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Chronic stress affects brain areas involved in learning and emotional responses. Although most studies have concentrated on the effect of stress on limbic-related brain structures, in this study we investigated whether chronic stress might induce impairments in diencephalic structures associated with limbic components of the stress response. Specifically, we analyzed the effect of chronic immobilization stress on the expression of sympathetic markers in the rat epithalamic pineal gland by immunohistochemistry and western blot, whereas the plasma melatonin concentration was determined by radioimmunoassay. We found that chronic stress decreased the expression of three sympathetic markers in the pineal gland, tyrosine hydroxylase, the p75 neurotrophin receptor and α-tubulin, while the same treatment did not affect the expression of the non-specific sympathetic markers Erk1 and Erk2, and glyceraldehyde-3-phosphate dehydrogenase. Furthermore, these results were correlated with a significant increase in plasma melatonin concentration in stressed rats when compared with control animals. Our findings indicate that stress may impair pineal sympathetic inputs, leading to an abnormal melatonin release that may contribute to environmental maladaptation. In addition, we propose that the pineal gland is a target of glucocorticoid damage during stress.
Several studies have shown that the hippocampus is involved in the regulation of the stress response via the hypothalamic-pituitary-adrenal (HPA) axis and is susceptible to stress-related damage evidenced, for example, in dendritic remodeling of CA3 pyramidal neurons and a decrease in adult neurogenesis in the dentate gyrus of rats (Magariños and McEwen 1995; McEwen and Chattarji 2004). Hippocampal volume reductions, which have been observed in patients with major depression, may be related to emotional and memory impairments (Sheline et al. 1996; Duman et al. 1999). More recently, it has been reported that, in addition to the hippocampus, the amygdala and prefrontal cortex in rats are also morphologically affected by stress (Vyas et al. 2002; Radley et al. 2004). These alterations may contribute to the cognitive deficits of major depression (Sapolsky 2001; McEwen and Chattarji 2004). In this study, we investigated whether other, non-telencephalic brain components could also be targets of stress-induced impairment. We chose a diencephalic component, the epithalamic pineal gland (PG), because this gland expresses a high density of the glucocorticoid receptor (Warembourg 1975; Meyer et al. 1998) and may be a target of stress-induced damage by glucocorticoids, the adrenal steroids secreted during stress (Sapolsky 2000).
The PG is a phototransducer neuroendocrine organ that plays a central role in the rhythmic secretion of melatonin, the night signal in all vertebrates (Simonneaux and Ribelayga 2003). Interactions between melatonin and the HPA system of the stress response have been demonstrated (Kellner et al. 1999). Melatonin receptors are present in brain areas that participate or are affected in the stress response, such as the adrenal glands and the hippocampus, whose activity is modulated by melatonin (Musshoff et al. 2002; Torres-Farfan et al. 2003). The PG receives photic information from the retina via the suprachiasmatic nucleus and the sympathetic neurons from the superior cervical ganglion (SCG), which, in this organ, is translated into hormonal information (Simonneaux and Ribelayga 2003). Rhythmic melatonin secretion from the PG has been related to important biological processes such as the modulation of neurotransmitter release, especially serotonin and dopamine (Simonneaux and Ribelayga 2003). In this line, melatonin has been associated with the regulation of cognitive and emotional processes such as memory and anxiety (Laudon et al. 1989; Boatright et al. 1994; Hemby et al. 2003).
In this context, we investigated the effects of chronic immobilization stress in rats on the expression of pineal sympathetic markers and plasma melatonin concentration, both of which were substantially modified.
- Top of page
- Materials and methods
In the present study, we analyzed the effect of chronic stress on pineal sympathetic markers and on plasma melatonin concentration in rats. Our results indicate that chronic stress decreased the expression of TH, p75(NTR) and α-tubulin (sympathetic markers), while the same treatment did not affect Erk1, Erk2 (non-specific sympathetic markers) and GAPDH expression (Figs 3 and 4). These results are compatible with sympathetic denervation of the PG. In addition, stress significantly increased plasma melatonin concentration during the dark phase (23.00 hours, p < 0.05) (Fig. 5). This may result from stimulation of pinealocyte β-receptors by the high levels of circulating norepinephrine (NE), which is secreted from the adrenal medullae during stress (Vogel and Jensh 1988).
