TMEM132D, a new candidate for anxiety phenotypes: evidence from human and mouse studies.

The lifetime prevalence of panic disorder (PD) is up to 4% worldwide and there is substantial evidence that genetic factors contribute to the development of PD. Single-nucleotide polymorphisms (SNPs) in TMEM132D, identified in a whole-genome association study (GWAS), were found to be associated with PD in three independent samples, with a two-SNP haplotype associated in each of three samples in the same direction, and with a P-value of 1.2e-7 in the combined sample (909 cases and 915 controls). Independent SNPs in this gene were also associated with the severity of anxiety symptoms in patients affected by PD or panic attacks as well as in patients suffering from unipolar depression. Risk genotypes for PD were associated with higher TMEM132D mRNA expression levels in the frontal cortex. In parallel, using a mouse model of extremes in trait anxiety, we could further show that anxiety-related behavior was positively correlated with Tmem132d mRNA expression in the anterior cingulate cortex, central to the processing of anxiety/fear-related stimuli, and that in this animal model a Tmem132d SNP is associated with anxiety-related behavior in an F2 panel. TMEM132D may thus be an important new candidate gene for PD as well as more generally for anxiety-related behavior.

Long-term anxiolytic and antidepressant-like behavioural effects of tiagabine, a selective GABA transporter-1 (GAT-1) inhibitor, coincide with a decrease in HPA system activity in C57BL/6 mice.

Gamma-aminobutyric acid (GABA) system plays a pivotal role in the pathophysiology of anxiety and mood disorders. This study was aimed to assess the anxiolytic and antidepressant-like properties of tiagabine, an inhibitor of the GABA transporter-1 (GAT-1), after acute and chronic administration in C57BL/6JOlaHsD mice with paroxetine as a positive control. In first experiments, the acute administration of tiagabine (7.5 mg/kg, orally [PO]) and paroxetine (10 mg/kg PO) induced anxiolytic effects in the elevated plus maze test and the modified hole board test and an antidepressant-like effect in the forced swim test. Chronic application of tiagabine (7.5 mg/kg PO) and paroxetine (10 mg/kg PO) for 22 days revealed an anxiolytic and antidepressant-like efficacy of tiagabine only. In a further experiment, we analysed the impact of chronic tiagabine versus paroxetine treatment on the hypothalamic-pituitary-adrenocortical (HPA) system regulation. GAT-1 blockade induced a setpoint-shift of the stress hormone system toward lower levels as indicated by decreased plasma corticosterone concentrations and attenuated gene expression levels of corticotropin-releasing factor in the paraventricular nucleus of the hypothalamus and of hippocampal steroid receptors. This data indicate that both acute and long-term anxiolytic and antidepressant-like properties of brain GAT-1 inhibition coincide with a reduction in HPA system activity in mice.

The GABA transporter 1 (SLC6A1): a novel candidate gene for anxiety disorders.

Recent evidence suggests that the GABA transporter 1 (GAT-1; SLC6A1) plays a role in the pathophysiology and treatment of anxiety disorders. In order to understand the impact of genetic variation within SLC6A1 on pathological anxiety, we performed a case-control association study with anxiety disorder patients with and without syndromal panic attacks. Using the method of sequential addition of cases, we found that polymorphisms in the 5' flanking region of SLC6A1 are highly associated with anxiety disorders when considering the severity of syndromal panic attacks as phenotype covariate. Analysing the effect size of the association, we observed a constant increase in the odds ratio for disease susceptibility with an increase in panic severity (OR approximately 2.5 in severely affected patients). Nominally significant association effects were observed considering the entire patient sample. These data indicate a high load of genetic variance within SLC6A1 on pathological anxiety and highlight GAT-1 as a promising target for treatment of anxiety disorders with panic symptoms.

Consequences of chronic social stress on behaviour and vasopressin gene expression in the PVN of DBA/2OlaHsd mice--influence of treatment with the CRHR1-antagonist R121919/NBI 30775.

