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Interest in cannabinoids' therapeutic potential in mental health is growing, supported by evidence of the involvement of the endocannabinoid system in psychiatric disorders such as anxiety, depression, and addiction. While the major cannabinoids cannabidiol (CBD) and Δ9-tetrahydrocannabinol (Δ9-THC) have been more extensively researched, approximately 120 minor cannabinoids from the cannabis plant have been identified. Although some displayed promising pharmacological profiles, research on their application for psychiatric disorders is fragmented. This systematic review evaluates, for the first time, both preclinical and clinical studies exploring minor cannabinoids' therapeutic potential in psychiatric disorders. 22 preclinical studies and one clinical study were included, investigating various minor cannabinoids in substance use disorders, anxiety disorders, depressive disorders, trauma and stressor-related disorders, psychotic disorders, neurodevelopmental disorders, and eating disorders. Despite the heterogeneous results and the moderate to high risk of bias in several articles, certain compounds demonstrate promise for further investigation. Δ8-tetrahydrocannabidivarin (Δ8-THCV) exhibited potential for nicotine addiction; Δ9-tetrahydrocannabidivarin (Δ9-THCV) for psychotic-like symptoms; cannabidiolic acid methyl ester (CBDA-ME) alleviated anxiety and depression-like symptoms, and cannabidivarin (CBDV) autism spectrum disorder-like symptoms., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Background: The pathophysiology of several neurodegenerative and neuropsychiatric disorders is linked to an altered immune system. However, it is often unclear how the immune system specifically affects these disorders since neuroimmune interactions are very complex. In this paper, we introduce an adjusted version of the adverse outcome pathway (AOP) approach from toxicology to the field of neuroimmunology. A review of the effect of TNF-α on fear learning deficits is used as a worked example to demonstrate how an AOP approach can help identify gaps of knowledge and crucial steps in the pathophysiology of neuroimmunological disorders., Methods: The AOP was constructed in five steps. First, the adverse outcome was formulated clearly and specifically. Second, the link between the molecular initiating event and the adverse outcome was established with a preliminary literature search in the Medline database. Third, a systematic literature search was performed in which we identified 95 relevant articles. Fourth, the main biological processes and relevant key events were identified. Fifth, the links between key events were determined and an AOP network was constructed., Results: We identified three pathways through which TNF-α may affect fear learning. First, TNF-α receptor activation increases NF-κB levels which increases oxidative stress levels and reduces the activity of glutamate transporters. This alters the synaptic plasticity which is associated with impaired fear acquisition, consolidation, and fear extinction. Second, activation of TNF-α receptors increases the expression and capacity of the serotonin transporter which is linked to impaired fear acquisition, expression, and extinction. Third, TNF-α receptor 1 activation can induce necroptosis, leading to neuroinflammation which is linked to fear learning deficits., Conclusion: To successfully apply the AOP approach in neuroimmunology we recommend defining adverse outcomes more precisely, establishing stronger connections between key events from various biological processes, incorporating feedforward and feedback loops, and identifying more mechanistic knowledge in later key events. These adjustments are needed to map the complex processes within the field of neuroimmunology and to identify gaps of knowledge., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

