The Hebb synapse has been postulated to serve as a mechanism subserving both regulation of synaptic strength in the adult nervous system (long-term potentiation and depression) and developmental activity-dependent plasticity. According to this model, pre- and postsynaptic temporal concordance of activity results in strengthening of connections, while discordant activity results in synapse weakening. Evidence is presented that proteases and protease inhibitors may be involved in modification of synaptic strength. This leads to a modification of the Hebb assumptions, namely that postsynaptic activity results in protease elaboration with a consequent general reduction of synaptic connections to the active postsynaptic element. Further, presynaptic activity, if strong enough, induces local release of a protease inhibitor, such as protease nexin I, which neutralizes proteolytic activity and produces a relative preservation of the active input. This formulation produces many of the effects of the classical Hebbian construction, but the protease/inhibitor model suggests additional specific mechanistic features for activity-dependent plasticity.

We have shown recently that the rapid decline in corticotrophin-releasing hormone (CRH) transcription following activation by stress is associated with induction and binding to the CRH promoter of the repressor isoforms of cAMP responsive element modulator (CREM), inducible cAMP early repressor (ICER). The ability of ICER to inhibit CRH transcription was examined in the hypothalamic cell line, 4B, which expresses CRH. Co-transfection of the inhibitory isoforms of CREM, ICER I and II and CREMbeta, and CRH promoter-luciferase constructs in 4B cells blunted basal and forskolin-stimulated CRH promoter activity, an effect which was abolished by mutation of the CRE of the CRH promoter. Western blot analyses and electromobility gel-shift and super-shift showed increases in endogenous ICER after 3 h of incubation with forskolin. Consistent with an inhibitory effect of CREM on CRH transcription, chromatin immunoprecipitation assays in cells transfected with ICER I revealed recruitment of CREM by the CRH promoter in conjunction with decreases in Pol II association. The study shows that generation of ICER following prolonged stimulation with forskolin, or transfection of an ICER expression vector in hypothalamic cell lines expressing CRH, is associated with CREM binding to the CRH promoter and transcriptional repression. The data support the hypothesis that induction of repressor isoforms of CREM is part of an intracellular feedback mechanism contributing to the termination of CRH transcription during stimulation.

Recent studies in vitro have shown that the CREB co-activator, Transducer of Regulated CREB activity (TORC), is required for transcriptional activation of the corticotropin releasing hormone (CRH) gene. To determine the physiological importance of TORC 2 regulating CRH transcription during stress, we examined the localization of TORC 2 in CRH neurons, and the relationship between changes in CRH hnRNA, nuclear translocation of TORC 2, and binding of TORC 2 to the CRH promoter. Immunohistochemistry revealed TORC 2 immunoreactivity (irTORC 2) in the dorsolateral (magnocellular) and dorsomedial (parvocellular) regions of the hypothalamic paraventricular nucleus (PVN). While staining was mostly cytosolic in basal conditions, there was a marked increase in nuclear irTORC 2 in the dorsomedial region at 30 min restraint, concomitant with increases in CRH hnRNA levels. Levels of nuclear irTORC 2 and CRH hnRNA had returned to basal 4h after stress. Double staining immunohistochemistry showed TORC 2 co-staining in 100% of detected CRH neurons, and nuclear translocation following 30 min restraint in 61%. Cellular distribution of TORC 2 in the dorsolateral PVN was unaffected by restraint. Chromatin immunoprecipitation (ChIP) experiments showed recruitment of TORC 2 and phospho-CREB by the CRH promoter following 30min restraint, but 4h after stress only phospho-CREB was associated with the CRH promoter. The demonstration that TORC 2 translocates to the nucleus of hypothalamic CRH neurons and interacts with the CRH promoter in conjunction with the activation of CRH transcription during restraint stress, provides strong evidence for the involvement of TORC 2 in the physiological regulation of CRH transcription.