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- Materials and methods
Accumulating evidences underlie the importance of the interplay between environmental and genetic factors in contributing to the risk to develop mental illness. Brain-derived neurotrophic factor (BDNF) and its Tyrosine receptor kinase B (TrkB) receptor play a fundamental contribution to brain development and plastic adaptations to life events. In the present study, the potential for the BDNF/TrkB contribution in increasing vulnerability to negative social experiences was assessed by subjecting TrkB.T1 overexpressing mice to a chronic social defeat model. TrkB.T1 mice overexpress the dominant-negative truncated splice variant of TrkB receptor leading to decreased BDNF signaling. After repeated social defeat, mice were assessed in a longitudinal study for behavioral, physiological, endocrine and immune responses potentially related to psychiatric endophenotypes. TrkB.T1 overexpression corresponded to smaller changes in metabolic parameters such as body weight, food intake, feed efficiency and peripheral ghrelin levels compared with wild-type (wt) littermates following social defeat. Interestingly, 4 weeks after the last defeat, TrkB.T1 overexpressing mice exhibited more consistent social avoidance effects than what observed in wt subjects. Finally, previously unreported effects of TrkB mutations could be observed on lymphoid organ weight and on peripheral immune biomarker levels, such as interleukin-1α and regulated on activation, normal, T-cell expressed, and secreted (RANTES), thus suggesting a systemic role of BDNF signaling in immune function. In conclusion, the present data support a contribution of TrkB to stress vulnerability that, given the established role of TrkB in the response to antidepressant treatment, calls for further studies addressing the link between stress susceptibility and variability in drug efficacy.
Brain-derived neurotrophic factor (BDNF) is a neurotrophin extensively involved in synaptic plasticity and in dynamic phenomena occurring in the brain such as neuronal maturation, dendritic remodeling and formation of synaptic contacts (Schinder & Poo 2000). Binding of BDNF to its high affinity receptor TrkB initiates a cascade of events leading to modulation of three major intracellular signaling pathways, the phosphoinosite-3-kinase/Akt, the phospholipase C and the extracellular signal-regulated kinase–mitogen-activated kinase pathways (Shaltiel et al. 2007). These events are directly involved in the mode of action of psychotropic drugs (Chen & Manji 2006; Picchini et al. 2004; Rantamäki et al. 2007), suggesting that different psychiatric conditions may derive from impairments in BDNF/TrkB signaling. An impressive amount of data supports this hypothesis: decreased BDNF and TrkB expression has been found in suicide subjects (Dwivedi et al. 2003); a functional single nucleotide BDNF polymorphism in humans has been associated to different mental disorders (Gratacos et al. 2007) and to vulnerability to the depressogenic effects of early life stress (Gatt et al. 2009); BDNF has been shown to play a major role in the mood-improving actions of antidepressants (Chen et al. 2001; Nibuya et al. 1995); finally, TrkB receptors need to be present in hippocampal neural progenitor cells for the neurogenic and behavioral actions of antidepressant treatments (Li et al. 2008).
To assess the functional role of BDNF/TrkB signaling in phenotypes related to mood disturbances, TrkB.T1 dominant-negative overexpressing mice showing reduced TrkB brain signaling (Saarelainen et al. 2000, 2003) were tested in a chronic social defeat model. The social defeat model is particularly appropriate due to the essential role of central BDNF in regulating the response to social stress in the mesolimbic dopamine pathway and to its link to sustained hippocampal epigenetic regulation, both of which mediate the long-term neural and behavioral plasticity observed in response to antidepressant treatment (Berton et al. 2006; Tsankova et al. 2006, 2007). This model offers the possibility to assess the importance of a ‘gene × environment' interaction, as stress exposure might unveil a greater vulnerability to adult social stress in TrkB.T1 mutant mice. Interestingly, using a similar social stress model, increased stress susceptibility was observed in mice carrying a partial genetic deficiency in the serotonin transporter (SERT) (Bartolomucci et al. 2010). In line with these findings, clinical data from human subjects suggest a link between a functional polymorphism in the promoter region of SERT and increased risk of developing depression following life stress experience (Caspi et al. 2003). A common single gene polymorphism (val66met) interfering with BDNF activity-dependent secretion has been described (Egan et al. 2003) and is considered potentially relevant for susceptibility to psychiatric disorders (Gratacos et al. 2007). Interestingly, for the same polymorphism an interaction between early life stress and cognitive and brain structure parameters was shown, leading to more severe depressive symptoms (Gatt et al. 2009). Therefore, the TrkB.T1 mice were chronically defeated to evaluate the potential increased vulnerability of deficient TrkB signaling to social stress in behavioral, physiological, endocrine and immune parameters relevant to psychiatric endophenotypes.
