It is well known that both chronic stress and dysfunctional noradrenergic systems are causatively involved in the pathophysiology of major depressive disorder. Animal studies have revealed that prolonged exposure to various stressors resulted in the motivational, neuroendocrine, anhedonic, and behavioral characteristics observed in human depression (Brady 1994; Sapolsky 1996). On the other hand, deficiencies of noradrenergic transmission have been thought to be one factor for the etiology of depression (Schatzberg and Schildkraut 1995; Charney 1998). Furthermore, many lines of evidences have revealed that the interaction between chronic stress and the noradrenergic system may contribute to the development of depression. For example, animal studies have shown that the brain noradrenergic system is rapidly activated by different stressors (Korf et al. 1973; Anisman and Sklar 1979; Abercrombie and Jacobs 1987; Ritter et al. 1998), which results in an increase in norepinephrine (NE) release from noradrenergic terminals (Pacak et al. 1995; Smagin et al. 1997; Rosario and Abercrombie 1999), and this can lead to an overall reduction of brain NE levels (Weiss et al. 1980; Carboni et al. 2010). Nevertheless, the underlying mechanisms are far from being fully understood. Exploring the molecular links of the interaction between chronic stress and alteration of the noradrenergic system is worthwhile for elucidating the biological basis of depression and identifying new treatments.
Stress triggers multiple biological reactions in different organisms and systems including the release of several stress-related hormones. Among them, as the final effector of the hypothalamus–pituitary–adrenal (HPA) axis, glucocorticoids have been implicated in most of the reported stress-induced physiological changes in brains (McEwen, 1999) through their ubiquitously distributed intracellular receptors (Bamberger et al. 1996). It has been suggested that prolonged stress-induced hypersecretion of glucocorticoids may form part of the intrinsic mechanism underlying the development of depression (Carrasco and Van de Kar, 2003). Thus, understanding glucocorticoid-induced modulation on neural systems may further clarify the relationship between stress and depression.
Generally, the central noradrenergic system is one of the targets modulated by glucocorticoids (Dallman et al. 2006). The noradrenergic system acts as an arousal and alerting system to enhance organismic function and behaviors. Therefore, interaction between glucocorticoid and noradrenergic systems may play an important integrative function in coping and adaptation to stress. Both the NE transporter (NET) and dopamine β-hydroxylase (DBH, EC 220.127.116.11) are the important endophenotype of the noradrenergic system. NET has the primary function for reuptake of NE from presynaptic terminals of noradrenergic nerves, by which NE transmission is inactivated at the synapse (Barker and Blakely 1995). DBH is an enzyme that catalyzes the oxidation of dopamine to NE (Friedman and Kaufman 1965). Both NET and DBH play an essential role for maintaining the transformational homeostasis and normal functions of the noradrenergic system. Therefore, the modulation of glucocorticoids on the noradrenergic system may be mediated by affecting the expression of these two phenotypes.
In a previous study, we found that chronic social defeat (CSD) significantly increased mRNA and protein levels of the NET in the locus coeruleus (LC), hippocampus, frontal cortex, and amygdala. The latter three regions are the projection areas of the LC neurons. CSD-induced increases in NET expression were abolished by adrenalectomy or treatment with corticosteroid receptor antagonists, suggesting the involvement of corticosterone (CORT) and corticosteroid receptors in this up-regulation (Chen et al. 2012). Furthermore, CSD also increased the expression of DBH in the same brain regions (Fan et al. 2013). To further verify that CORT secreted during stressful events accounts for this effect, adult Fischer 344 rats were treated with a dose relevant to stress-induced plasma CORT concentration, and the expression of NET and DBH in the LC as well as the main NE terminal regions were examined. Meanwhile, two behavioral tasks, elevated T-maze and open-field, were performed to examine whether there is a parallel behavioral change after CORT treatment. This findings demonstrate that chronic treatment with CORT up-regulated expression of NET and DBH in the LC and its terminal regions, which was mediated through corticosteroid receptors. The expressional alteration of NET and DBH induced by CORT was accompanied by increased stressful behavior. These results are similar to those observed in the CSD model, and indicated that there is an integrative interaction between chronic stress, through CORT, and the noradrenergic system.