Accumulating evidence suggests that corticotropin-releasing hormone (CRH) neurocircuitry modulate the neuroendocrine and behavioural phenotypes in depression and anxiety. Thus, the administration of the selective CRH-receptor 1 (CRHR1)-antagonist R121919/NBI 30775 has proven its ability to act as an anxiolytic in rats. It is still unclear whether vasopressinergic neuronal circuits, which are known to be involved in the regulation of emotionality, are affected by R121919/NBI 30775. Using DBA/2OlaHsd mice, we investigated the effects of chronic social defeat and concomitant treatment with R121919/NBI 30775 on 1) the behavioural profile in the modified hole board test and 2) in-situ hybridization analysis-based expression of arginine vasopressin (AVP) and CRH mRNA in both the hypothalamic paraventricular nucleus and supraoptic nucleus. The results suggest that chronic social defeat leads to increased avoidance behaviour and reduction in directed exploration, general exploration, and locomotion. Chronic treatment with the CRHR1-antagonist was effective in reversing the directed exploration to control level. The dissection of the antagonist-treated group into responders and non-responders using the parameter time spent on board revealed further positive effects of R121919/NBI 30775 on avoidance behaviour and locomotion. Behavioural changes were accompanied by alterations in AVP gene expression in the paraventricular nucleus. Taken together, the anxiolytic action of the CRHR1 antagonist was found in a subgroup of animals only, and further studies have to be done to clarify the inter-individual biological differences in response patterns to this compound to optimise its application under clinical conditions.

Corticotropin-releasing factor, vasopressin and receptor systems in depression and anxiety.

Affective disorders tend to be chronic and life-threatening diseases: suicide is estimated to be the cause of death in 10-15% of individuals with major depressive disorders. Major depression is one of the most prevalent and costly brain diseases with up to 20% of the worldwide population suffering from moderate to severe forms of the disease. Only 50% of individuals with depression show full remission in response to currently available antidepressant drug therapies which are based on serendipitous discoveries made in the 1950s. Previously underestimated, other severe depression-associated deleterious health-related effects have increasingly been recognized. Epidemiological studies have provided substantial evidence that patients with depression have a 2-4-fold increased risk both of developing cardiovascular disease and of mortality after experiencing a myocardial infarction. The majority of patients suffering from affective disorders have measurable shifts in their stress hormone regulation as reflected by elevated secretion of central and peripheral stress hormones or by altered hormonal responses to neuroendocrine challenge tests. In recent years, these alterations have increasingly been translated into testable hypotheses addressing the pathogenesis of illness. Refined molecular technologies and the creation of genetically engineered mice have allowed to specifically target individual genes involved in regulation of corticotropin releasing factor (CRF) and vasopressin (AVP) system elements. The cumulative evidence makes a strong case implicating dysfunction of these systems in the etiology and pathogenesis of depression and pathological anxiety. Translation of these advances into novel therapeutic strategies has already been started.

Endocrine disturbances in depression.

Depression is one of the most common psychiatric disorders. For a long time, clinicians suspected a causal link between depression and the endocrine system. The most frequently occurring endocrine abnormality in depressed subjects is hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. CRH and AVP are likely to play a substantial role in the pathophysiology of this disorder, and their receptors appear to be a specific target for future antidepressant drugs. Depression also affects the hypothalamic-pituitary-GH (HPGH) and -thyroid (HPT) axes. Alterations in the reproductive system may also play a role in the pathology of depression. In addition, there is increasing evidence that leptin and neurosteroids, such as DHEA, are implicated in mood disorders.

Mutagenesis and knockout models: hypothalamic-pituitary-adrenocortical system.