- Top of page
- Materials and methods
In the present study, we characterized the behavioral, physiological, endocrine and immune response to chronic psychosocial stress in mice overexpressing a truncated ‘dominant-negative’ isoform of the BDNF receptor TrkB.T1 that represents a natural splicing variant of the receptor lacking the kinase domain (Saarelainen et al. 2000). TrkB.T1 has been shown to be upregulated in pathological states such as human Alzheimer's disease (Ferrer et al. 1999), is involved in pain processing (Renn et al. 2009) and is promoted by stress and various environmental factors (Jeanneteau et al. 2008). Mutant mice utilized in the present study display an overexpression of TrkB.T1 isoform in the hippocampus, several cortical layers, thalamus and amygdale, leading to a decrease in TrkB signaling in several, but not all (e.g. striatum), areas of the brain (Saarelainen et al. 2000). Importantly, the Thy1 promoter targets the expression of TrkB.T1 transgene to postnatal neurons (expression starts at early postnatal days) (Saarelainen et al. 2000). As a result of this modification, an inhibition in the activation of the TrkB kinase, a decrease in BDNF signaling and in BDNF hippocampal levels (∼60%) was detected in TrkB.t1 mice, whereas no differences from wt could be observed, at least in our experimental conditions, in basal metabolic function and general activity.
Therefore, to analyze the interaction between genotype and environment, TrkB.T1 overexpressing mice were subjected to a chronic social stress procedure. Social defeat effects elicited robust effects on metabolic parameters, more pronouncedly and persistently in wt compared with TrkB.T1 mice that, in turn, exhibited a greater resistance to defeat-induced metabolic alterations. The present results in wt mice are in agreement with the growing data showing obesity inducing adaptations to defeat stress whose underlying mechanisms have only recently begun to be elucidated (Chuang et al. 2010), and that confer further translational relevance to the social defeat model. A mounting literature indicates that obesity is one of the most important environmental risk factors for developing mental disorders (Richardson et al. 2003; Vieweg et al. 2006), and recent epidemiological evidences support the comorbidity between depressive and metabolic disorders (Beydoun & Wang 2009; Chuang et al., 2010; Goldbacher et al., 2009; Golden et al., 2008; Ma & Xiao, 2009). A metabolic role is also played by BDNF and TrkB that are implicated in the neuroendocrine control of mammalian feeding behavior and energy homeostasis (Tsao et al. 2008; Wisse & Schwartz 2003). Mice (Xu et al. 2003) or humans (Yeo et al. 2004) with defective tyrosine kinase receptor TrkB display excessive appetite, reduced energy expenditure and obesity. The expression of BDNF in the ventromedial hypothalamus, an important component of the central nervous system (CNS) energy homeostasis circuitry, is reduced after food deprivation (Kernie et al. 2000; Xu et al. 2003); mice with genetically reduced BDNF expression also develop obesity and hyperphagic behavior (Fox & Byerly 2004; Kernie et al. 2000; Lyons et al. 1999; Unger et al. 2007). Although the mechanism by which TrkB regulates feeding behavior and weight is complex and not well understood, one explanation for the lack of overt hyperphagia and obesity phenotype in TrkB.T1 mice presently reported should take into consideration compensatory changes in CNS regions critical for feeding behavior and body weight homeostasis. Furthermore, the hyperphagic/obese phenotype produced by BDNF deficiency is age-dependent, as its occurrence is reported mostly in mature animals (above 5 months old) (Fox & Byerly 2004; Kernie et al. 2000; Lyons et al. 1999) wherein it has been related to a late reduction in serotonin levels (Rios et al. 2001). Nevertheless, an altered metabolic homeostasis in TrkB.T1 compared with wt mice is supported by the observed increased levels of active ghrelin, consistent with the proposed role of BDNF as a metabotrophin (Gomez-Pinilla et al. 2008). Ghrelin is secreted from the stomach generally in response to energy depletion related to decreased food intake (Ariyasu et al. 2001; Kojima et al. 1999) and, interestingly, it can also bind hippocampal receptors (Guan et al. 1997) with profound effects on hippocampal synaptic plasticity (Diano et al. 2006), thus linking metabolic and cognitive systems. Given the established cognitive deficits of TrkB.T1 mice (Saarelainen et al. 2000) and the altered ghrelin secretion observed in the present experiments, further studies should be performed to identify the mediators involved in the cross-talk between energy homeostasis and high brain functions.