Previous reports have shown that chronic immobilization stress increases dendritic arborization in stellate neurons of the basolateral amygdala, which could be the cellular substrate of the enhancement in anxiety-like behavior in the elevated plus-maze (Fig. 2a) (Vyas et al. 2002; McEwen and Chattarji 2004). The same stress treatment did not affect spontaneous motor activity (Fig. 1), indicating that poor performance in the elevated plus-maze is related to an increase in anxiety in stressed rats. In this line, some reports suggest that different types of maze and the inter-test interval may have an impact on behavioral performance (Bellani et al. 2006). Other reports suggest that in mice, the testing interval in various types of maze has no impact on performance (Paylor et al. 2006). We believe that testing motor activity in rats does not influence performance in the elevated plus-maze because the motor activity test is a non-aversive task in relation to other tests such as the water maze (Morris 1984). Conversely, if the elevated plus-maze test is performed before the motor activity test, it is possible that the plus-maze test affects the performance of the motor activity test, because the plus-maze has been reported to be anxiogenic (Rodgers et al. 1996; Ouagazzal et al. 1999). Moreover, chronic immobilization stress produced reduction in percentage body weight gain, significant adrenal hypertrophy and acute gastric lesions (Fig. 2b). These results are similar to those from previous reports using the same signs as stress markers (Magariños and McEwen 1995; Vyas et al. 2002).
Having established the efficacy of our stress regime, the novel and interesting observations of the present study came from our analyses of the effect of chronic immobilization stress on the expression of sympathetic markers in the rat PG and the plasma melatonin concentration. In this line, destruction of sympathetic terminals in the PG with 6-hydroxydopamine, or bilateral superior cervical ganglionectomy, does not block but rather, potentiates the increases in melatonin content and AA-NAT activity in the PG following physical immobilization in rats (Lynch et al. 1973, 1977). Conversely, stress-induced increase in pineal AA-NAT activity was blocked by bilateral adrenalectomy (Lynch et al. 1977). These findings suggest that synthesis of melatonin in the PG may be regulated both by the pineal sympathetic innervation and via the circulating NE secreted from the adrenal medulla. Stress increased the levels of circulating NE in rats (Vogel et al. 1988), and the stress-related high plasma melatonin concentration is probably produced through stimulation of pinealocyte β-receptors by the high levels of circulating NE. We believe that our immunohistochemical and western blot studies suggest that stress could induce sympathetic denervation of the PG. It is difficult to produce direct evidence that stress produces sympathetic denervation in the PG. Loss of nerve terminals could be assessed by measuring labeled NE uptake as a function of uptake blockers or alpha 2-adrenergic treatments. However, it is difficult to determine which cell types are in fact contributing to the differences in NE uptake because, in addition to the pineal sympathetic neurons, the phagocytes (Balter and Schwartz 1977; Cosentino et al. 1999), peptidergic neuron-like cells (Al-Damluji and Kopin 1996) and pineal neurons (Simonneaux and Ribelayga 2003) of the PG take up NE. Secondly, stress may affect the expression of pineal adrenergic receptors or the interaction between NE and the pineal adrenergic receptors. Finally, it is difficult to discriminate in these experiments whether or not stress is affecting NE metabolism in the pineal sympathetic axon terminals, for example, affecting monoamine oxidase expression which may induce an increase in NE release from the axon terminals. Another proposed method for providing functional evidence that stress produces pineal sympathetic denervation is to inject NE into the rat, which could stimulate the pineal gland if it is truly denervated, as indicated by melatonin production or arylalkylamine N-acetyltransferase activity. For this experiment, a new stressor would be applied when the NE was injected, possibly affecting the expression of several neuropeptides that regulate melatonin secretion in the brain or induce an increase of NE in the adrenal glands. Thus, this procedure might produce changes in melatonin secretion independently of sympathetic innervation.
In general, the regulation of the synthesis and secretion of melatonin in the PG is complex and not completely understood. In addition to the dense sympathetic innervation of the PG, other fibers originating from various central structures (especially the habenular nuclei, paraventricular nucleus, thalamic intergeniculate leaflet, dorsal raphe and lateral hypothalamus) innervate the rodent PG (Simonneaux and Ribelayga 2003). Therefore, various neural, endocrine and paracrine inputs participate in the regulation of melatonin synthesis and release. As the melatonin levels measured in the stress group at the other time points in the dark phase did not differ from the control, we propose that some of these inputs could inhibit the synthesis or release of melatonin at these time points, leading to a cancellation of the activating effects of circulating NE.
