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Besides inducing transcription by activating nuclear receptors, corticosterone (CORT) acts rapidly to alter cellular activity through multiple signaling cascades. Pharmacology-based experiments presented here show that nanomolar doses of CORT activate several pathways in primary hippocampal cultures within 20 minutes, a time-frame that excludes genomic mechanisms mediated by classical glucocorticoid (GR) and mineralocorticoid (MR) nuclear receptors. Moreover, none of these effects were subject to inhibition by either a series of structurally-unrelated antagonists of the classical GR and MR or by inhibitors of translation. Accordingly, a major aim of this work was to identify mechanisms proximal to the cell membrane that could potentially mediate these rapid actions of CORT. Initial time-course studies showed that 10 nM CORT rapidly triggers protein tyrosine kinase (PTK) activation that can be blocked by the small molecule inhibitor, PP2. It was also found that Src kinase is rapidly activated by CORT through tyrosine phosphorylation at Y416 and dephosphorylation at Y527. These events were subject to regulation by another key PTK member, Pyk2 (proline-rich tyrosine kinase 2). A series of experiments designed to study the potential mechanisms that regulate Pyk2, revealed that CORT increases the phosphorylation status of at least three tyrosine sites within Pyk2, namely, Y402, Y579/580 and Y881. Further, pharmacological probing uncovered requisite roles for other signaling pathways, such as the classical PLC-PKC pathway and the “novel” PKA and PKB pathways. Interestingly, further investigations revealed that the rapid CORT-induced activation of Pyk2/Src occurs in a G-protein dependent manner. This, together with the observation that membrane-impermeable BSA-conjugated CORT (CORT-BSA) produces similar responses profile to that obtained with CORT, led to pilot experiments to probe whether GPR30, a recently-described G protein-coupled receptor that appears to transduce signals from other steroid ligands, might be the putative receptor for CORT. Support for this view was provided by the finding that the rapid actions of CORT and CORT-BSA on Pyk2/Src activation could be blocked by a novel inhibitor of GPR30. Nevertheless, further studies will be needed to establish the role of GPR30 more firmly. Other studies sought to identify downstream targets of Pyk2/Src. Results show that Pyk2/Src regulates activation of c-Abl (another PTK) and RhoA, both of which are regulators of a number of cellular processes, including actin cytoskeleton remodeling. Furthermore, it was found that CORT triggers phosphorylation of the NR2B subunit of NMDAR, increases surface expression of NMDAR and activates downstream MAP kinases; all of these events depend on Src, one of whose direct substrates is the NR2B subunit. In a summary, the evidence presented in this dissertation suggests that, by acting via a G protein-coupled receptor, rather than through classical nuclear receptor mechanisms, CORT rapidly activates a series of intracellular signaling cascades that lie proximal to the neuronal plasma membrane; Pyk2/Src are early kinases involved and beyond these divergent pathways come into play, ultimately influencing functions ranging from rearrangements of the actin cytoskeleton, spine structure, scaffold protein clustering and function, to synaptic plasticity and transcriptional regulation.

Glial loss in the hippocampus has been suggested as a factor in the pathogenesis of stress-related brain disorders that are characterized by dysregulated glucocorticoid (GC) secretion. However, little is known about the regulation of astrocytic fate by GC. Here, we show that astrocytes derived from the rat hippocampus undergo growth inhibition and display moderate activation of caspase 3 after exposure to GC. Importantly, the latter event, observed both in situ and in primary astrocytic cultures is not followed by either early- or late-stage apoptosis, as monitored by stage I or stage II DNA fragmentation. Thus, unlike hippocampal granule neurons, astrocytes are resistant to GC-induced apoptosis; this resistance is due to lower production of reactive oxygen species (ROS) and a greater buffering capacity against the cytotoxic actions of ROS. We also show that GC influence hippocampal cell fate by inducing the expression of astrocyte-derived growth factors implicated in the control of neural precursor cell proliferation. Together, our results suggest that GC instigate a hitherto unknown dialog between astrocytes and neural progenitors, adding a new facet to understanding how GC influence the cytoarchitecture of the hippocampus., S. Yu was supported by a fellowship from the Max Planck Society. This study was supported by the Max Planck Institute of Psychiatry and by grants from the German Academic Exchange Service (DAAD) and the Portuguese Rectors' Conference (CRUP), and an Integrated Project grant from the European Commission (CRESCENDO, Contract No. LSHM-CT-2005-01852). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.