Potential pleiotropic beneficial effects of adjuvant melatonergic treatment in posttraumatic stress disorder.

Loss of circadian rhythmicity fundamentally affects the neuroendocrine, immune, and autonomic system, similar to chronic stress and may play a central role in the development of stress-related disorders. Recent articles have focused on the role of sleep and circadian disruption in the pathophysiology of posttraumatic stress disorder (PTSD), suggesting that chronodisruption plays a causal role in PTSD development. Direct and indirect human and animal PTSD research suggests circadian system-linked neuroendocrine, immune, metabolic and autonomic dysregulation, linking circadian misalignment to PTSD pathophysiology. Recent experimental findings also support a specific role of the fundamental synchronizing pineal hormone melatonin in mechanisms of sleep, cognition and memory, metabolism, pain, neuroimmunomodulation, stress endocrinology and physiology, circadian gene expression, oxidative stress and epigenetics, all processes affected in PTSD. In the current paper, we review available literature underpinning a potentially beneficiary role of an add-on melatonergic treatment in PTSD pathophysiology and PTSD-related symptoms. The literature is presented as a narrative review, providing an overview on the most important and clinically relevant publications. We conclude that adjuvant melatonergic treatment could provide a potentially promising treatment strategy in the management of PTSD and especially PTSD-related syndromes and comorbidities. Rigorous preclinical and clinical studies are needed to validate this hypothesis.

GABA-independent GABAA receptor openings maintain tonic currents.

Activation of GABA(A) receptors (GABA(A)Rs) produces two forms of inhibition: phasic inhibition generated by the rapid, transient activation of synaptic GABA(A)Rs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of perisynaptic or extrasynaptic GABA(A)Rs, which can detect extracellular GABA. Such tonic GABA(A)R-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABA(A) receptor openings. This tonic GABA(A)R conductance is resistant to the competitive GABA(A)R antagonist SR95531 (gabazine), which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker, picrotoxin. When slices are perfused with 200 nm GABA, a concentration that is comparable to CSF concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABA(A)Rs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations.

A functional role for both -aminobutyric acid (GABA) transporter-1 and GABA transporter-3 in the modulation of extracellular GABA and GABAergic tonic conductances in the rat hippocampus.

Tonic γ-aminobutyric acid (GABA)A receptor-mediated signalling controls neuronal network excitability in the hippocampus. Although the extracellular concentration of GABA (e[GABA]) is critical in determining tonic conductances, knowledge on how e[GABA] is regulated by different GABA transporters (GATs) in vivo is limited. Therefore, we studied the role of GATs in the regulation of hippocampal e[GABA] using in vivo microdialysis in freely moving rats. Here we show that GAT-1, which is predominantly presynaptically located, is the major GABA transporter under baseline, quiescent conditions. Furthermore, a significant contribution of GAT-3 in regulating e[GABA] was revealed by administration of the GAT-3 inhibitor SNAP-5114 during simultaneous blockade of GAT-1 by NNC-711. Thus, the GABA transporting activity of GAT-3 (the expression of which is confined to astrocytes) is apparent under conditions in which GAT-1 is blocked. However, sustained neuronal activation by K(+)-induced depolarization caused a profound spillover of GABA into the extrasynaptic space and this increase in e[GABA] was significantly potentiated by sole blockade of GAT-3 (i.e. even when uptake of GAT-1 is intact). Furthermore, experiments using tetrodotoxin to block action potentials revealed that GAT-3 regulates extrasynaptic GABA levels from action potential-independent sources when GAT-1 is blocked. Importantly, changes in e[GABA] resulting from both GAT-1 and GAT-3 inhibition directly precipitate changes in tonic conductances in dentate granule cells as measured by whole-cell patch-clamp recording. Thus, astrocytic GAT-3 contributes to the regulation of e[GABA] in the hippocampus in vivo and may play an important role in controlling the excitability of hippocampal cells when network activity is increased.

Continuous Free Cortisol Profiles in Healthy Men.

