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 1825 kg/m(2)) received placebo (saline), 250 mu g 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.

Context: The pituitary-adrenal axis had historically been considered a representative model for circadian rhythms. A recently developed portable collection device has provided the opportunity to evaluate free cortisol profiles using the microdialysis approach in individuals free to conduct their day-to-day activities in their own surroundings. Methods: Two separate experiments were conducted in healthy male volunteers. The total and subcutaneous (SC) free cortisol levels were measured at 10-minute intervals for a 24-hour period in one experiment, and the SC free cortisol levels were measured at 20-minute interval for 72 consecutive hours in free-living individuals in the second experiment. Results: The characteristic circadian rhythm was evident in both serum total and SC free cortisol, with the lowest levels achieved and maintained in the hours surrounding sleep onset and the peak levels occurring in every individual around waking. In all free-living individuals, the circadian rhythm was consistent across the 72-hour period, despite a wide range of activities. All the participants also showed increased cortisol after the consumption of lunch. The lowest levels during all 24-hour periods were observed during the hours after lights off, at the onset of sleep. Conclusions: To the best of our knowledge, the present study is the first to report up to three consecutive 24-hour measurements of SC free cortisol in healthy individuals. We believe our study is a landmark study that paves the way for ambulatory monitoring of free cortisol profiles continuously for a period of 72 hours in free-living individuals performing their day-to-day activities whether healthy or with diseases involving the hypothalamic-pituitary-adrenal axis.

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.

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.

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.

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.

Hormonal systems are major regulators of metabolic and cognitive function and many of these, including the critical stress-responsive hypothalamic-pituitary-adrenal (HPA) axis, release their constituent hormones in a circadian manner. This circadian rhythmicity is made up from an underlying approximate hourly ultradian rhythm. In order to understand the importance of both circadian and ultradian rhythms in man it is important to be able to carry out multiple sampling studies over extended periods in a subject's home setting, which is the most meaningful physiological setting for homeostatically important hormones. This study has developed a novel automated sampling system that, when used in combination with a microdialysis system, collects timed samples of microdialysis fluid over a full 24 h in individuals going about their normal everyday activity. The apparatus has the capacity to provide sufficient sample volumes to measure changes in hormone concentration over 24 h, including the important period when subjects are asleep.

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Key points The extracellular concentration of the neurotransmitter γ‐aminobutyric acid (GABA) is critical in determining GABAA receptor‐mediated tonic conductance in the hippocampus. Two GABA transporters (GAT‐1 and GAT‐3) are present in the CA3 and dentate gyrus of the hippocampus. The expression of GAT‐3 is confined to astrocytes and its role in the regulation of GABAergic neurotransmission is unclear. Using microdialysis and specific GAT uptake inhibitors we show that not only GAT‐1 but also GAT‐3 contributes to the regulation of hippocampal extracellular concentrations of GABA in rats under in vivo conditions. We further found that changes in extracellular concentrations of GABA resulting from both GAT‐1 and GAT‐3 inhibition precipitate supra‐additive changes in tonic conductance in dentate granule cells in vitro. These results help us to understand the mechanisms underlying the regulation of GABAergic tonic conductance in the hippocampus and can help to develop improved therapeutic strategies for neurological and psychiatric disorders. Abstract 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.

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.

In the adult cerebral cortex, GABA transporters (GATs) are expressed by both neurons and astrocytes. GAT-1 immunoreactivity is found in axon terminals of GABAergic neurons and astrocytes, while GAT-3 immunolabeling occurs only in the latter. The present study was designed to determine whether the expression of GAT-1 and GAT-3 in the adult rat cerebrum changes after needle lesion and colchicine infusion. Following a needle puncture or a saline injection, immunolabeling for GAT-1 and GAT-3 was slightly increased in an area around the needle track. Not only was the neuropil labeling for both GATs increased, but also a few neuronal somata were found to be immunoreactive for GAT-1. Colchicine injections induced a striking increase in immunolabeling for both GATs in the neuropil in an area adjacent to the needle path and surrounding it. A homologous region of the contralateral hemisphere also showed a moderate increase of immunoreactivity in the neuropil for both GATs. Furthermore, this contralateral site showed many neuronal somata immunolabeled for GAT-1. These changes were mainly detected during the first 5 days following intracortical lesions. These results indicate that (1) the upregulation of GAT-1 and GAT-3 in cortical interneurons and astrocytes is caused by both mechanical and chemical factors associated with the injections; (2) increased GAT-1 and GAT-3 expression contralateral to the site of colchicine injection is mediated by transcellular signaling across the corpus callosum; and (3) the lesion-induced GAT expression may play a protective role by helping to balance excitatory and inhibitory neuronal activities.