An increasing amount of evidence suggests that immune responses may affect trophoblast functioning, which in turn may play a role in gestational disorders and fetal development. This systematic review offers the first summary of in vitro studies on the trophoblast response to immunological triggers, in conjunction with a risk of bias analysis. A search in Pubmed and Embase yielded 110 relevant studies. Primary trophoblasts were the most commonly used cell type, but trophoblast subtypes were not always defined. Similarly, the exact natures of trophoblast cell lines were sometimes unclear. Cytokines and Toll-like receptor agonists were often used as interventions, but most studies focused on a select few substances such as tumor necrosis factor-α and lipopolysaccharide. In regard to the outcome parameters, some important trophoblast functions, such as hormone production and barrier formation were underrepresented. Whether or not risk of bias was high varied strongly between types of bias. Risk of selection bias, for example, was usually low. However, none of the included studies mentioned blinding or plate randomization. Only a select few studies mentioned passage numbers, use of vehicle control or conflict of interest. In conclusion, better characterization of trophoblast subtypes and a broader range of studied interventions and outcome parameters would contribute to a more complete understanding of trophoblast responses to immune stimuli. Additionally, researchers are encouraged to replicate experiments and pay close attention when setting up and writing down methodologies, in order to improve the reproducibility and translatability of their work., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this article., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Rationale: Selective serotonin reuptake inhibitors (SSRIs) are the first choice of treatment for anxiety-like disorders. However, which aspects of anxiety are affected by SSRIs is not yet fully understood., Objective: We aimed to systematically review the effect of six clinically effective SSRIs on four aspects of unconditioned anxiety: approach-avoidance behaviour (elevated plus maze), repetitive behaviour (marble burying), distress behaviour (ultrasonic vocalization), and activation of the autonomous nervous system (stress-induced hyperthermia)., Methods: We identified publications by searching Medline and Embase databases and assessed the risk of bias. A random effects meta-analysis was performed and moderator effects were analysed with Bayesian penalized meta-regression., Results: Our search yielded 105 elevated plus maze, 63 marble burying, 11 ultrasonic vocalization, and 7 stress-induced hyperthermia articles. Meta-analysis suggested that SSRIs reduce anxiety-like behaviour in the elevated plus maze, marble burying and ultrasonic vocalization test and that effects are moderated by pre-existing stress conditions (elevated plus maze) and dose dependency (marble burying) but not by duration of treatment or type of SSRI. The reporting quality was low, publication bias was likely, and heterogeneity was high., Conclusion: SSRIs seem to reduce a broad range of unconditioned anxiety-associated behaviours. These results should be interpreted with caution due to a high risk of bias, likely occurrence of publication bias, substantial heterogeneity and limited moderator data availability. Our review demonstrates the importance of including bias assessments when interpreting meta-analysis results. We further recommend improving the reporting quality, the conduct of animal research, and the publication of all results regardless of significance., (© 2024. The Author(s).)

The first 1000 days of life is a critical period of development in which adverse circumstances can have long-term consequences for the child's health. Maternal immune activation is associated with increased risk of neurodevelopmental disorders in the child. Aberrant immune responses have been reported in individuals with neurodevelopmental disorders. Moreover, lasting effects of maternal immune activation on the offspring's immune system have been reported. Taken together, this indicates that the effect of maternal immune activation is not limited to the central nervous system. Here, we explore the impact of maternal immune activation on the immune system of the offspring. We first describe the development of the immune system and provide an overview of reported alterations in the cytokine profiles, immune cell profiles, immune cell function, and immune induction in pre-clinical models. Additionally, we highlight recent research on the impact of maternal COVID-19 exposure on the neonatal immune system and the potential health consequences for the child. Our review shows that maternal immune activation alters the offspring's immune system under certain conditions, but the reported effects are conflicting and inconsistent. In general, epigenetic modifications are considered the mechanism for fetal programming. The available data was insufficient to identify specific pathways that may contribute to immune programming. As a consequence of the COVID-19 pandemic, more research now focuses on the possible health effects of maternal immune activation on the offspring. Future research addressing the offspring's immune response to maternal immune activation can elucidate specific pathways that contribute to fetal immune programming and the long-term health effects for the offspring., (© 2024. The Author(s).)

Rationale and Objectives: Fear conditioning is an important aspect in the pathophysiology of anxiety disorders. The fear-potentiated startle test is based on classical fear conditioning and over the years, a broad range of drugs have been tested in this test. Synthesis of the available data may further our understanding of the neurotransmitter systems that are involved in the expression of conditioned fear., Methods: Following a comprehensive search in Medline and Embase, we included 68 research articles that reported on 103 drugs, covering 56 different drug classes. The systematic review was limited to studies using acute, systemic drug administration in naive animals., Results: Qualitative data synthesis showed that most clinically active anxiolytics, but not serotonin-reuptake inhibitors, reduced cued fear. Anxiogenic drugs increased fear potentiation in 35% of the experiments, reduced fear potentiation in 29% of the experiments, and were without effect in 29% of the experiments. Meta-analyses could be performed for five drug classes and showed that benzodiazepines, buspirone, 5-HT 1A agonists, 5-HT 1A antagonists, and mGluR2,3 agonists reduced cued conditioned fear. The non-cued baseline startle response, which may reflect contextual anxiety, was only significantly reduced by benzodiazepines and 5-HT 1A antagonists. No associations were found between drug effects and methodological characteristics, except for strain., Conclusions: The fear-potentiated startle test appears to have moderate to high predictive validity and may serve as a valuable tool for the development of novel anxiolytics. Given the limited available data, the generally low study quality and high heterogeneity additional studies are warranted to corroborate the findings of this review., (© 2023. The Author(s).)