As mentioned above, immobilization stress induces functional impairment in the rat pinealocytes (Martinez et al. 1992; Milin et al. 1996; Milin 1998). In our view, the PG may be a target site for glucocorticoid damage during stress because, like other regions such as the hippocampus that are sensitive to stress, this gland expresses a high density of glucocorticoid receptor, which is also higher than other regions that are not affected by glucocorticoids, such as the intermediate and posterior lobes of the pituitary (Warembourg 1975; Meyer et al. 1998). Prolonged glucocorticoid secretion during chronic stress may have deleterious effects in the PG. Proposed mechanisms include inhibition of glucose transport, and a faster decline of ATP concentrations and metabolism in pinealocytes, similar to that proposed in hippocampal stress damage (Sapolsky 2000). Sympathetic denervation may be another of the deleterious effects of stress on the PG, that is normally highly irrigated and innervated by sympathetic neurons of the SCG. Chronic stress increases the synthesis and release of NE from the adrenal medulla and activates the sympathetic-adrenergic-noradrenergic system of the stress response (Vogel et al. 1988; Tafet and Bernardini 2003). The net effect of both changes may be an increase in NE concentration between sympathetic axon terminals and the NE receptor-expressing pinealocyte plasmatic membranes. We propose that the increase in NE surpasses the NE uptake capacity of the sympathetic axon terminal and the biodegradation capacity of NE through o-metilation catalyzed by catechol-o-methyltransferase in the PG, increasing the NE catechol group oxidation and free radical production, and favoring the retraction or neurodegeneration of sympathetic neurons. A similar process is found with l-dopa and dopamine (Dagnino-Subiabre et al. 2000a,b).
Impairments in the pineal sympathetic innervation and the rhythmic secretion of melatonin may, in turn, affect the information transmitted to brain areas that regulate the limbic-HPA and sympathetic-adrenergic-noradrenergic systems, because the hippocampus, an important regulator of both, has high levels of melatonin receptors (Musshoff et al. 2002). In vivo experiments of SCG ganglionectomy and pineal sympathetic denervation with 6-hydroxy-dopamine in mammals showed several effects, such as alterations in the normal photoperiodic control of reproduction and in the immune system (Cardinali et al. 1981a,b; Aldegunde et al. 1985; Magnusson and Kanje 1998). Different models of chronic stress induced similar effects associated with in vivo alterations of melatonin secretion (Lynch et al. 1977; Vollrath and Welker 1988; Persengiev et al. 1991). If the PG is denervated by chronic stress, the light and darkness information received by the retina may not reach other cells of the body because a key neuronal circuit that translates the neurochemical message of the photoperiod into a hormonal message is interrupted. Likewise, pineal denervation may have enormous biological implications, because rhythmic melatonin secretion participates in the modulation of several biological processes, such as the regulation of cellular metabolism by the circadian cycle (Simonneaux and Ribelayga 2003) and the release of neurotransmitters, e.g. serotonin and dopamine (Simonneaux and Ribelayga 2003). These changes may be a related to alterations in brain areas that are enriched in melatonin receptors and that are regulated by melatonin, like the hippocampus and amygdala (Miguez et al. 1991a,b; Musshoff et al. 2002).
There is evidence, especially at the clinical level, that stress induces depressive symptoms and is an important cause of the development of several depressive disorders, including major depression (Gold et al. 1988; Post 1992; Tafet and Bernardini 2003). Sleep disturbances such as insomnia (Jindal and Thase 2004), and reduced nocturnal peak of pineal melatonin secretion, are almost always present in depressed patients (Pacchierotti et al. 2001). It is possible that in major depression, decreased melatonin levels are a consequence of the low plasma NE concentration observed (Koenigsberg et al. 2005).
In conclusion, chronic stress specifically decreased sympathetic marker expression in the PG and increased circulating melatonin levels in rats. Both alterations may affect the capacity to process the emotional interpretation of external stimuli by the hippocampus and amygdala. Furthermore, this study proposes that the PG may be a target of glucocorticoid damage during stress, and that the stress-related alterations in rhythmic melatonin secretion may play a key role in the development of stress-related disorders. If this is true, medical treatment to achieve normal rhythmic melatonin secretion could become an effective approach to prevent the development and/or progression of some of the depressive symptoms in chronically stressed subjects.