Hyperactivity of central neuropeptidergic circuits such as the corticotropin-releasing hormone (CRH) and vasopressin (AVP) neuronal systems is thought to play a causal role in the etiology and symptomatology of anxiety disorders. Indeed, there is increasing evidence from basic science that chronic stress-induced perturbation of CRH and AVP neurocircuitries may contribute to abnormal neuronal communication in conditions of pathological anxiety. Anxiety disorders aggregate in families, and accumulating evidence supports the notion that the major source of familial risk is genetic. In this context, refined molecular technologies and the creation of genetically engineered mice have allowed us to specifically target individual genes involved in the regulation of the elements of the CRH (e.g., CRH peptides, CRH-related peptides, their receptors, binding protein). During the past few years, studies performed in such mice have complemented and extended our knowledge. The cumulative evidence makes a strong case implicating dysfunction of CRH-related systems in the pathogenesis of anxiety disorders and depression and leads us beyond the monoaminergic synapse in search of eagerly anticipated strategies to discover and develop better therapies.

Challenge studies in anxiety disorders.

In psychiatry, the use of pharmacological challenges in panic disorder is unique in that the clinical phenomenon of central interest (i.e., the panic attack) can be provoked readily and assessed in the clinical laboratory setting. During the past 20 years pharmacological challenge studies have increased our knowledge concerning the neurobiology of panic disorder remarkably and may ultimately result in novel and more causal treatment strategies. Moreover, the differences in sensitivity to certain panicogens such as serotonergic agents, lactate, carbon dioxide and cholecystokinin tetrapeptide are likely to be fruitful in serving as biological markers of subtypes of panic disorders and should be a major focus of research, as the identification of reliable endophenotypes is currently one of the major rate-limiting steps in psychiatric genetic studies.

Repetitive transcranial magnetic stimulation increases the release of dopamine in the nucleus accumbens shell of morphine-sensitized rats during abstinence.

Recent studies in rodents have shown that withdrawal from chronic drug abuse is associated with a significant decrease in dopamine (DA) release in mesolimbic structures, especially in the shell region of the nucleus accumbens. Since the DA system is known to play an important role in reward processes, a withdrawal-associated impairment in mesolimbic DA-mediated transmission could possibly implicate reward deficit and thus enhance vulnerability to drug craving and relapse. We have previously demonstrated that acute repetitive transcranial magnetic stimulation (rTMS) has a modulatory effect on DA release in several areas of the rat brain, including dorsal striatum, hippocampus, and nucleus accumbens shell. In the present study, we investigated the possible use of rTMS as a tool in re-establishing the dysregulated DA secretion observed during withdrawal in morphine-sensitized male Sprague-Dawley rats. Using intracerebral microdialysis, we monitored the effects of acute rTMS (20 Hz) on the intra-accumbal release-patterns of DA in freely moving animals that were subjected to a morphine sensitization scheme for a period of 8 days. We provide first evidence that acute rTMS (20 Hz) is able to increase DA concentration in the shell region of the nucleus accumbens in both control animals and morphine-sensitized rats during abstinence. The DA release in morphine-sensitized rats was significantly higher than in controls. rTMS, therefore, might gain a potential therapeutic role in the treatment of dysphoric and anhedonic states during drug withdrawal in humans.

The high-affinity non-peptide CRH1 receptor antagonist R121919 attenuates stress-induced alterations in plasma oxytocin, prolactin, and testosterone secretion in rats.

Evidence from basic and clinical research suggests that hyperactivity of central corticotropin-releasing hormone (CRH) circuits contributes to causality and course of affective disorders. Therefore, CRH receptor antagonists have attracted attention as potential therapeutics. We could previously show that the novel high-affinity non-peptide CRH 1 receptor antagonist R121919 significantly inhibits stress-induced corticotropin release and displays anxiolytic effects in rats selectively bred for high anxiety-related behavior. These animals are characterized by their innate hyper-reactivity of the hypothalamic-pituitary-adrenocortical system linked to an increased emotionality and therefore are suitable for the evaluation of CRH 1 receptor antagonists. Here we show that in addition to its effects on anxiety-related behavior and corticotropin secretion, R121919 attenuates the stress-induced release of corticosterone, prolactin, and oxytocin. Moreover, the decrease in plasma testosterone following exposure to stress is abolished by R121919. Our data indicate that antagonism of CRH 1 receptors may prevent stress-associated endocrine alterations.

Repetitive transcranial magnetic stimulation increases the release of dopamine in the mesolimbic and mesostriatal system.

