Christiane Brandwein

IL-6 knockout mice exhibit resistance to stress-induced development of depression-like behaviors.

Cytokine-dependent mechanisms in the CNS have been implicated in the pathogenesis of depression. Interleukin-6 is upregulated in depressed patients and dowregulated by antidepressants. It is, however, unknown whether IL-6 is involved in the pathogenesis of depression. We subjected IL-6-deficient mice (IL-6(-/-)) to depression-related tests (learned helplessness, forced swimming, tail suspension, sucrose preference). We also investigated IL-6 in the hippocampus of stressed wild-type mice. IL-6(-/-) mice showed reduced despair in the forced swim, and tail suspension test, and enhanced hedonic behavior. Moreover, IL-6(-/-) mice exhibited resistance to helplessness. This resistance may be caused by the lack of IL-6, because stress increased IL-6 expression in wild-type hippocampi. This suggests that IL-6 is a component in molecular mechanisms in the pathogenesis of depression. IL-6(-/-) mice represent tools to study IL-6-dependent signaling pathways in the pathophysiology of depression in vivo. Moreover, these mice may support the screening of compounds for depression by altering cytokine-mediated signaling.

AMPA receptor subunit 1 (GluR-A) knockout mice model the glutamate hypothesis of depression

Recent evidence indicates that glutamate homeostasis and neurotransmission are altered in major depressive disorder, but the nature of the disruption and the mechanisms by which it contributes to the syndrome are unclear. Glutamate can act via AMPA, NMDA, or metabotropic receptors. Using targeted mutagenesis, we demonstrate here that mice with deletion of the main AMPA receptor subunit GluR‐A represent a depression model with good face and construct validity, showing behavioral and neurochemical features of depression also postulated for human patients. GluR‐A−/− mice display increased learned helplessness, decreased serotonin and norepinephrine levels, and disturbed glutamate ho‐meostasis with increased glutamate levels and increased NMDA receptor expression. These results correspond well with current concepts regarding the role of AMPA and NMDA receptors in depression, postulating that compounds that augment AMPA receptor signaling or decrease NMDA receptor functions have antidepressant effects. GluR‐A−/− mice represent a model to investigate the pathophysiology underlying the depressive phenotype and to identify changes in neural plasticity and resilience evoked by the genetic alterations in glutamatergic function. Furthermore, GluR‐A−/− mice may be a valuable tool to study biological mechanisms of AMPA receptor modulators and the efficacy of NMDA antagonists in reducing behavioral or biochemical changes that correlate with increased helplessness.—Chourbaji, S., Vogt, M. A., Fumagalli, F., Sohr, R., Frasca, A., Brandwein, C, Hörtnagl, H., Riva, M. A., Sprengel, R., Gass, P. AMPA receptor subunit 1 (GluR‐A) knockout mice model the glutamate hypothesis of depression. FASEB J. 22, 3129–3134 (2008)

Depression-prone mice with reduced glucocorticoid receptor expression display an altered stress-dependent regulation of brain-derived neurotrophic factor and activity-regulated cytoskeleton-associated protein

Increasing evidence suggests that depression is characterised by impaired brain plasticity that might originate from the interaction between genetic and environmental risk factors. Hence, the aim of this study was to investigate changes in neuroplasticity following exposure to stress, an environmental condition highly relevant to psychiatric disorders, in glucocorticoid receptor-deficient mice (GR +/−), a genetic model of predisposition to depression. Specifically, we have analysed the neurotrophin brain-derived neurotrophic factor (BDNF) and the immediate-early gene activity-regulated cytoskeletal-associated protein (Arc), two closely related molecules that can contribute to neuroplastic and morphological changes observed in depression. We found a region-specific influence of the GR-genotype on BDNF levels both under basal and stress conditions. Steady-state levels of BDNF mRNA were unchanged in hippocampus while up-regulated in frontal lobe of GR+/− mice. Following exposure to an acute stress, increased processing from pro- to mature BDNF was observed in hippocampal synaptosomes of wild-type mice, but not in GR mutants. Furthermore, the stress-dependent modulation of Arc was impaired in the hippocampus of GR+/− mice. These results indicate that GR+/− mice show overt differences in the stress-induced modulation of neuroplastic proteins, which may contribute to pathologic conditions that may originate following gene x environment interaction.

