Gianluigi Guidotti

Glucocorticoid Receptor and FKBP5 Expression Is Altered Following Exposure to Chronic Stress: Modulation by Antidepressant Treatment

Major depression is thought to originate from the interaction between susceptibility genes and adverse environmental events, in particular stress. The hypothalamus–pituitary–adrenal (HPA) axis is the major system involved in stress response and its dysregulation is an important element in the pathogenesis of depression. The stress response is therefore finely tuned through a series of mechanisms that control the trafficking of glucocorticoid receptors (GRs) to the nucleus, including binding to the chaperone protein FKBP5 and receptor phosphorylation, suggesting that these elements may also be affected under pathologic conditions. On these bases, we investigated FKBP5 and GR expression and phosphorylation in the hippocampus (ventral and dorsal) and in the prefrontal cortex of rats exposed to chronic mild stress (CMS) and we analyzed the effect of a concomitant antidepressant treatment. We found that animals exposed to CMS show increased expression of FKBP5 as well as enhanced cytoplasmic levels of GR, primarily in ventral hippocampus and prefrontal cortex. Chronic treatment with the antidepressant duloxetine is able to normalize such alterations, mainly in the prefrontal cortex. Moreover, we demonstrate that CMS-induced alterations of GR trafficking and transcription may be sustained by changes in receptor phosphorylation, which are also modulated by pharmacological intervention. In summary, while GR-related changes after CMS might be relevant for the depressive phenotype, the ability of antidepressant treatment to correct some of these alterations may contribute to the normalization of HPA axis dysfunctions associated with stress-related disorders.

Reduced neuroplasticity in aged rats: a role for the neurotrophin brain-derived neurotrophic factor

Aging is a physiological process characterized by a significant reduction of neuronal plasticity that might contribute to the functional defects observed in old subjects. Even if the neurobiological mechanisms that contribute to such impairment remain largely unknown, a role for neurotrophic molecules, such as the neurotrophin brain-derived neurotrophic factor (BDNF), has been postulated. On this basis, the purpose of this study was to provide a detailed investigation of the BDNF system, at transcriptional and translational levels, in the ventral and dorsal hippocampus and in the prefrontal cortex of middle-aged and old rats, compared with in adult animals. The expression of major players in BDNF regulation and response, including the transcription factors, calcium-responsive transcription factor, cyclic adenosine monophosphate (cAMP) responsive element-binding protein (CREB), and neuronal Per Arnt Sim (PAS) domain protein 4, and the high-affinity receptor tropomyosin receptor kinase B (TrkB), was also analyzed. Our results demonstrate that the BDNF system is affected at different levels in aged rats with global impairment including reduced transcription, impaired protein synthesis and processing, and decreased activation of the TrkB receptors. These modifications might contribute to the cognitive deficits associated with aging and suggest that pharmacological strategies aimed at restoring reduced neurotrophism might be useful to counteract age-related cognitive decline.

Stress-Induced Changes of Hippocampal NMDA Receptors: Modulation by Duloxetine Treatment

It is now well established that the glutamatergic system contributes to the pathophysiology of depression. Exposure to stress, a major precipitating factor for depression, enhances glutamate release that can contribute to structural abnormalities observed in the brain of depressed subjects. On the other hand, it has been demonstrated that NMDA antagonists, like ketamine, exert an antidepressant effect at preclinical and clinical levels. On these bases, the purpose of our study was to investigate whether chronic mild stress is associated with specific alterations of the NMDA receptor complex, in adult rats, and to establish whether concomitant antidepressant treatment could normalize such deficits. We found that chronic stress increases the expression of the obligatory GluN1 subunit, as well as of the accessory subunits GluN2A and GluN2B at transcriptional and translational levels, particularly in the ventral hippocampus. Concomitant treatment with the antidepressant duloxetine was able to normalize the increase of glutamatergic receptor subunit expression, and correct the changes in receptor phosphorylation produced by stress exposure. Our data suggest that prolonged stress, a condition that has etiologic relevance for depression, may enhance glutamate activity through post-synaptic mechanisms, by regulating NMDA receptors, and that antidepressants may in part normalize such changes. Our results provide support to the notion that antidepressants may exert their activity in the long-term also via modulation of the glutamatergic synapse.

Modulation of the inflammatory response in rats chronically treated with the antidepressant agomelatine

Growing evidence suggests that the activation of the inflammatory/immune system contributes to depression pathogenesis, a hypothesis that might hold strong clinical implication. Indeed more than 30% of depressed patients fail to achieve remission, which poses the necessity to identify systems that may represent novel targets for medications. Accordingly, goal of this study was to evaluate the ability of the antidepressant agomelatine to modulate specific components of the immune response in the rat brain following an inflammatory challenge with lipopolysaccharide (LPS). To this aim, adult male rats were chronically treated with agomelatine before being acutely challenged with LPS 16 h after the last drug administration. Rats were sacrificed 2, 6, or 24h after the challenge and several components of the inflammatory response have been investigated by using real-time PCR or ELISA. We found that agomelatine significantly reduced the LPS-induced up-regulation of the pro-inflammatory cytokines interleukin-1β and interleukin-6 in the rat brain as well as at peripheral level. At central level, these effects are associated to the inhibition of NF-κB translocation as well as to alterations of mechanisms responsible for microglia activation. In addition, we found that agomelatine was also able to alter the expression of enzymes related to the kynurenine pathway that are thought to represent important mediators to inflammation-related depression. These data disclose novel properties that may contribute to the therapeutic effect of agomelatine providing evidence for a crucial role of specific components of the immune/inflammatory system in the antidepressant response and thereby in depression etiopathology.