Repetitive transcranial magnetic stimulation (rTMS) is suggested to be a potentially useful treatment in major depression. In order to optimize rTMS for therapeutic use, it is necessary to understand the neurobiological mechanisms involved, particularly the nature of the neurochemical changes induced. Using intracerebral microdialysis in urethane-anesthetized and conscious adult male Wistar rats, we monitored the effects of acute rTMS (20 Hz) on the intrahippocampal, intraaccumbal and intrastriatal release patterns of dopamine and its metabolites (homovanillic acid, 3,4-dihydroxyphenylacetic acid). The stimulation parameters were adjusted according to the results of accurate MRI-based computer-assisted reconstructions of the current density distributions induced by rTMS in the rat brain, ensuring stimulation of frontal brain regions. In the dorsal hippocampus, the shell of the nucleus accumbens and the dorsal striatum the extracellular concentration of dopamine was significantly elevated in response to rTMS. Taken together, these data provide the first in vivo evidence that acute rTMS of frontal brain regions has a modulatory effect on both the mesolimbic and the mesostriatal dopaminergic systems. This increase in dopaminergic neurotransmission may contribute to the beneficial effects of rTMS in the treatment of affective disorders and Parkinson's disease.

Transcranial magnetic stimulation as a therapeutic tool in psychiatry: what do we know about the neurobiological mechanisms?

Potential therapeutic properties of repetitive transcranial magnetic stimulation (rTMS) have been suggested in several psychiatric disorders such as depression, mania, obsessive-compulsive disorder, posttraumatic stress disorder and schizophrenia. By inducing electric currents in brain tissue via a time-varying strong magnetic field, rTMS has the potential to either directly or trans-synaptically modulate neuronal circuits thought to be dysfunctional in these psychiatric disorders. However, in order to optimize rTMS for therapeutic use, it is necessary to understand the neurobiological mechanisms involved, particularly the nature of the changes induced and the brain regions affected. Compared to the growing number of clinical studies on its putative therapeutic properties, the studies on the basic mechanisms of rTMS are surprisingly scarce. rTMS currently still awaits clinical routine administration although,there is compelling evidence that it causes changes in neuronal circuits as reflected by behavioural changes and decreases in the activity of the hypothalamic-pituitary-adrenocortical system. Both alterations suggest regional changes in neurotransmitter/neuromodulator release, transsynaptic efficiency, signaling pathways and in gene transcription. Together, these changes are, in part, reminiscent of those accompanying antidepressant drugs.

Expression of CRHR1 and CRHR2 in mouse pituitary and adrenal gland: implications for HPA system regulation.

Deficiency of corticotropin-releasing hormone receptor I (CRHR1) reduces anxiety-related behavior in mice and severely impairs the stress response of the hypothalamic-pituitary-adrenocortical (HPA) system. Most recently, we could show that severe emotional stressors induce a significant rise in plasma ACTH even in mice deficient for the CRHR1 (Crhr1-1-) which is, however, not accompanied by an increase in plasma corticosterone concentration, suggesting that CRHR1 might be directly involved in the regulation of adrenal corticosterone release. We therefore used the Crhr1-1- mouse model to clarify the potential role of adrenal CRHR1 in the regulation of the HPA system and, in particular, of corticosterone secretion. In Crhr1-/- mice, intravenous ACTH administration failed to stimulate corticosterone secretion despite a significant upregulation of ACTH receptor mRNA levels in the adrenal cortex of these mutants. Further, by means of RT-PCR and in situ hybridization analyses, we could provide first evidence that both CRHR1 and CRHR2 are expressed in the mouse pituitary and adrenal cortex. Stimulation of pituitary CRHR2 does not induce ACTH secretion either in vitro or in vivo. Our data strongly suggest that CRHR1 plays a crucial role in the release of corticosterone from the adrenal cortex, independently of pituitary function. The existence of an intra-adrenal CRH/CRHR1 regulatory system which contributes to the corticosteroid secretory activity adds to the complexity of HPA system regulation and stress hormone homeostasis.