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- Material and methods
Our previous study demonstrated that chronic social defeat in rats up-regulated the expression of the noradrenergic phenotype in the LC and its terminal regions (Chen et al. 2012). In this study, we attempted to mimic the stress state through chronic administration of CORT to rats. Results showed that CORT ingestion significantly increased both mRNA and protein levels of NET/DBH in the rat LC. The same treatment also increased NET protein levels in LC projection regions such as the hippocampus, frontal cortex, and amygdala, as well as DBH protein levels in the hippocampus and amygdala. Elevated NET and DBH expression in most of these areas (except for NET protein levels in the LC) was abolished by treatment with combination of corticosteroid receptor antagonist mifepristone and spironolactone. Also, treatment with mifepristone alone prevented CORT-induced increases of NET expression and DBH protein levels in the LC. Furthermore, this CORT-induced expressional up-regulation of the noradrenergic phenotypes was accompanied by specific anxiety-like behavioral alterations, as demonstrated by inhibitory avoidance of the open arm and reduced center zone entries. Taken together, these studies confirmed that there is a functional linkage between chronic stress and the noradrenergic system through action of CORT on the expression of noradrenergic phenotypes.
Dysregulation in the function of the HPA axis activity and central noradrenergic system is a common feature of many stress-related mental disorders including major depression and anxiety disorders (Bunney and Davis 1965; Thomson and Craighead 2008). Animal studies have demonstrated that stress-induced activation of the LC-NE system (Abercrombie and Jacobs 1987) alters the release and metabolism of NE in the noradrenergic neuronal cell bodies and their terminal regions (Pacak et al. 1995; Smagin et al. 1997). However, whether an abnormal HPA axis, represented by hypercortisolemia, and a disturbed noradrenergic system has a causative relationship is not completely clear. Our previous studies demonstrated that chronic stress increased expression of NET (Chen et al. 2012) and DBH (Fan et al. 2013) in the LC and its main projection regions, indicating that chronic stress activated the noradrenergic system by action on noradrenergic phenotypes. To further clarify this relationship, this study was carried out through oral administration of CORT to mimic the chronic stress. Results revealed that CORT ingestion up-regulated NET and DBH expression in most central noradrenergic neurons (except for DBH protein levels in the frontal cortex, discussed below). The results not only confirmed the similarity between the CORT ingestion and the CSD models, but also lead us to postulate that elevated NET (increased reuptake of intracellular NE) and DBH (increase NE synthesis to compensate the stress-induced release of NE) induced by CORT ingestion may be a necessary response for integrating homeostasis, an important adaptive modification of the central NE system reacted to chronic stress (Goddard et al. 2010). It may be worth mentioning that tyrosine hydroxylase as a rate-limiting enzyme plays an important role for the biosynthesis of NE. However, DBH also is a key factor to determine the rate of NE synthesis (Kobayashi et al. 1994; Kim et al. 2002), as disruption of the DBH gene has been reported to block the synthesis of NE (Sabban 2007; Kvetnansky et al. 2008). This study demonstrated that CORT ingestion markedly increased mRNA and protein levels of DBH in the LC, and DBH protein levels in the hippocampus, and amygdala. These results indicate that in response to CORT exposure, up-regulated expression of DBH in the LC and some terminal regions may increase synthesis of NE. However, as NE concentration in the brain has not been measured in this study, this notion has to be verified by further experiments.