Stress-resistant mice overexpressing glucocorticoid receptors display enhanced BDNF in the amygdala and hippocampus with unchanged NGF and serotonergic function

Dysfunctional glucocorticoid receptor (GR) signaling has been shown to be involved in the pathogenesis of depressive behavior in mice and humans. In accordance with this hypothesis GR overexpressing mice are less susceptible to develop depressive-like behavior when subjected to stressful events. Here, we analyzed GR overexpressing mice for morning and evening content of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and the tissue levels of serotonin and its metabolite 5-hydroxyindoleacetic acid) in brain areas suspected to be involved in stress adaptation. BDNF concentrations in the hippocampus and amygdala/piriform cortex were significantly enhanced in GR overexpressing mice (by maximally +103%) compared to wildtype animals. Diurnal variations, as detected for NGF in the hypothalamus, for BDNF in the frontal cortex and striatum and for serotonergic function in the frontal cortex and hypothalamus, were not affected by the genotype. In conclusion, GR overexpression-dependent increases of hippocampal and amygdala BDNF content presumably represent a dynamic correlate of enhanced stress resistance.

Suitability of tamoxifen-induced mutagenesis for behavioral phenotyping

Tamoxifen-induced mutagenesis via the so-called CreER(T2) fusion enzyme is a key technology for the inducible gene knockout in the adult murine brain. However, it requires a subchronic transient treatment with high doses of the non-selective estrogen receptor antagonist tamoxifen. It has been shown earlier that acute tamoxifen treatment causes behavioral alterations, while the long-term behavioral effects of tamoxifen in mice are so far unknown. Therefore C57BL/6 male mice, a common strain used for targeted mutagenesis and behavioral analyses, were subjected to a tamoxifen treatment protocol as used for inducible mutagenesis in vivo, and analyzed for effects on general behavior (locomotion, exploration), emotional behavior (anxiety, depression) and on learning and memory after a drug-free interval period of 4 weeks. The results demonstrate that a test for depression-like behavior, i.e. the Forced Swim Test, is affected even more than 4 weeks after tamoxifen treatment. In contrast, in all other tests, tamoxifen treated mice showed unaltered behaviors, indicating that the currently established 5-day protocol of tamoxifen treatment (40 mg/kg bid) for inducible mutagenesis has no or little effects on the behavior of C57BL/6 male mice after a latency period of 4 weeks. These results are important for all studies using tamoxifen-induced mutagenesis since this protocol obviously does not evoke alterations in general behaviors such as locomotion, exploration or anxiety-like behaviors, which might confound more complex behavioral analyses, nor does it affect standard tests for learning and memory, such as Morris Water Maze, contextual and cued Fear Conditioning and T-Maze learning.

Differential regulation of nerve growth factor and brain-derived neurotrophic factor in a mouse model of learned helplessness

Stress-induced helplessness in rodents constitutes a well-defined model to investigate neurobiological mechanisms of depression. Neurotrophins like nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF) have both been shown to be involved in neurobiological changes of physiological and pathological reactions to stress. In this study we investigated NGF and BDNF protein levels in the frontal cortex and hippocampus in mice treated with an established model of inducible helplessness via electric footshocks compared to untreated controls at various times (0 h up to 14 days after treatment). NGF levels were transiently decreased by one forth in the frontal cortex of shocked mice at 6 h after the stress treatment, whereas BDNF levels remained unchanged in the brain areas investigated throughout the time course. In addition, frontal cortex BDNF levels showed a significantly higher concentration in the right compared to the left hemisphere (up to 3-fold). This effect was detectable independently of treatment, namely in shocked and control mice at any time point measured. In conclusion, a transient decrease of frontal NGF constitutes the most striking correlate of neurobiological changes in this animal model of stress-induced change of behaviour. Interhemispherical differences of BDNF content in the frontal cortex are a new finding that might reflect intracerebral side dominance. Thus, subsequent studies of frontal cortex BDNF expression should carefully consider an interhemispherical variance to avoid misinterpretation.

Differential regulation of neurotrophins and serotonergic function in mice with genetically reduced glucocorticoid receptor expression.