Differential analysis of behavior and diazepam-induced alterations in C57BL/6N and BALB/c mice using the modified hole board test.

A variety of test procedures are used in preclinical research on behavioral pharmacology and to dissociate behavioral differences or pharmacologically induced behavioral alterations several independent tests are usually performed. In the present study we introduce a modified hole board procedure for mice which allows us to investigate a variety of behavioral parameters such as anxiety, risk assessment, exploration, locomotion, food-intake inhibition, novelty seeking, and arousal by using only one test. The modified hole board was established by investigating the behavior of two inbred mouse strains, C57BL/6 and BALB. Significant differences in terms of locomotor activity, general exploration, and other parameters were found. Moreover, strain-specific exploration strategies could be detected in the modified hole board. Further, the test was validated by investigating the effects of diazepam as standard anxiolytic on the behavior in both mouse strains. Acute administration of diazepam (1 and 3 mg/kg) induced strong sedative effects in a dose-dependent manner in C57BL/6 mice. In BALB mice, the lower dosage of diazepam showed an activating and anxiolytic action while the 3 mg dosage revealed a slight sedative but still anxiolytic effect in these animals. Taken together, the results demonstrate that the modified hole board enables to differentially investigate behavioral phenotypes and also pharmacologically-induced behavioral alterations in mice. Therefore, this new strategy allows to reduce the number of experimental animals and the time needed, thus, representing an effective screening-tool for behavioral investigations.

Hyperactivity of CRH neuronal circuits as a target for therapeutic interventions in affective disorders.

Increasing evidence suggests that the neuroendocrine changes seen in psychiatric patients, especially in those suffering from affective disorders, may be causally related to the psychopathology and course of these clinical conditions. The most robustly confirmed neuroendocrine finding among psychiatric patients with affective disorders is hyperactivity of the hypothalamic-pituitary-adrenocortical (HPA) system, resulting from hyperactive hypothalamic corticotropin-releasing hormone (CRH) neurons. A large body of preclinical and clinical evidence suggests that both genetic and environmental factors contribute to the development of these HPA system abnormalities. Further, normalization of HPA system regulation was shown to be a prerequisite for favorable treatment response and stable remission among depressives. Preclinical data based on animal models including selectively bred rat lines and mouse mutants support the notion that CRH neurons are hyperactive also in neuroanatomical regions that are involved in behavioral regulation but are located outside the neuroendocrine system. This raises the question of whether more direct interventions such as CRH receptor antagonists would open a new lead in the treatment of stress-related disorders such as depression, anxiety and sleep disorders. Recent clinical observations support this possibility.

Neuroendocrine and behavioral effects of repetitive transcranial magnetic stimulation in a psychopathological animal model are suggestive of antidepressant-like effects.

The neuroendocrine and behavioral effects of repetitive transcranial magnetic stimulation (rTMS) were investigated in two rat lines selectively bred for high and low anxiety-related behavior. The stimulation parameters were adjusted according to the results of accurate computer-assisted and magnetic resonance imaging-based reconstructions of the current density distributions induced by rTMS in the rat and human brain, ensuring comparable stimulation patterns in both cases. Adult male rats were treated in two 3-day series under halothane anesthesia. In the forced swim test, rTMS-treatment induced a more active coping strategy in the high anxiety-related behavior rats only (time spent struggling; 332% vs. controls), allowing these animals to reach the performance of low anxiety-related behavior rats. In contrast, rTMS-treated low anxiety-related behavior rats did not change their swimming behavior. The development of active coping strategies in high anxiety-related behavior rats was accompanied by a significantly attenuated stress-induced elevation of plasma corticotropin and corticosterone concentrations. In summary, the behavioral and neuroendocrine effects of rTMS of frontal brain regions in high anxiety-related behavior rats are comparable to the effects of antidepressant drug treatment. Interestingly, in the psychopathological animal model repetitive transcranial magnetic stimulation induced changes in stress coping abilities in the high-anxiety line only.

The anxiolytic effect of the CRH(1) receptor antagonist R121919 depends on innate emotionality in rats.