CONTEXT: In humans, approximately 95% of circulating cortisol is bound to corticosteroid-binding globulin and albumin. It is only the free fraction that is biologically active and can activate signaling pathways via glucocorticoid hormone receptors in cells. Microdialysis is a well-established technique that enables the sampling of molecules in different compartments of the body, including extracellular fluid. This is the first study validating a rapid sampling microdialysis method measuring free cortisol in the subcutaneous and blood compartments of healthy volunteers. METHODS: Healthy nonsmoking volunteers (42 men, aged 18-24 years; body mass index 18-25 kg/m2) received placebo (saline), 250 µg Synacthen, or 1 mg dexamethasone with 10-minute sampling to measure total and free cortisol (subcutaneous, intravenous, and saliva) for an hour before and 4 hours after administration. RESULTS: Following stimulation by Synacthen, total serum cortisol and free cortisol in both compartments rose significantly, achieving and maintaining maximum levels between 2 and 3 hours following the stimulus. A decline in cortisol levels was evident after the administration of dexamethasone or placebo, but there was a clear pulsatile activity around lunchtime in the latter group, which was prominent in the blood compartment (total and free cortisol). There was good correlation between serum total and free cortisol (subcutaneous and intravenous) in the Synacthen and dexamethasone groups with no significant delay (less than 5 minutes) between total and free cortisol. CONCLUSIONS: This seminal study demonstrated the dynamic responses of total blood cortisol and microdialysis derived free cortisol in blood, subcutaneous tissue, and saliva in men.

Corticosterone levels in the brain show a distinct ultradian rhythm but a delayed response to forced swim stress.

Circulating corticosterone levels show an ultradian rhythm resulting from the pulsatile release of glucocorticoid hormone by the adrenal cortex. Because the pattern of hormone availability to corticosteroid receptors is of functional significance, it is important to determine whether there is also a pulsatile pattern of corticosterone concentration within target tissues such as the brain. Furthermore, it is unclear whether measurements of plasma corticosterone levels accurately reflect corticosterone levels in the brain. Given that the hippocampus is a principal site of glucocorticoid action, we investigated in male rats hippocampal extracellular corticosterone concentrations under baseline and stress conditions using rapid-sampling in vivo microdialysis. We found that hippocampal extracellular corticosterone concentrations show a distinct circadian and ultradian rhythm. The PULSAR algorithm revealed that the pulse frequency of hippocampal corticosterone is 1.03 +/- 0.07 pulses/h between 0900 and 1500 h and is significantly higher between 1500 and 2100 h (1.31 +/- 0.05). The hippocampal corticosterone response to stress is stressor dependent but resumes a normal ultradian pattern rapidly after the termination of the stress response. Similar observations were made in the caudate putamen. Importantly, simultaneous measurements of plasma and hippocampal glucocorticoid levels showed that under stress conditions corticosterone in the brain peaks 20 min later than in plasma but clears concurrently, resulting in a smaller exposure of the brain to stress-induced hormone than would be predicted by plasma hormone concentrations. These data are the first to demonstrate that the ultradian rhythm of corticosterone is maintained over the blood-brain barrier and that tissue responses cannot be reliably predicted from the measurement of plasma corticosterone levels.

Water temperature determines neurochemical and behavioural responses to forced swim stress: an in vivo microdialysis and biotelemetry study in rats.