The neurotrophin and serotonin (5-HT) hypotheses of depression were studied in a mouse model of reduced glucocorticoid receptor (GR) function (GR(+/-) mice), which recently has been proven as a murine model of predisposition for depressive behaviour under stressful conditions. In this model we studied diurnal changes in neurotrophins and serotonergic function in candidate brain regions mediating depressive behaviour. Morning and evening levels of brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), 5-HT and its metabolite 5-hydroxyindoleacetic acid (5-HIAA) were analyzed in representative brain regions of GR(+/-) and wildtype mice. The diurnal variation of hippocampal BDNF in wildtypes with higher levels in the morning was absent in GR(+/-) mice. Hypothalamus and parietal cortex displayed enhanced BDNF levels in GR(+/-) mice. In the frontal cortex, striatum and hypothalamus NGF increased from morning to evening in both genotypes, with an exaggeration in GR(+/-) mice. The diurnal variation of 5-HT levels and turnover did not differ significantly between genotypes. It was only in the hypothalamus that the evening level of 5-HIAA was lower in GR(+/-) mice than in wildtype mice. In conclusion, the present data indicate a contribution of altered BDNF and NGF protein levels to the predisposition for depressive behaviour in the GR(+/-) mouse model of depression, but argue against an eminent role of the serotonergic system.

Activation of glucocorticoid receptors increases 5-HT2A receptor levels

Major depression is associated with both dysregulation of the hypothalamic pituitary adrenal axis and serotonergic deficiency, not the least of the 5-HT2A receptor. However, how these phenomena are linked to each other, and whether a low 5-HT2A receptor level is a state or a trait marker of depression is unknown. In mice with altered glucocorticoid receptor (GR) expression we investigated 5-HT2A receptor levels by Western blot and 3H-MDL100907 receptor binding. Serotonin fibre density was analyzed by stereological quantification of serotonin transporter immunopositive fibers. To establish an effect of GR activation on 5-HT2A levels, mature organotypic hippocampal cultures were exposed to corticosterone with or without GR antagonist mifepristone and mineralocorticoid receptor (MR) antagonist spironolactone. In GR under-expressing mice, hippocampal 5-HT2A receptor protein levels were decreased (26.3 +/- 1.6%, p < 0.05) and frontal 5-HT2A receptor binding was decreased (20 +/- 15%, p < 0.01) as compared to wild-type mice. Conversely, in over-expressing GR mice hippocampal 5-HT2A receptor protein levels were increased (60.8 +/- 4.0%, p = 0.0001) and 5-HT2A receptor binding was increased in dorsal hippocampus (77 +/- 35%, p < 0.05) as compared to wild-type mice. No difference in serotonin fibre density was observed in the GR over-expressing mice, while the GR under-expressing mice showed lower serotonergic innervation in the frontal cortex area. An effect of GR activation on 5-HT2A receptor levels was further corroborated by the culture studies as long-term exposure of 3 microM corticosterone to organotypic hippocampal cultures increased 5-HT2A receptor levels (p < 0.05). The corticosterone-induced 5-HT2A receptor up-regulation was blocked by addition of either spironolactone or mifepristone.

Evaluation of effects of previous exposure to an acute stressor before testing for depression-like behaviours in mice

Test batteries are an essential and broadly used tool for behavioural phenotyping, especially with regard to mouse models of particular diseases, such as depression. Facing the problem of an often limited number of mutant animals, it therefore seems crucial to develop and optimise such test batteries in terms of an ideal throughput of subjects. This study aimed to characterize several common stressors, which are used for the investigation of depressive-like features with regard to their capability of each of them to affect performance in a subsequent behavioural test. Here we investigated swim-, restraint- and footshock-stress in male C57/BL6 mice, focusing on post-stress corticosterone elevations as well as potential effects on the behavioural level. The stressors increased circulating corticosterone levels when assessed 1 h after exposure. On the behavioural level, no test interactions could be detected, which suggests, that in general, combining these test conditions in experiments with a restricted availability of animals seems to be rather unproblematic.

Morc1 knockout evokes a depression-like phenotype in mice

Morc1 gene has recently been identified by a DNA methylation and genome-wide association study as a candidate gene for major depressive disorder related to early life stress in rodents, primates and humans. So far, no transgenic animal model has been established to validate these findings on a behavioral level. In the present study, we examined the effects of a Morc1 loss of function mutation in female C57BL/6N mice on behavioral correlates of mood disorders like the Forced Swim Test, the Learned Helplessness Paradigm, O-Maze and Dark-Light-Box. We could show that Morc1(-/-) mice display increased depressive-like behavior whereas no behavioral abnormalities regarding locomotor activity or anxiety-like behavior were detectable. CORT plasma levels did not differ significantly between Morc1(-/-) mice and their wildtype littermates, yet - surprisingly - total Bdnf mRNA-levels in the hippocampus were up-regulated in Morc1(-/-) animals. Although further work would be clarifying, Morc1(-/-) mice seem to be a promising epigenetically validated mouse model for depression associated with early life stress.