Stressful life events are potent factors that trigger, induce or exacerbate episodes of depression. During chronic stress, several hormones and systems are involved. Stress-induced release of glucocorticoids, as the final effector of the HPA axis, and subsequent activation of corticosteroid receptors in the brain play a crucial role in mounting the adaptive response to stress. Development of a model that produces a persistent, varied behavioral alteration would be a major advance for understanding the neurobiology of depression. To extend our previous work using CSD animal models, non-invasive CORT ingestion was performed to verify the previous observation. The results showed that oral administration of 100 μg/mL CORT for 3 weeks resulted in a blood concentration of CORT about 56 μg/dl, a level corresponding to the stress status found in animal studies (Sapolsky et al. 1995). Also, sucrose consumption, an analog of ‘anhedonia-like’ symptoms in stress paradigm, exhibited a very similar pattern to that in stress model rats (Chen et al. 2012), indicating that chronic non-invasive administration of CORT can mimic the CSD model to induce “anhedonia-like” depressive-like behavior. It is worth pointing out that while reduced sucrose consumption in the third week of CORT treatment was absolutely lower than that in the controls (p < 0.05), it did not reach the statistical significance when compared to the baseline before the treatment. Nevertheless, sucrose consumption in the chronic social defeat rat model showed a consistently decreased state throughout CSD (Chen et al. 2012). This discrepancy between the CORT ingestion and CSD regime may be accounted by the different experimental paradigm used in these two studies. In the previous study, a paradigm of 4 week stress with variable defeat session schedules was used for the purpose to avoid the habituation of rats to a regular schedule. In contrast, in this study, CORT was administrated daily. A habituation may occur during this period. The curve of the sucrose consumption can verify this phenomenon: the lowest sucrose consumption rate appeared in the first week of CORT ingestion, which seemed to be attenuated with time.
In this study, CORT ingestion significantly increased expression of NET in the LC region and its major terminal regions, as well as the expression of DBH in the most of these regions. However, no significant changes were found in DBH protein levels in the frontal cortex after CORT ingestion. In contrast, CSD regimen up-regulated DBH protein levels in all major terminal regions (Fan et al. 2013). Currently we do not have a satisfactory explanation for this discrepancy of DBH protein levels between this study and previous CSD experiment. While the technical factors related to measurement may be considered, the similar phenomenon has been reported previously. For example, chronic stress significantly reduced expression of brain-derived neurotrophic factors in the frontal cortex (Roceri et al. 2004; Mao et al. 2010; Li et al. 2012), whereas chronic administration of CORT had no significant effect on mRNA and protein levels of the same factor in the same brain region (Jacobsen and Mork 2006). One of the possibilities for such different effect on DBH expression between chronic stress and CORT treatment may be explained by the fact that chronic stress is involved by more hormones and signal transduction pathways (Kwon et al. 2007). Multiple hormones and their activated signal transduction pathways may produce relatively stronger effects on the DBH gene, which has several cis- and trans-regulatory elements in the prompter regions (Shaskus et al. 1992; Afar et al. 1996), than that caused by CORT treatment alone. In addition, given that DBH expression levels are lower in cortical regions (Schroeter et al. 2000), the effect from CORT ingestion on DBH in the frontal cortex may be smaller than other regions, although DBH expression there still shows a potential enhancement.
Regarding behavior, the corticosteroid receptor antagonists increased overall horizontal activity. Although there are no studies to show chronic effects of the combination of these antagonists on behavioral activity, one study has shown that an acute dose of mifepristone had no effect on behavioral activity (De Vries et al. 1996). These antagonists have been shown to alleviate motor impairments caused by stress and CORT treatment, whereas either antagonist alone produced no significant differences compared to controls (Jadavji et al. 2011). Studies using the immunohistochemical technique have shown the presence of glucocorticoid receptors in the motor cortex, basal ganglia, and cerebellum (Ahima & Harlan 1990, 1991), thus rendering these motor brain regions sensitive to the actions of glucocorticoid receptors. In contrast, mineralocorticoid receptors in the motor system appear to be restricted to cortical areas, but are present in the motor cortex (Roland et al. 1995). Therefore, blockade of both receptors appears to produce a synergistic effect on specific behavioral activity in the status of stress or CORT treatment. Regardless, the task dependent effects of the CORT antagonist cocktail emphasizes that these tasks measure different types of behaviors related to stressful events, but also highlight the complexity of behavioral testing. A recent review (Ramos 2008) nicely summarizes and emphasizes the importance of different behavioral tasks related to emotionality, anxiety, and stress to provide a comprehensive profile of the effects of stress on behavior.