Forced swimming is a behavioural stress model increasingly used to investigate the neurocircuitry of stress responses. Although forced swim stress clearly is a psychological stressor (anxiety, panic), its physical aspects are often neglected. There are indications that behavioural and neurochemical responses to swim stress depend on the water temperature. Thus, we investigated the responsiveness of hippocampal serotonergic neurotransmission (important in the coordination of stress responses), and of behaviour and core body temperature to forced swimming at different water temperatures (19, 25 and 35 degrees C). In vivo microdialysis and biotelemetry in freely-behaving rats were used. Dialysates were analysed for serotonin (5-HT) and its metabolite 5-HIAA (5-hydroxyindoleacetic acid) by HPLC with electrochemical detection. Forced swimming in water at 25 and 19 degrees C decreased core body temperature by 8 and 12 degrees C, respectively. A rapid and pronounced increase in hippocampal 5-HT and 5-HIAA was found in rats that swam at 35 degrees C, whereas biphasic responses in 5-HT and 5-HIAA were observed at 25 and 19 degrees C. Also swim stress behaviour and post-stress home cage behaviour depended on the water temperature. Comparing the serotonergic and core body temperature changes revealed that a combination of two different 5-HT and 5-HIAA responses seems to shape the neurotransmitter response. Swimming-induced increases in hippocampal extracellular concentrations of 5-HT and 5-HIAA occurred at all water temperatures, but these increases were temporarily quenched, or concentrations were transistently decreased, when core body temperature fell below 31 degrees C in water at 25 or 19 degrees C. These data demonstrate that water temperature is a key factor determining the impact of forced swim stress on behaviour and neurochemistry, and underscore that changes in these parameters should be interpreted in the light of the autonomic responses induced by this stressor.

Stress and the brain: solving the puzzle using microdialysis.

Aberrant functioning of the hypothalamic-pituitary-adrenocortical (HPA) axis seems to be involved in depression and anxiety. However, the mechanisms underlying the relationship between stress and mental illness are not completely resolved yet. The therapeutical efficacy of selective serotonin re-uptake inhibitors and benzodiazepines points to a key role of serotonin and gamma-aminobutyric acid (GABA) in depression and anxiety. Thus, it can be hypothesised that stress-induced changes in serotonin and GABA contribute to a dysregulation of the HPA axis and to the development of psychiatric disorders in susceptible subjects. It will, therefore, be crucial to increase our understanding of the effects of stress on serotonin and GABA. Various refinements have made in vivo microdialysis an extremely powerful method to study the highly dynamic neurotransmitter responses in stress physiology and behaviour. Furthermore, microdialysis can also be used to measure free corticosterone levels in the brain and, thus, HPA axis activity and neurotransmission can be monitored concomitantly. Here we review the effects of acute and chronic stress on serotonin and GABA, as assessed by microdialysis, in the hippocampus; a brain structure critically involved in the behavioural and neuroendocrine responses to stress. From the microdialysis data discussed, it can be concluded that both serotonin and GABA in the hippocampus are highly responsive to stress, but also that these responses are shaped by the exact nature of the stressor, i.e. the balance between the psychological and physical aspects of the stressful challenge.

The endogenous cannabinoid system affects energy balance via central orexigenic drive and peripheral lipogenesis.

The cannabinoid receptor type 1 (CB1) and its endogenous ligands, the endocannabinoids, are involved in the regulation of food intake. Here we show that the lack of CB1 in mice with a disrupted CB1 gene causes hypophagia and leanness. As compared with WT (CB1+/+) littermates, mice lacking CB1 (CB1-/-) exhibited reduced spontaneous caloric intake and, as a consequence of reduced total fat mass, decreased body weight. In young CB1-/- mice, the lean phenotype is predominantly caused by decreased caloric intake, whereas in adult CB1-/- mice, metabolic factors appear to contribute to the lean phenotype. No significant differences between genotypes were detected regarding locomotor activity, body temperature, or energy expenditure. Hypothalamic CB1 mRNA was found to be coexpressed with neuropeptides known to modulate food intake, such as corticotropin-releasing hormone (CRH), cocaine-amphetamine-regulated transcript (CART), melanin-concentrating hormone (MCH), and preproorexin, indicating a possible role for endocannabinoid receptors within central networks governing appetite. CB1-/- mice showed significantly increased CRH mRNA levels in the paraventricular nucleus and reduced CART mRNA levels in the dorsomedial and lateral hypothalamic areas. CB1 was also detected in epidydimal mouse adipocytes, and CB1-specific activation enhanced lipogenesis in primary adipocyte cultures. Our results indicate that the cannabinoid system is an essential endogenous regulator of energy homeostasis via central orexigenic as well as peripheral lipogenic mechanisms and might therefore represent a promising target to treat diseases characterized by impaired energy balance.