Rationale: Selective serotonin reuptake inhibitors (SSRIs) are considered first-line medication for anxiety-like disorders such as panic disorder, generalized anxiety disorder, and post-traumatic stress disorder. Fear learning plays an important role in the development and treatment of these disorders. Yet, the effect of SSRIs on fear learning are not well known., Objective: We aimed to systematically review the effect of six clinically effective SSRIs on acquisition, expression, and extinction of cued and contextual conditioned fear., Methods: We searched the Medline and Embase databases, which yielded 128 articles that met the inclusion criteria and reported on 9 human and 275 animal experiments., Results: Meta-analysis showed that SSRIs significantly reduced contextual fear expression and facilitated extinction learning to cue. Bayesian-regularized meta-regression further suggested that chronic treatment exerts a stronger anxiolytic effect on cued fear expression than acute treatment. Type of SSRI, species, disease-induction model, and type of anxiety test used did not seem to moderate the effect of SSRIs. The number of studies was relatively small, the level of heterogeneity was high, and publication bias has likely occurred which may have resulted in an overestimation of the overall effect sizes., Conclusions: This review suggests that the efficacy of SSRIs may be related to their effects on contextual fear expression and extinction to cue, rather than fear acquisition. However, these effects of SSRIs may be due to a more general inhibition of fear-related emotions. Therefore, additional meta-analyses on the effects of SSRIs on unconditioned fear responses may provide further insight into the actions of SSRIs., (© 2023. The Author(s).)

The endocannabinoid system is a promising candidate for anxiolytic therapy, but translation to the clinic has been lagging. We meta-analyzed the evidence for anxiety-reduction by compounds that facilitate endocannabinoid signaling in humans and animals. To identify areas of specific potential, effects of moderators were assessed. Literature was searched in Pubmed and Embase up to May 2021. A placebo/vehicle-control group was required and in human studies, randomization. We excluded studies that co-administered other substances. Risk of bias was assessed with SYRCLE's RoB tool and Cochrane RoB 2.0. We conducted three-level random effects meta-analyses and explored sources of heterogeneity using Bayesian regularized meta-regression (BRMA). The systematic review yielded 134 studies. We analyzed 120 studies (114 animal, 6 human) that investigated cannabidiol (CBD, 61), URB597 (39), PF-3845 (6) and AM404 (14). Pooled effects on conditioned and unconditioned anxiety in animals (with the exception of URB597 on unconditioned anxiety) and on experimentally induced anxiety in humans favored the investigational drugs over placebo/vehicle. Publication year was negatively associated with effects of CBD on unconditioned anxiety. Compared to approach avoidance tests, tests of repetitive-compulsive behavior were associated with larger effects of CBD and URB597, and the social interaction test with smaller effects of URB597. Larger effects of CBD on unconditioned anxiety were observed when anxiety pre-existed. Studies reported few side effects at therapeutic doses. The evidence quality was low with indications of publication bias. More clinical trials are needed to translate the overall positive results to clinical applications., Competing Interests: Conflict of interest The authors have no conflicting interests to declare., (Copyright © 2023. Published by Elsevier B.V.)

Background: Preclinical research suggests that cannabidiol (CBD) may have therapeutic potential in pathological anxiety. Dosing guidelines to inform future human studies are however lacking., Aim: We aimed to predict the therapeutic window for anxiety-reducing effects of CBD in humans based on preclinical models., Methods: We conducted two systematic searches in PubMed and Embase up to August 2021, into pharmacokinetic (PK) and pharmacodynamic (PD) data of systemic CBD exposure in humans and animals, which includes anxiety-reducing and potential side effects. Risk of bias was assessed with SYRCLE's RoB tool and Cochrane RoB 2.0. A control group was an inclusion criterion in outcome studies. In human outcome studies, randomisation was required. We excluded studies that co-administered other substances. We used the IB-de-risk tool for a translational integration of outcomes., Results: We synthesised data from 87 studies. For most observations (70.3%), CBD had no effect on anxiety outcomes. There was no identifiable relation between anxiety outcomes and drug levels across species. In all species (humans, mice, rats), anxiety-reducing effects seemed to be clustered in certain concentration ranges, which differed between species., Discussion: A straightforward dosing recommendation was not possible, given variable concentration-effect relations across species, and no consistent linear effect of CBD on anxiety reduction. Currently, these results raise questions about the broad use as a drug for anxiety. Meta-analytic studies are needed to quantitatively investigate drug efficacy, including aspects of anxiety symptomatology. Acute and (sub)chronic dosing studies with integrated PK and PD outcomes are required for substantiated dose recommendations.