Although the impairment in TrkB signaling induced in either TrkB.T1 and BDNF+/− mice (MacQueen et al. 2001; Monteggia et al. 2004; Saarelainen et al. 2003) does not lead to depressive-like phenotypes in basal conditions, TrkB.T1 defeated mice exhibited a very consistent social avoidance. Differential coping styles are reported following social defeat in mice (Krishnan et al. 2007); coherently, in the present study a dichotomic response was shown within defeated wt mice, although, because of the small sample size, experimental subjects could not be separated into resilient/susceptible subpopulations of meaningful size. On the other hand, a homogeneously aversive response (i.e. diminished time in interaction zone) was observed in defeated TrkB.T1 mice toward the confined aggressor. BDNF signaling has been repeatedly linked to defeat-induced social avoidance and particularly to a susceptible phenotype (Berton et al. 2006; Krishnan et al. 2007). BDNF levels in specific brain areas, such as the nucleus accumbens (NAc), have been linked to the development of susceptible vs. unsusceptible phenotypes. Bilateral intra-NAc infusions of BDNF enhanced susceptibility (Krishnan et al. 2007), whereas a knockdown of BDNF within the ventral tegmental area (VTA) was shown to prevent defeat-induced avoidance (Berton et al. 2006). These data are consistent with a model wherein susceptibility to a defeat-induced avoidant phenotype is caused by upregulation of VTA neuronal activity, which results in increased BDNF signaling within the NAc. The apparent discrepancy with our findings, showing a consistent avoidance induced in TrkB.T1 mice expressing diminished hippocampal BDNF levels, is resolved by taking into consideration the exquisite region-specificity of BDNF functional role in modulating depression-like behaviors (Wang et al. 2008). BDNF increases in hippocampus have been shown to attenuate depression-related phenotypes (Shirayama et al. 2002; Siuciak et al. 1997), whereas hippocampal TrkB.T1 overexpression or BDNF deletion may prevent the efficacy of antidepressant medications (Monteggia et al. 2004; Saarelainen et al. 2003). In agreement with the current results, increased hippocampal levels of BDNF have been recently linked to attenuated defeat-induced social avoidance in mice (Yan et al. 2010). The directionality of these effects and the related region-specificity are shared with other factors implicated in the phenomenology of depression (i.e. cAMP response element binding protein) (Nibuya et al. 1996; Pliakas et al. 2001). Together with the present data, these studies suggest a differential pathophysiology within the VTA-NAc than within the hippocampus.
The well-established consequences of defeat on rodent physiology were confirmed in the present study by persistently enlarged adrenal glands and decreased reproductive organ weight (Raab et al. 1985; Razzoli et al. 2010; Selye 1950; Van Kampen et al. 2002). Several abnormalities were also observed in TrkB.T1 internal organ anatomy, such as spleen and thymus that presented decreased sizes, whose mechanisms are unclear, given the CNS expression of the TrkB.T1 transgene. On the other hand, peripheral changes are observed following experimental disruptions of brain areas characterized by rich neurotrophic activity as in other preclinical models such as the olfactory bulbectomy (Connor et al. 2000; Komori et al. 2002). Similar peripheral alterations are also reported in clinically depressed patients (Weisse 1992), thus further corroborating the existence of a cross-talk between central and peripheral systems in stress-related disorders to explain the observed immunomodulatory effect observed in TrkB.T1 mice. It is noteworthy that BDNF is considered a stress-responsive intercellular messenger modifying hypothalamus–pituitary–adrenal axis activity, as suggested in depressed patients carriers of the Met/Met genotype (Derijk 2009; Schüle et al. 2006) and through its action on stress-related pathways it could exert a peripheral effect on immune function.
An imbalanced immune function due to TrkB.T1 transgene is also supported by the reported immune biomarker results. Only a limited number of the parameters were altered at the end of the experimental procedure, as IL-1α and RANTES levels were found to be decreased in mutant mice. Further studies should be aimed at assessing in a more dynamic fashion the immune system response of TrkB.T1 mice, or after subjecting them to systemic challenge, thus leading to a better understanding of the functional relevance of the highlighted immune alterations.
In conclusion, our results support a role of neurotrophin mediated signaling in contributing to the vulnerability to social stress; furthermore, a complex phenotype depending on the overexpression of TrkB.T1 receptor and the consequent decreased hippocampal BDNF has been highlighted jointly with peripheral alterations in parameters related to the metabolic and immune function that demand further investigation. In consideration of the involvement of BDNF/TrkB pathway in response to the currently available antidepressant treatments, such studies bear the potential to further advance the knowledge of the neuroplastic mechanisms linked to stress vulnerability and ultimately to the risk of psychiatric disorders and their comorbidities.