Voluntary exercise impacts on the rat hypothalamic-pituitary-adrenocortical axis mainly at the adrenal level.

INTRODUCTION: Evidence is accumulating that the regular performance of exercise is beneficial for stress coping. However, the hypothalamic-pituitary-adrenocortical (HPA) axis of voluntarily exercising rats has never been comprehensively investigated. METHODS: Therefore, male Sprague-Dawley rats were given access to a running wheel in their home cage for 4 weeks in which they ran 4-7 km per night. RESULTS: After 4 weeks, the exercising animals showed significantly less body weight gain, less abdominal fat tissue, decreased thymus weight, and increased adrenal weight (relative to body weight). Furthermore, tyrosine hydroxylase (TH) mRNA levels were selectively increased in the right adrenal medulla indicating an increase in sympathoadrenomedullary capacity in exercising rats. No changes were observed in paraventricular corticotropin-releasing hormone (CRH), arginine-vasopressin (AVP) and oxytocin mRNA levels. Mineralocorticoid receptor (MR) mRNA levels in hippocampus and glucocorticoid receptor (GR) mRNA levels in frontal cortex, parvocellular paraventricular nucleus and anterior pituitary were unchanged, whereas GR mRNA levels were increased in distinct hippocampal cell layers. Early morning baseline levels of plasma ACTH and corticosterone were similar in both groups. Interestingly, the response to different stressful stimuli (e.g. forced swimming, novelty) revealed that the exercising rats showed stressor-specific changes in HPA hormone responses. Forced swimming evoked a markedly enhanced response in corticosterone levels in the exercising rats. In contrast, if rats were exposed to a novel environment, exercising rats showed a much lower response in corticosterone than the control animals. However, the response in ACTH to either stressor was comparable between groups. Thus, in exercising rats physically demanding stressors evoke enhanced glucocorticoid responses whereas mild psychologically stressful stimuli such as novelty result in an attenuated glucocorticoid response. Interestingly, this attenuated hormone response corresponded with the observation that the exercising rats showed less anxious behaviour in the novelty situation. CONCLUSIONS: The differential responses in plasma corticosterone levels to different types of stress in the face of comparable responses in ACTH levels underscore the existence of critical regulatory control mechanisms at the level of the adrenal gland. We have hypothesized that changes in the sympathoadrenomedullary input may play an important role in these distinct glucocorticoid responses to stress. Our previous studies have shown similar changes in voluntarily exercising mice. Therefore, we conclude that the effects of exercise on the organism are not species-specific. Thus, our observations may have translational implications for the human situation.

Glucocorticoids, epigenetic control and stress resilience.

Glucocorticoid hormones play a pivotal role in the response to stressful challenges. The surge in glucocorticoid hormone secretion after stress needs to be tightly controlled with characteristics like peak height, curvature and duration depending on the nature and severity of the challenge. This is important as chronic hyper- or hypo-responses are detrimental to health due to increasing the risk for developing a stress-related mental disorder. Proper glucocorticoid responses to stress are critical for adaptation. Therefore, the tight control of baseline and stress-evoked glucocorticoid secretion are important constituents of an organism's resilience. Here, we address a number of mechanisms that illustrate the multitude and complexity of measures safeguarding the control of glucocorticoid function. These mechanisms include the control of mineralocorticoid (MR) and glucocorticoid receptor (GR) occupancy and concentration, the dynamic control of free glucocorticoid hormone availability by corticosteroid-binding globulin (CBG), and the control exerted by glucocorticoids at the signaling, epigenetic and genomic level on gene transcriptional responses to stress. We review the beneficial effects of regular exercise on HPA axis and sleep physiology, and cognitive and anxiety-related behavior. Furthermore, we describe that, possibly through changes in the GABAergic system, exercise reduces the impact of stress on a signaling pathway specifically in the dentate gyrus that is strongly implicated in the behavioral response to that stressor. These observations underline the impact of life style on stress resilience. Finally, we address how single nucleotide polymorphisms (SNPs) affecting glucocorticoid action can compromise stress resilience, which becomes most apparent under conditions of childhood abuse.