Preclinical research suggests that enhancing CB1 receptor agonism may improve fear extinction. In order to translate this knowledge into a clinical application we examined whether cannabidiol (CBD), a hydrolysis inhibitor of the endogenous CB1 receptor agonist anandamide (AEA), would enhance the effects of exposure therapy in treatment refractory patients with anxiety disorders. Patients with panic disorder with agoraphobia or social anxiety disorder were recruited for a double-blind parallel randomised controlled trial at three mental health care centres in the Netherlands. Eight therapist-assisted exposure in vivo sessions (weekly, outpatient) were augmented with 300 mg oral CBD (n = 39) or placebo (n = 41). The Fear Questionnaire (FQ) was assessed at baseline, mid- and post-treatment, and at 3 and 6 months follow-up. Primary analyses were on an intent-to-treat basis. No differences were found in treatment outcome over time between CBD and placebo on FQ scores, neither across (β = 0.32, 95% CI [-0.60; 1.25]) nor within diagnosis groups (β = -0.11, 95% CI [-1.62; 1.40]). In contrast to our hypotheses, CBD augmentation did not enhance early treatment response, within-session fear extinction or extinction learning. Incidence of adverse effects was equal in the CBD (n = 4, 10.3%) and placebo condition (n = 6, 15.4%). In this first clinical trial examining CBD as an adjunctive therapy in anxiety disorders, CBD did not improve treatment outcome. Future clinical trials may investigate different dosage regimens., Competing Interests: Declaration of Competing Interest All authors report no biomedical financial interests or potential conflicts of interest., (Copyright © 2022. Published by Elsevier B.V.)

Radiotherapy (RT) is a cornerstone treatment strategy for brain tumours. Besides cytotoxicity, RT can cause disruption of the blood-brain barrier (BBB), resulting in an increased permeability into the surrounding brain parenchyma. Although this effect is generally acknowledged, it remains unclear how and to what extent different radiation schemes affect BBB integrity. The aim of this systematic review and meta-analysis is to investigate the effect of photon RT regimens on BBB permeability, including its reversibility, in clinical and preclinical studies. We systematically reviewed relevant clinical and preclinical literature in PubMed, Embase, and Cochrane search engines. A total of 69 included studies (20 clinical, 49 preclinical) were qualitatively and quantitatively analysed by meta-analysis and evaluated on key determinants of RT-induced BBB permeability in different disease types and RT protocols. Qualitative data synthesis showed that 35% of the included clinical studies reported BBB disruption following RT, whereas 30% were inconclusive. Interestingly, no compelling differences were observed between studies with different calculated biological effective doses based on the fractionation schemes and cumulative doses; however, increased BBB disruption was noted during patient follow-up after treatment. Qualitative analysis of preclinical studies showed RT BBB disruption in 78% of the included studies, which was significantly confirmed by meta-analysis (p < 0.01). Of note, a high risk of bias, publication bias and a high heterogeneity across the studies was observed. This systematic review and meta-analysis sheds light on the impact of RT protocols on BBB integrity and opens the discussion for integrating this factor in the decision-making process of future RT, with better study of its occurrence and influence on concomitant or adjuvant therapies., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2022 The Authors.)

Trace Amine-Associated Receptor 1 (TAAR1) is a potential target for the treatment of depression and other CNS disorders. However, the precise functional roles of TAAR1 to the actions of clinically used antidepressants remains unclear. Herein, we addressed these issues employing the TAAR1 agonist, o-phenyl-iodotyramine (o-PIT), together with TAAR1-knockout (KO) mice. Irrespective of genotype, systemic administration of o-PIT led to a similar increase in mouse brain concentrations. Consistent with the observation of a high density of TAAR1 in the medial preoptic area, o-PIT-induced hypothermia was significantly reduced in TAAR1-KO mice. Furthermore, the inhibition of a prepulse inhibition response by o-PIT, as well as its induction of striatal tyrosine hydroxylase phosphorylation and elevation of extracellular DA in prefrontal cortex, were all reduced in TAAR1-KO compared to wildtype mice. O-PIT was active in both forced-swim and marble-burying tests, and its effects were significantly blunted in TAAR1-KO mice. Conversely, the actions on behaviour and prefrontal cortex dialysis of a broad suite of clinically used antidepressants were unaffected in TAAR1-KO mice. In conclusion, o-PIT is a useful tool for exploring the hypothermic and other functional antidepressant roles of TAAR1. By contrast, clinically used antidepressants do not require TAAR1 for expression of their antidepressant properties.