Developmental influence of the serotonin transporter on the expression of npas4 and GABAergic markers: modulation by antidepressant treatment

Alterations of the serotonergic system are involved in the pathophysiology of mood disorders and represent an important target for its pharmacological treatment. Genetic deletion of the serotonin transporter (SERT) in rodents leads to an anxious and depressive phenotype, and is associated with reduced neuronal plasticity as indicated by decreased brain-derived neurotrophic factor (Bdnf) expression levels. One of the transcription factors regulating Bdnf is the neuronal PAS domain protein 4 (Npas4), which regulates activity-dependent genes and neuroprotection, and has a critical role in the development of GABA synapses. On the basis of these premises, we investigated the expression of Npas4 and GABAergic markers in the hippocampus and prefrontal cortex of homozygous (SERT−/−) and heterozygous (SERT+/−) knockout rats, and analyzed the effect of long-term duloxetine treatment on the expression of these targets. We found that Npas4 expression was reduced in both the brain structures of adult SERT+/− and SERT−/− animals. This effect was already present in adolescent SERT−/−, and could be mimicked by prenatal exposure to the antidepressant fluoxetine. Moreover, SERT−/− rats showed a strong impairment of the GABAergic system, as indicated by the reduction of several markers, including the vesicular transporter (Vgat), glutamic acid decarboxylase-67 (Gad67), the receptor subunit GABA A receptor, gamma 2 (GABAA-γ2), and calcium-binding proteins that label subgroups of the GABAergic neurons. Interestingly, chronic treatment with the antidepressant duloxetine was able to restore the physiological levels of Npas4 and GABAergic markers in SERT−/− rats, although some differences in the modulation of GABAergic genes exist between hippocampus and prefrontal cortex. Our results demonstrate that SERT knockout rats, an animal model of mood disorders, have reduced Npas4 expression that correlates with decreased expression of Bdnf exon I and IV. These changes lead to an impairment of the GABAergic system that may contribute to the anxious and depressive phenotype associated with inherited SERT downregulation.

Exposure to early life stress regulates Bdnf expression in SERT mutant rats in an anatomically selective fashion.

Although the causes of psychiatric disorders are not fully understood, it is well established that mental illness originates from the interaction between genetic and environmental factors. In this regard, compelling evidence demonstrates that depression can be the consequence of altered, and often maladaptive, response to adversities during pre‐ and early post‐natal life. In this study, we investigated the impact of chronic maternal separation (MS) on the expression of the neurotrophin brain‐derived neurotrophic factor (BDNF) in serotonin transporter (SERT) knockout rats in the ventral and dorsal hippocampus as well as the ventromedial and dorsomedial prefrontal cortex (PFC). We found that both SERT deletion and the MS led to an overall reduction in Bdnf expression in the ventral hippocampus and the ventromedial PFC, whereas in the dorsal hippocampus and in the dorsomedial PFC, we observed a significant increase in the neurotrophin gene expression after MS exposure, specifically in the heterozygous SERT rats. In summary, we show that the modulation of Bdnf expression in SERT mutant rats exposed to MS reflects the complex functional consequences of this gene–environment interaction with a clear distinction between the ventral and the dorsal subfields of the hippocampus and of the PFC.

Early life stress and serotonin transporter gene variation interact to affect the transcription of the glucocorticoid and mineralocorticoid receptors, and the co-chaperone FKBP5, in the adult rat brain

The short allelic variant of the serotonin transporter (5-HTT) promoter-linked polymorphic region (5-HTTLPR) has been associated with the etiology of major depression by interaction with early life stress (ELS). A frequently observed endophenotype in depression is the abnormal regulation of levels of stress hormones such as glucocorticoids. It is hypothesized that altered central glucocorticoid influence on stress-related behavior and memory processes could underlie the depressogenic interaction of 5-HTTLPR and ELS. One possible mechanism could be the altered expression of the genes encoding the glucocorticoid and mineralocorticoid receptors (GR, MR) and their inhibitory regulator FK506-binding protein 51 (FKBP5) in stress-related forebrain areas. To test this notion, we exposed heterozygous (5-HTT+/−) and homozygous (5-HTT−/−) serotonin transporter knockout rats and their wildtype littermates (5-HTT+/+) to daily 3 h maternal separations from postnatal day 2 to 14. In the medial prefrontal cortex (mPFC) and hippocampus of the adult male offspring, we found that GR, MR, and FKBP5 mRNA levels were affected by ELS × 5-HTT genotype interaction. Specifically, 5-HTT+/+ rats exposed to ELS showed decreased GR and FKBP5 mRNA in the dorsal and ventral mPFC, respectively. In contrast, 5-HTT+/− rats showed increased MR mRNA levels in the hippocampus and 5-HTT−/− rats showed increased FKBP5 mRNA in the ventral mPFC after ELS exposure. These findings indicate that 5-HTT genotype determines the specific adaptation of GR, MR, and FKBP5 expression in response to early life adversity. Therefore, altered extra-hypothalamic glucocorticoid signaling should be considered to play a role in the depressogenic interaction of ELS and 5-HTTLPR.