Association of Body Mass Index with Depression, Anxiety and Suicide-An Instrumental Variable Analysis of the HUNT Study.

OBJECTIVE: While high body mass index is associated with an increased risk of depression and anxiety, cumulative evidence indicates that it is a protective factor for suicide. The associations from conventional observational studies of body mass index with mental health outcomes are likely to be influenced by reverse causality or confounding by ill-health. In the present study, we investigated the associations between offspring body mass index and parental anxiety, depression and suicide in order to avoid problems with reverse causality and confounding by ill-health. METHODS: We used data from 32,457 mother-offspring and 27,753 father-offspring pairs from the Norwegian HUNT-study. Anxiety and depression were assessed using the Hospital Anxiety and Depression Scale and suicide death from national registers. Associations between offspring and own body mass index and symptoms of anxiety and depression and suicide mortality were estimated using logistic and Cox regression. Causal effect estimates were estimated with a two sample instrument variable approach using offspring body mass index as an instrument for parental body mass index. RESULTS: Both own and offspring body mass index were positively associated with depression, while the results did not indicate any substantial association between body mass index and anxiety. Although precision was low, suicide mortality was inversely associated with own body mass index and the results from the analysis using offspring body mass index supported these results. Adjusted odds ratios per standard deviation body mass index from the instrumental variable analysis were 1.22 (95% CI: 1.05, 1.43) for depression, 1.10 (95% CI: 0.95, 1.27) for anxiety, and the instrumental variable estimated hazard ratios for suicide was 0.69 (95% CI: 0.30, 1.63). CONCLUSION: The present study's results indicate that suicide mortality is inversely associated with body mass index. We also found support for a positive association between body mass index and depression, but not for anxiety.

Effects of long-term voluntary exercise on the mouse hypothalamic-pituitary-adrenocortical axis.

We studied the effects of long-term (i.e. 4 wk) voluntary exercise on the hypothalamic-pituitary-adrenocortical (HPA) axis in male mice. Voluntary exercise was provided by giving mice access to a running wheel, in which they indeed ran for about 4 km/d. Exercising mice showed similar body weights as control animals but presented less abdominal fat, lighter thymuses, and heavier adrenal glands. Exercise resulted in asymmetric structural changes in the adrenal glands. Whereas control mice had larger left than right adrenals, this condition was abolished in exercising animals, mainly because of enlargement of the right adrenal cortex. Tyrosine hydroxylase mRNA expression in the adrenal medullas of exercising mice was increased. In exercising mice, early-morning baseline plasma ACTH levels were decreased, whereas plasma corticosterone levels at the start of the dark phase were twice as high as those in control animals. To forced swimming and restraint stress, exercising mice responded with higher corticosterone levels than those of the control animals but with similar ACTH levels. However, if exposed to a novel environment, then exercising mice presented decreased ACTH responses. Interestingly, exercising mice showed a decreased corticosterone response to novelty only when the novel environment contained a functioning running wheel. Glucocorticoid receptor levels were unchanged, whereas mineralocorticoid receptor levels were decreased, in hippocampus of exercising animals. Corticotropin-releasing factor mRNA levels in the paraventricular nucleus were lower in exercising mice. Thus, voluntary exercise results in complex, adaptive changes at various levels within the HPA axis as well as in sympathoadrenomedullary and limbic/neocortical afferent control mechanisms. These changes seem to underlie the differential responsiveness of the HPA axis to physical vs. emotional challenges.

Effect of chronic administration of selective glucocorticoid receptor antagonists on the rat hypothalamic-pituitary-adrenocortical axis.