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.

Varenicline, the most successful smoking cessation aid, is a selective partial agonists at α4β2* nicotinic receptors. Its efficacy is likely to be shared by other drugs with similar receptor action, including cytisine. The present study aimed to characterize behavioral effects of cytisine compared with nicotine using locomotor activity tests, intracranial self-stimulation of ventral tegmental area (discrete-trial threshold current intensity titration procedure), drug discrimination (0.6 mg/kg nicotine from vehicle), physical dependence (osmotic minipumps delivering 6 mg/kg/day of nicotine) and intravenous nicotine self-administration (0.01 mg/kg per infusion) in adult Wistar rats. Cytisine (1–3 mg/kg) partially substituted for nicotine and at the highest dose tended to antagonize nicotine's discriminative stimulus effects. Nicotine (0.05–0.4 mg/kg), but not cytisine (0.3–3 mg/kg), lowered ICSS thresholds and cytisine dose-dependently reversed effects of nicotine. Nicotine (0.15–0.6 mg/kg), but not cytisine (0.3–3 mg/kg), stimulated locomotor activity and cytisine (3 mg/kg) fully reversed these effects of nicotine. Acute pretreatment with nicotine (0.15–0.6 mg/kg), but not cytisine (0.3–3 mg/kg), reinstated extinguished nicotine self-administration. Continuous infusion of nicotine induced physical dependence, as indicated by reduced rates of food-reinforced responding induced by a challenge dose of mecamylamine. At the highest tested dose (3 mg/kg), cytisine tended to reduce response rates irrespective of whether the rats were continuously exposed to nicotine or saline. Cytisine behaves like a weak partial agonist, mimicking effects of nicotine to a limited degree. Although cytisine reversed several effects of nicotine, it seemed to have a reduced potential to produce withdrawal signs in nicotine-dependent subjects.

Levels of stress perceived by people in our modern society seem to have risen considerably, a phenomenon which may be related to the increased incidence of stress-related mental health problems such as depression and anxiety disorders. Whereas the body is well equipped to deal with single, acute stressful challenges, regulatory systems may derail when stress is of a repeated or prolonged nature. Two of the systems that appear to be malfunctioning in psychiatric disease are the hypothalamic-pituitary-adrenocortical (HPA) axis (i.e., the stress hormone axis) and the serotonin (5-HT) system. This chapter will focus on the effects of stress on serotonergic neurotransmission. We will first discuss how stress affects the activity of the serotonergic cell bodies in the rostral (midbrain/pontine) raphe nuclei (the dorsal and median raphe nucleus), followed by an overview of the effects of stress on 5-HT synthesis. The dorsal and median raphe nuclei project to major limbic forebrain structures involved in the coordination of the stress response, such as the frontal cortex, hippocampus, amygdala and hypothalamus. Therefore, we will subsequently highlight the effects of stress on the extracellular levels of 5-HT in these structures as assessed by in vivo microdialysis. Next, the (aberrant) functioning of serotonergic neurotransmission in animal models with changes in HPA axis regulation will be discussed, followed by an excursion into the beneficial effects of moderate exercise on stress coping mechanisms.The reviewed data clearly demonstrate that the neurotransmitter 5-HT is not only highly responsive to acute stress; its responses are also intricately coordinated in a stressor-dependent and brain region-specific manner. This notion holds true for the regulation of the firing rate of serotonergic neurons, the activity and expression of tryptophan hydroxylase (the rate-limiting enzyme in the synthesis of 5-HT) and the extracellular levels of 5-HT in terminal regions. Furthermore, these data show that the 5-HT system can become compromised during situations of aberrant functioning of the HPA axis for example, due to changes in the corticotropin-releasing factor system or in corticosteroid receptor function. Most data presented here are obtained during and following acute stressful challenges. This information is of great relevance for our understanding of the relationship between stress and 5-HT. However, there is unfortunately only very limited information available on the effects of repeated and chronic stress. With respect to mental health disease (and animal welfare), it will therefore be essential to make the next step and increase our knowledge on how prolonged stress may perturb serotonergic neurotransmission. Finally, as stress will not suddenly 'disappear' from our society, and the costs of stress-related disease are burdening the health systems of many countries, it is important to study the mechanisms underlying adequate coping with stress and stress resilience, and to find ways to improve and support such mechanisms - for example, by exercise.

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.