not CCK-2R receptors. The role of CCK in olfactory-GnRH-1 system development was evaluated using nasal explants, after assessing that the in vivo expression of both CCK and CCK receptors was mimicked in this in vitro model. Exogenous application of CCK (10-7 M) reduced both olfactory axon outgrowth and migration of GnRH-1 cells. This inhibition was mediated by CCK-1R receptors. Moreover, CCK-1R but not CCK-2R antagonism caused a shift in the location of GnRH-1 neurons, increasing the distance that the cells migrated. GnRH-1 neuronal migration in mice carrying a genetic deletion of either CCK-1R or CCK-2R receptor genes was also analyzed. At embryonic day 14.5, the total number of GnRH-1 cells was identical in wild-type and mutant mice; however, the number of GnRH-1 neurons within forebrain was significantly greater in CCK-1R Ϫ/Ϫ embryos, consistent with an accelerated migratory process. These results indicate that CCK provides an inhibitory influence on GnRH-1 neuronal migration, contributing to the appropriate entrance of these neuroendocrine cells into the brain, and thus represent the first report of a developmental role for CCK.

Excitotoxicity mediated via the (S)-a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of receptor for L-glutamate contributes to various neuropathologies involving acute brain injury and chronic degenerative disorders. In this study, AMPA-induced neuronal injury and staurosporine (STS)-mediated apoptosis were compared in primary neuronal cultures of murine cerebral cortex by analyzing indices up-and downstream of mitochondrial activation. AMPAmediated apoptosis involved induction of Bax, loss of mitochondrial transmembrane potential (DY m ), early release of cytochrome c (cyt c), and more delayed release of second mitochondrial activator of caspases (SMAC), Omi, and apoptosis-inducing factor (AIF) with early calpain and minor late activation of caspase 3. STS-induced apoptosis was characterized by a number of differences, a more rapid time course, non-involvement of DY m , and relatively early recruitment of SMAC and caspase 3. The AMPA-induced rise in intracellular calcium appeared insufficient to evoke DY m as release of cyt c preceded mitochondrial depolarization, which was followed by the cytosolic translocation of SMAC, Omi, and AIF. Bax translocation preceded cyt c release for both stimuli inferring its involvement in apoptotic induction. Inclusion of the broad spectrum caspase inhibitor zVAD-fmk reduced the AMPA-induced release of cyt c, SMAC, and AIF, while only affecting the redistribution of Omi and AIF in the STS-treated neurons. Only AIF release was affected by a calpain inhibitor (calpastatin) which exerted relatively minor effects on the progression of cellular injury. AMPA-mediated release of apoptogenic proteins was more hierarchical relative to STS with its calpain activation and caspase-dependent AIF redistribution arguing for a model with cross-talk between caspase-dependent/independent apoptosis. Keywords: (S)-a-amino-3-hydroxy-5-methylisoxazole-4-propionate, apoptosis, Bax, calpain, caspase 3, mitochondrial membrane potential, mitochondrial pro-apoptotic proteins.