The effects of the selective glucocorticoid receptor (GR) antagonists ORG 34850, ORG 34116, and ORG 34517 on the rat hypothalamic-pituitary-adrenocortical (HPA) system were investigated. To assess the potency of the compounds to occupy GR in the brain and pituitary, we applied a single acute subcutaneous (s.c.) injection (10 mg/kg). ORG 34517 was most potent to occupy GR in the anterior pituitary and distinct brain areas, whereas all compounds were unable to occupy mineralocorticoid receptor (MR). Chronic administration of ORG 34850, ORG 34116, and ORG 34517 (20 mg/kg/day) for 1, 3, and 5 weeks resulted only in minor changes in brain GR levels. However, profound increases of hippocampal MR were observed virtually at all time points. Treatment with ORG 34850 and ORG 34116 elicited episodic increases in HPA axis activity, whereas ORG 34517 did not cause any changes in HPA activity. Thus, the GR antagonists exert distinct effects on the HPA axis, which may be pertinent for the proposed antidepressant activity of these compounds.

Altered serotonergic neurotransmission but normal hypothalamic-pituitary-adrenocortical axis activity in mice chronically treated with the corticotropin-releasing hormone receptor type 1 antagonist NBI 30775.

Antagonists of the corticotropin-releasing hormone receptor type 1 (CRH-R1) are regarded as promising tools for the treatment of stress-related psychiatric disorders. Owing to the intricate relationship between CRH and serotonin (5-HT), we studied the effects of chronic oral treatment of C57Bl6/N mice with the CRH-R1 antagonist NBI 30775 (formerly known as R121919) on hippocampal serotonergic neurotransmission during basal (on 15th day of treatment) and stress (forced swimming; on 16th day of treatment) conditions by in vivo microdialysis. Given the important role of CRH in the regulation of hypothalamic-pituitary-adrenocortical (HPA) axis activity and behavior, the effects of NBI 30775 on dialysate-free corticosterone levels, and on home cage and forced swimming-related behavior were also assessed. Chronic administration of NBI 30775 (18.4+/-0.9 mg/kg/day) did not result in alterations in food consumption and body weight. NBI 30775 caused complex changes in hippocampal serotonergic neurotransmission. Whereas no effects on the diurnal rhythms of 5-HT and its metabolite 5-hydroxyindoleacetic acid were found, the responses of the neurotransmitter and its metabolite to 10 min of forced swim stress were reduced and prolonged, respectively. NBI 30775 did not change free corticosterone levels over the diurnal rhythm. Moreover, NBI 30775-treated mice showed a similar forced swim stress-induced increase in corticosterone as observed in the control group. No effects of NBI 30775 on home cage, and swim stress-related active behaviors (climbing, swimming) and immobility were found. Thus, whereas chronic antagonism of CRH-R1 did not compromise HPA axis performance and behavior, distinct changes in serotonergic neurotransmission developed. Owing to the important role of 5-HT in the pathophysiology of mood and anxiety disorders, the latter observation may contribute to the therapeutical efficacy of CRH-R1 antagonists in these illnesses.

Circadian and ultradian rhythms of free glucocorticoid hormone are highly synchronized between the blood, the subcutaneous tissue, and the brain.

Total glucocorticoid hormone levels in plasma of various species, including humans, follow a circadian rhythm that is made up from an underlying series of hormone pulses. In blood most of the glucocorticoid is bound to corticosteroid-binding globulin and albumin, resulting in low levels of free hormone. Although only the free fraction is biologically active, surprisingly little is known about the rhythms of free glucocorticoid hormones. We used single-probe microdialysis to measure directly the free corticosterone levels in the blood of freely behaving rats. Free corticosterone in the blood shows a distinct circadian and ultradian rhythm with a pulse frequency of approximately one pulse per hour together with an increase in hormone levels and pulse height toward the active phase of the light/dark cycle. Similar rhythms were also evident in the subcutaneous tissue, demonstrating that free corticosterone rhythms are transferred from the blood into peripheral target tissues. Furthermore, in a dual-probe microdialysis study, we demonstrated that the circadian and ultradian rhythms of free corticosterone in the blood and the subcutaneous tissue were highly synchronized. Moreover, free corticosterone rhythms were also synchronous between the blood and the hippocampus. These data demonstrate for the first time an ultradian rhythm of free corticosterone in the blood that translates into synchronized rhythms of free glucocorticoid hormone in peripheral and central tissues. The maintenance of ultradian rhythms across tissue barriers in both the periphery and the brain has important implications for research into aberrant biological rhythms in disease and for the development of improved protocols for glucocorticoid therapy.