CORT appears to have increased anxiety as measured on the elevated T-maze, which was not alleviated by the corticosteroid receptor antagonist cocktail. This may be because of the fact that the cocktail was given only once daily, but oral administration of CORT was continuing throughout the day over the entire three-week period. Thus, the antagonist cocktail did not sufficiently block the effects of CORT on anxiety as tested on the elevated T-maze. It was noteworthy that elevated T-maze tasks showed that either mifepristone or spironolactone in the absence of CORT ingestion resulted in a significant increase in Avoidance tests (Table 2), indicating that these antagonists alone may have potential anxiogenic effects. Although these results may explain the reason why the cocktail failed to reverse CORT-induced anxiety-like behavior (Table 3), they are in conflict to the available reports in the literature. It was reported that treatment with spironolactone or other mineralocorticoid receptor antagonists, either by infusion into the brain or systematic administration, exerted anxiolytic effect, but not for glucocorticoid receptor antagonists (Korte et al. 1995; Smythe et al. 1997; Bitran et al. 1998; Hlavacova et al. 2010). Other studies also showed that treatment with mifepristone had no any anxiogenic effects (Calfa et al. 2006; Auger and Forbes-Lorman 2008). Currently, we do not have a satisfactory explanation for such conflict. Different species of rats (Fischer 344 in this study verse Wistar or others in those reports) may account for these differences, as it is well established that individual rats exhibit marked differences in behavioral responses to a novel environment (Kabbaj et al. 2000). Also, another attributable factor for such conflict may be the difference in the treatment period: 21 days in this study verse 3–11 days in those reports. Since biochemical measurements in this study showed that these antagonist administered alone did not cause a significant alteration for expression of NET and DBH in the absence of CORT, these potential anxiogenic effects may not be related to these phenotypes in the brain. As the study to use antagonists alone to test their behavioral effects is limited, further studies are warranted to pursue this issue.
The reason of possible non-involvement of noradrenergic phenotypes in these antagonists-induced anxiogenic effects is not known, as even the degree that noradrenergic systems modulate behavioral state remained ambiguous. The LC-NE system is a critical component of the neural architecture. As such, it appears reasonable to propose that dysregulation of this system might contribute to dysregulation of a variety of cognitive and affective processes, resulting in specific behavioral alteration such as anxiety. However, the data in the literature show some difference. For example, rats with 6-hydroxydopamine-induced LC ablation did not show any signs of impairment in learning and performance of feat-motivated tasks (Mason and Fibiger 1979). Also, noradrenaline depleted rats were more reluctant to leave a familiar place and took longer to consume the food pellets in an unfamiliar place, suggesting an increase in fear following the lesion. Moreover, there was no difference in the behavioral parameters (elevated plus-maze, light–dark box and open-field test) between DBH (−/−) and DBH (+/−) mice (Cryan et al. 2001; Marino et al. 2005). Therefore, while we think the alteration of CORT-induced noradrenergic phenotype alterations possibly account for the specific behavioral changes in this study, there is another notion that the LC-NE system may be viewed as a general and global modulator of neuronal circuits that guide behavioral action (Itoi 2008; Itoi and Sugimoto 2010).
In summary, this study demonstrates that non-invasive CORT administration significantly up-regulated NET and DBH expression in the LC and its main terminal regions and it was mediated by corticosteroid receptors, a similar phenomenon to that of CSD regime as reported previously (Chen et al. 2012). In addition, the CORT ingestion not only induced anhedonia-like behavior but also anxiety behavioral alteration. Taken together with previous observations, the results of this study indicate that CORT plays a primary role in the chronic stress-induced activates the LC-NE system, which may be related to the pathophysiology of stress-precipitated psychiatric disorders.