Effects of acute stress on brain-derived neurotrophic factor in the hippocampus of transgenic mouse model of depression

Brain-Derived Neurotrophic Factor (BDNF) is a member of the neurotrophin family which includes a group of molecules important for the development and the maintenance of the nervous system. Since BDNF is highly expressed in the hippocampus, the action and regulation of this neurotrophin in this area has become subject of intense study. The gene codifying for BDNF is a stress responsive gene and alterations in its expression may be important in regulating some of the physiological and pathophysiological effects of chronic and acute stress in the hippocampus. Different studies show that several types of stress reduce BDNF expression in the hippocampus of control animals [Smith et al., 1995] and these works led to a neurotrophic hypothesis of depression [Nestler et al., 2002]. Nevertheless, the effect of stress on BDNF gene expression may differ between a "normal" and a "pathological" brain. In our study, we used transgenic mice with glucocorticoid receptor impaired (GRi) expression created [Pepin et al., 1992], as a tool to study the neuroendocrine changes observed in stress-related disorders, such as major depression. This GRi mouse model is characterized by dysfunctional glucocorticoid inhibitory feedback and an excessive activation of the hypothalamic pituitar~adrenal (HPA) axis, that can be restored by antidepressant drugs' treatment. The hypothesis was tested that a single period of 30 minutes of restraint stress affects BDNF expression in the hippocampus of GRi mice differently than in wildtype (WT) mice. Using RNase protection assay and in situ hybridization we had assessed the BDNF mRNA hippocampal levels, while the levels of BDNF and its precursor, pro-BDNF were analyzed by western blotting. Our results indicated that 30 minutes of restraint enhanced BDNF mRNA expression in the CA3 hippocampal subregion of GRi mice; the same stress procedure induced also a statistically significant increase of pro- BDNF level in hippocampus of GRi mice. No effect of acute stress was observed in the WT at the level of the expression of BDNE Moreover, we evaluated the effects of restraint on signalling pathways implicated in the regulation of BDNF expression (mitogen-activated protein kinase and calcium/calmodulin-dependent kinase cascades) that converge on the phosphorylation of CREB that we found down-regulated in the hippocampus of GRi mice and up-regulated in WT mice. Our data suggest that, in the presence of psychophysiological stress (restraint stress), GRi mice display altered hippocampal regulation in BDNF gene expression. Thus, life-long central GR dysfunction may negatively affects neural functioning by limiting the capacity to cope with change or acute stress, which could be a predisposing or determining factor in depression. Understanding the mechanisms underlying the induction of BDNF mRNA and accumulation of pro-BDNF in the hippocampus of GRi mice, may help to clarify the molecular basis of action of this neurotrophin and contribute to the development of new strategies reducing the vulnerability of neurons to stress, thus preventing neuropathological alterations in the hippocampus.Brain-Derived Neurotrophic Factor (BDNF) is a memberof the neurotrophin family which includes a groupof molecules important for the development and themaintenance of the nervous system. Since BDNF is highlyexpressed in the hippocampus, the action and regulationof this neurotrophin in this area has become subject of intense study. The gene codifying for BDNF is a stressresponsivegene and alterations in its expression may beimportant in regulating some of the physiological andpathophysiological effects of chronic and acute stress inthe hippocampus. Different studies show that several typesof stress reduce BDNF expression in the hippocampusof control animals [Smith et al., 1995] and these worksled to a neurotrophic hypothesis of depression [Nestleret al., 2002]. Nevertheless, the effect of stress on BDNFgene expression may differ between a "normal" and a"pathological" brain.In our study, we used transgenic mice with glucocorticoidreceptor impaired (GRi) expression created [Pepinet al., 1992], as a tool to study the neuroendocrinechanges observed in stress-related disorders, such as majordepression. This GRi mouse model is characterized bydysfunctional glucocorticoid inhibitory feedback and anexcessive activation of the hypothalamic pituitar~adrenal(HPA) axis, that can be restored by antidepressant drugs'treatment. The hypothesis was tested that a single periodof 30 minutes of restraint stress affects BDNF expressionin the hippocampus of GRi mice differently than in wildtype(WT) mice. Using RNase protection assay and insitu hybridization we had assessed the BDNF mRNAhippocampal levels, while the levels of BDNF and itsprecursor, pro-BDNF were analyzed by western blotting.Our results indicated that 30 minutes of restraintenhanced BDNF mRNA expression in the CA3 hippocampalsubregion of GRi mice; the same stress procedureinduced also a statistically significant increase of pro-BDNF level in hippocampus of GRi mice. No effectof acute stress was observed in the WT at the levelof the expression of BDNE Moreover, we evaluated theeffects of restraint on signalling pathways implicated inthe regulation of BDNF expression (mitogen-activatedprotein kinase and calcium/calmodulin-dependent kinasecascades) that converge on the phosphorylation of CREBthat we found down-regulated in the hippocampus of GRimice and up-regulated in WT mice.Our data suggest that, in the presence of psychophysiologicalstress (restraint stress), GRi mice displayaltered hippocampal regulation in BDNF gene expression.Thus, life-long central GR dysfunction may negativelyaffects neural functioning by limiting the capacity to copewith change or acute stress, which could be a predisposingor determining factor in depression. Understanding themechanisms underlying the induction of BDNF mRNAand accumulation of pro-BDNF in the hippocampus ofGRi mice, may help to clarify the molecular basis of actionof this neurotrophin and contribute to the development ofnew strategies reducing the vulnerability of neurons tostress, thus preventing neuropathological alterations in thehippocampus