A rapid release of corticosteroid-binding globulin from the liver restrains the glucocorticoid hormone response to acute stress.

A strict control of glucocorticoid hormone responses to stress is essential for health. In blood, glucocorticoid hormones are for the largest part bound to corticosteroid-binding globulin (CBG), and just a minor fraction of hormone is free. Only free glucocorticoid hormone is able to exert biological effects, but little is known about its regulation during stress. We found, using a dual-probe in vivo microdialysis method, that in rats, the forced-swim stress-induced rise in free corticosterone (its major glucocorticoid hormone) is strikingly similar in the blood and in target compartments such as the subcutaneous tissue and the brain. However, in all compartments, the free corticosterone response was delayed by 20-30 min as compared with the total corticosterone response in the blood. We discovered that CBG is the key player in this delay. Swim stress evoked a fast (within 5 min) and profound rise in CBG protein and binding capacity in the blood through a release of the protein from the liver. Thus, the increase in circulating CBG levels after stress restrains the rise in free corticosterone concentrations for approximately 20 min in the face of mounting total hormone levels in the circulation. The stress-induced increase in CBG seems to be specific for moderate and strong stressors. Both restraint stress and forced swimming caused an increase in circulating CBG, whereas its levels were not affected by mild novelty stress. Our data uncover a new, highly dynamic role for CBG in the regulation of glucocorticoid hormone physiology after acute stress.

Distinct, time-dependent effects of voluntary exercise on circadian and ultradian rhythms and stress responses of free corticosterone in the rat hippocampus.

Previous work has shown that allowing rats to voluntarily exercise in a running wheel for 4 wk modifies the hypothalamic-pituitary-adrenal axis and behavioral coping responses to stress. To investigate whether long-term voluntary exercise would also affect the free, biologically active fraction of corticosterone in the brain, we conducted an in vivo microdialysis study in the hippocampus of rats. We monitored both the baseline circadian and ultradian patterns of corticosterone in hippocampus dialysates over the diurnal cycle and the responses to forced swim and novelty stress at different stages of exercise. Exercise for 1 d, 2 d, or 1 wk did not affect baseline circadian and ultradian pulse parameters or stress-induced hippocampal free corticosterone concentrations suggesting that acute or short-term periods of exercise do not affect baseline and stress-induced hormone levels. Baseline hormone parameters in 4 wk exercised rats, however, showed significantly increased pulse amplitudes (+108%) and mean free corticosterone levels (+42%) between 1500 and 2100 h but not between 0900 and 1500 h. Surprisingly, although our previous work showed substantial changes in stress-evoked plasma (total) corticosterone responses in long-term exercised animals, no differences in stress-induced hippocampal free hormone responses could be observed between exercised and sedentary animals. This lack of differences was not caused by compensatory changes in plasma corticosteroid-binding-globulin binding levels in exercising rats. Thus, long-term exercising rats show anticipatory increases in glucocorticoid output before the start of the active phase. These rats also reveal the putative existence of a containment mechanism preventing overexposure of the brain to glucocorticoid hormones.

Effects of exposure to a predator on behaviour and serotonergic neurotransmission in different brain regions of C57bl/6N mice.

Clinical studies and animal models have provided evidence that stress and serotonin may play a role in the aetiology of psychiatric diseases such as depression and anxiety. In addition, reciprocal interactions between stress and serotonergic neurotransmission have been demonstrated. However, the relationships between stress, serotonin and behaviour are far from completely understood. In this integrative study, we aimed to elucidate the effect of the psychological stress model predator exposure on behaviour and serotonergic neurotransmission in mice. We used a high time-resolution microdialysis method to measure extracellular levels of serotonin (5-hydroxytryptamine, 5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) in the hippocampus, prefrontal cortex, lateral septum and caudate putamen of C57BL/6N [corrected] mice, before (08:30-10:30 h), during (10:30-11:00 h) and after exposure (11:00-14:00 h) to a rat. Detailed behavioural observations were also made. Rat exposure resulted in behavioural activation, with predominant risk-assessment activities, and in increases in hippocampal, cortical, septal but not striatal 5-HT and 5-HIAA. When rat exposure was repeated on the consecutive day, small behavioural differences and reductions in 5-HIAA levels, but no differences in the 5-HT response, as compared with the first exposure were observed. As increases in 5-HT often coincide with behavioural activation, it was particularly interesting to find that 5-HT also increased in periods when mice only made minor movements such as sniffing, and that an effect of predator stress was absent in the caudate putamen. Our results indicate that the presence of the rat leads to differential activation of serotonergic neurotransmission in higher brain structures, probably involved in the coping response to this potentially life-threatening situation.