Neuroadaptive changes after chronic lurasidone treatment : implication for mood and stress-related disorders

Background: Brain kappa-opioid receptors (KORs) have been implicated in the behavioral consequences of stress, such as drug seeking and depressive-like behavior. Previously, we have shown that systemic KOR antagonism produces anxiolytic-like effects in tests of both conditioned and unconditioned fear. The present studies were designed to further characterize interactions between KOR systems and stress-induced behavior using footshock as a stressor, the effects of which can be measured by potentiation of the acoustic startle reflex. This potentiation was investigated after systemic blockade or constitutive deletion of KORs. Recently, it has been shown that stress induces KOR phosphorylation in the nucleus accumbens, where KORs are expressed on the terminals of dopamine (DA) neurons originating in the ventral tegmental area (VTA). To characterize the involvement of this specific population of KORs we generated a conditional knockout in which KORs are selectively deleted in DA-containing neurons. Methods: To study the effects of KOR on stress-induced behaviors, mice were designed with loxP sites flanking exon 3 of the KOR gene. This arrangement enables gene inactivation by Cre recombinase and generation of our two KOR KO lines. Floxed mice were crossed to either a ubiquitous Cre line (EIIaCre) to generate constitutive KOR KOs or DATCre to generate conditional KOs lacking KORs specifically in DA transporter (DAT)-expressing cells, such as those found in the VTA and substantia nigra (SN). Knockout of the receptor was confirmed with autoradiography using a radiolabeled KOR agonist ([H]U-69593). Further, mRNA analyses were performed on brain tissue samples using qPCR to demonstrate specificity of deletion. To test the effect of KOR antagonism on shock-potentiated startle, mice were matched into groups with equivalent baseline startle and given an intraperitoneal injection of the KOR antagonist JDTic or vehicle 24 hr prior to testing to accommodate the slow onset and persistent actions of this drug. In the test session, mice received a baseline startle test followed by ascending footshock amplitudes (0.2 mA, 0.4 mA, and 0.8 mA) each followed by a startle test. The following day, mice were given a final startle test. KOR KO mice were subjected to the same behavioral protocol without administration of JDTic. Data were analyzed using appropriate ANOVAs and significant effects were analyzed using post hoc Bonferroni tests. Experiments were conducted in accordance with National Institutes of Health and McLean Hospital guidelines for the care and use of laboratory animals. Results: Autoradiography demonstrated a lack of KOR binding in constitutive KOR KO mice and reduced binding in conditional DATCre KOs. Further, qPCR demonstrated no detectable KOR mRNA in KOs and reduced KOR mRNA specifically in the VTA/SN of DATCre KOs. Blockade of KOR receptors by JDTic attenuated footshock induced increases in startle. When shocked mice were returned to the testing chamber on the following day, JDTic pretreated mice continued to show significantly decreased context conditioning. In comparison, constitutive KOR KO mice had similar levels of footshock-potentiated startle compared to littermate controls. Like JDTic treated mice, DATCre KOs showed significantly lower levels of potentiation than controls. Conclusions: These studies used two novel lines of KOR KO mice, as demonstrated by reduced levels of KOR mRNA and KOR binding, to follow up initial findings that systemic blockade of KORs with JDTic reduces startle potentiation after footshock. Interestingly, constitutive KOR KO mice show equivalent levels of footshock-potentiated startle compared to wild type littermates, whereas KOR deletion restricted to DAT expressing neurons (DATCre KO) was sufficient to reduce potentiated startle following the first two blocks of footshock. This pattern of results suggests that compensatory adaptations occurring outside brain DA systems may offset behavioral changes that are caused by KOR deletion within brain DA systems in constitutive KOR KOs. Overall, these data implicate KORs of midbrain dopaminergic cells in the manifestation of stress, and provide additional evidence that disruption of KOR function reduces stress-induced anxiety-like behavior. JDTic treated mice had acute blockade of KORs, while KOR KO mice had a lifelong absence of KORs. Thus, the finding that systemic KOR antagonism produces anti-stress effects that are not seen in the constitutive KOR KO mice suggests the importance of neuroadaptations occurring during brain development.