Hyperactivity of central corticotropin-releasing hormone (CRH) circuits appears to contribute to the symptomatology of affective and anxiety disorders and therefore CRH receptor antagonists have attracted attention as potential therapeutic agents. R121919, a novel high-affinity nonpeptide CRH(1) receptor antagonist, displaced (125)I-oCRH in rat pituitary, cortex and amygdala, but not in choroid plexus or cerebral blood vessels in vitro and in vivo, which is consistent with CRH(1) receptor antagonism. In vivo, R121919 significantly inhibited stress-induced corticotropin release in rats selectively bred for high- and low-anxiety-related behaviour but displayed anxiolytic effects in high-anxiety rats only. These data, corroborated by ex vivo receptor occupancy studies, suggest that this animal model is appropriate for the evaluation of CRH(1) receptor antagonists and that compounds such as R121919 will be beneficial whenever the central stress hormone system is hyperactive.

Repetitive transcranial magnetic stimulation induces active coping strategies and attenuates the neuroendocrine stress response in rats.

The effects of repetitive transcranial magnetic stimulation (rTMS) on various brain functions were investigated in adult male Wistar rats. The stimulation parameters were adjusted according to the results of accurate computer-assisted, magnetic resonance imaging-based reconstructions of the current density distributions induced by rTMS in the rat and human brain, ensuring comparable stimulation patterns in both cases. The animals were subjected to daily rTMS-treatment (three trains of 20 Hz; 2.5 s) for 8 weeks from the age of 4 weeks on. In the forced swim test these rats showed a more active stress coping strategy than the control rats. This was accompanied by a significantly attenuated stress-induced elevation of plasma ACTH concentrations. Pituitary changes accounting for the attenuation were ruled out by the corticotropin-releasing hormone test. Baseline concentrations of ACTH and corticosterone were indistinguishable in the two groups. No changes were found in the anxiety-related behavior of the rats on the elevated plus-maze or in behavior during the social interaction test. Accordingly, the binding characteristics of the benzodiazepine agonist [(3)H]flunitrazepam at the benzodiazepine/gamma-aminobutyric acid type A receptor complex were similar in the rTMS and control groups. In summary, chronic rTMS treatment of frontal brain regions in rats resulted in a change in coping strategy that was accompanied by an attenuated neuroendocrine response to stress, thus revealing parallels to the effects of antidepressant drug treatment.

Selective activation of the hypothalamic vasopressinergic system in mice deficient for the corticotropin-releasing hormone receptor 1 is dependent on glucocorticoids.

Deficiency of CRH receptor 1 (CRHR1) severely impairs the stress response of the hypothalamic-pituitary-adrenocortical (HPA) system and reduces anxiety-related behavior in mice. Intriguingly, in mice deficient for the CRHR1 (Crhr1-/-), basal plasma levels of ACTH are normal, suggesting the presence of compensatory mechanisms for pituitary ACTH secretion. We therefore studied the impact of the hypothalamic neuropeptides arginine vasopressin (AVP) and oxytocin (OXT) on HPA system regulation in homozygous and heterozygous Crhr1 mutants under basal and different stress conditions. Basal plasma AVP concentrations were significantly elevated in Crhr1-/- mice. AVP messenger RNA expression was increased in the paraventricular nucleus of Crhr1-/- mutants together with a marked increase in AVP-like immunoreactivity in the median eminence. Administration of an AVP V1-receptor antagonist significantly decreased basal plasma ACTH levels in mutant mice. After continuous treatment with corticosterone, plasma AVP levels in homozygous Crhr1-/- mice were indistinguishable from those in wild-type littermates, thus providing evidence that glucocorticoid deficiency is the major driving force behind compensatory activation of the vasopressinergic system in Crhr1-/- mice. Neither plasma OXT levels under several different conditions nor OXT messenger RNA expression in the paraventricular nucleus were different between the genotypes. Taken together, our data reveal a selective compensatory activation of the hypothalamic vasopressinergic, but not the oxytocinergic system, to maintain basal ACTH secretion and HPA system activity in Crhr1-/- mutants.