The selective glucocorticoid receptor antagonist ORG 34116 decreases immobility time in the forced swim test and affects cAMP-responsive element-binding protein phosphorylation in rat brain.

Glucocorticoid receptor (GR) antagonists can block the retention of the immobility response in the forced swimming test. Recently, we showed that forced swimming evokes a distinct spatiotemporal pattern of cAMP-responsive element-binding protein (CREB) phosphorylation in the dentate gyrus (DG) and neocortex. In the present study, we found that chronic treatment of rats with the selective GR antagonist ORG 34116 decreased the immobility time in the forced swim test, increased baseline levels of phosphorylated CREB (P-CREB) in the DG and neocortex and affected the forced swimming-induced changes in P-CREB levels in a time- and site-specific manner. Overall, we observed that, in control rats, forced swimming evoked increases in P-CREB levels in the DG and neocortex, whereas in ORG 34116-treated animals a major dephosphorylation of P-CREB was observed. These observations underscore an important role of GRs in the control of the phosphorylation state of CREB which seems to be of significance for the immobility response in the forced swim test and extend the molecular mechanism of action of GRs in the brain.

Differential monoaminergic, neuroendocrine and behavioural responses after central administration of corticotropin-releasing factor receptor type 1 and type 2 agonists.

Corticotropin-releasing factor (CRF) mediates various aspects of the stress response. To differentiate between the roles of CRF(1) and CRF(2) receptor subtypes in monoaminergic neurotransmission, hypothalamic-pituitary-adrenocortical axis activity and behaviour we compared the effects of CRF and urocortin 1 with those of the selective CRF(2) receptor ligands urocortin 2 and urocortin 3. In vivo microdialysis in the rat hippocampus was used to assess free corticosterone, extracellular levels of serotonin (5-HT) and noradrenaline (NA), and their metabolites 5-hydroxyindoleacetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG), respectively. Intracerebroventricular (i.c.v.) injection of CRF and urocortin 1, 2 and 3 (1.0 microg) increased hippocampal levels of 5-HT and 5-HIAA. CRF and urocortin 1 increased NA and MHPG, whereas urocortin 2 and urocortin 3 elevated MHPG, but not NA levels. CRF and the urocortins induced an immediate increase in behavioural activity. CRF and urocortin 1 mainly caused grooming and exploratory behaviour. In contrast, urocortin 2 and urocortin 3 both induced exploratory behaviour, but not grooming, and increased time spent eating food pellets. All urocortins, but not CRF, suppressed food intake 4-6 h after injection. Hippocampal free corticosterone levels were elevated by CRF, urocortin 1 and 3, but not by urocortin 2. The time courses of the CRF- and urocortin 1-induced responses were significantly prolonged as compared to those of the CRF(2) receptor ligands. The stimulatory changes evoked by CRF and urocortin 1 were present up to 4-6 h after injection, whereas the effects of urocortin 2 and urocortin 3 returned to baseline within 2.5 h after injection. Pre-treatment with the selective antagonist antisauvagine-30 (5.0 microg, i.c.v.) confirmed that the effects of urocortin 3 were CRF(2) receptor-mediated. The differential time course of the monoaminergic, neuroendocrine and behavioural effects of CRF and urocortin 1, as compared to urocortin 2 and urocortin 3, and the specific behavioural pattern induced by the CRF(2) receptor ligands, suggest a distinct role for CRF(2) receptors in the stress response.