The maternal polyinosinic:polycytidylic acid (poly(I:C)) animal model is frequently used to study how maternal immune activation may impact neuro development in the offspring. Here, we present the first systematic review and meta-analysis on the effects of maternal poly(I:C) injection on immune mediators in the offspring and provide an openly accessible systematic map of the data including methodological characteristics. Pubmed and EMBASE were searched for relevant publications, yielding 45 unique papers that met inclusion criteria. We extracted data on immune outcomes and methodological characteristics, and assessed the risk of bias. The descriptive summary showed that most studies reported an absence of effect, with an equal number of studies reporting an increase or decrease in the immune mediator being studied. Meta-analysis showed increased IL-6 concentrations in the offspring of poly(I:C) exposed mothers. This effect appeared larger prenatally than post-weaning. Furthermore, poly(I:C) administration during mid-gestation was associated with higher IL-6 concentrations in the offspring. Maternal poly(I:C) induced changes in IL-1β, Il-10 and TNF-α concentrations were small and could not be associated with age of offspring, gestational period or sampling location. Finally, quality of reporting of potential measures to minimize bias was low, which stresses the importance of adherence to publication guidelines. Since neurodevelopmental disorders in humans tend to be associated with lifelong changes in cytokine concentrations, the absence of these effects as identified in this systematic review may suggest that combining the model with other etiological factors in future studies may provide further insight in the mechanisms through which maternal immune activation affects neurodevelopment., Competing Interests: M.M. Tiemessen is an employee at Danone Nutricia Research. The other authors declare no conflict of interest., (© 2020 The Author(s).)

Various animal models are used to study pharmacokinetics (PK) of drugs in development. Human renal clearance (CLr) should be predictable through interpolation from animal data by allometric scaling. Based on this premise, we quantified interspecies differences in CLr, and related them to drug properties. Using PubMed and EMBASE, we systematically reviewed literature on human and animal CLr measures for 20 renally excreted drugs, calculated average fold errors, and quantified mean differences between animals and humans. Our results show that animal models are generally good predictors for human drug clearance using simple allometry, except for rats, with which human CLr is significantly overestimated., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Rationale: Fear conditioning is an important factor in the etiology of anxiety disorders. Previous studies have demonstrated a role for serotonin (5-HT) 1A receptors in fear conditioning. However, the relative contribution of somatodendritic 5-HT 1A autoreceptors and post-synaptic 5-HT 1A heteroreceptors in fear conditioning is still unclear., Objective: To determine the role of pre- and post-synaptic 5-HT 1A receptors in the acquisition and expression of cued and contextual conditioned fear., Methods: We studied the acute effects of four 5-HT 1A receptor ligands in the fear-potentiated startle test. Male Wistar rats were injected with the 5-HT 1A receptors biased agonists F13714 (0-0.16 mg/kg, IP), which preferentially activates somatodendritic 5-HT 1A autoreceptors, or F15599 (0-0.16 mg/kg, IP), which preferentially activates cortical post-synaptic 5-HT 1A heteroreceptors, with the prototypical 5-HT 1A receptor agonist R(+)8-OH-DPAT (0-0.3 mg/kg, SC) or the 5-HT 1A receptor antagonist WAY100,635 (0-1.0 mg/kg, SC)., Results: F13714 (0.16 mg/kg) and R(+)-8-OH-DPAT (0.03 mg/kg) injected before training reduced cued fear acquisition. Pre-treatment with F15599 or WAY100,635 had no effect on fear learning. In the fear-potentiated startle test, F13714 (0.04-0.16 mg/kg) and R(+)-8-OH-DPAT (0.1-0.3 mg/kg) reduced the expression of cued and contextual fear, whereas F15599 had no effect. WAY100,635 (0.03-1.0 mg/kg) reduced the overall startle response., Conclusions: The current findings indicate that activation of somatodendritic 5-HT 1A autoreceptors reduces cued fear learning, whereas 5-HT 1A receptors seem not involved in contextual fear learning. Moreover, activation of somatodendritic 5-HT 1A autoreceptors may reduce cued and contextual fear expression, whereas we found no evidence for the involvement of cortical 5-HT 1A heteroreceptors in the expression of conditioned fear.

Background: Phobic anxiety disorders are among the most prevalent psychiatric disorders and are burdensome in terms of loss of quality of life and work productivity. Evidence-based treatments are relatively successful in the majority of patients, especially exposure therapy. However, a substantial subset of patients fails to achieve or stay in remission. Preclinical and genetic research have yielded evidence that the cannabinoid system is involved in the extinction of fear, presumed to underlie the beneficial effects of exposure therapy in phobic disorders. A cannabinoid constituent that may enhance endocannabinoid signaling is cannabidiol (CBD), a non-psychoactive component of cannabis. Hence, the addition of CBD to exposure therapy is expected to strengthen effects of treatment. To determine the added benefit of CBD on exposure therapy, we conduct a randomized controlled trial, in which patients in whom previous treatment as usual has not yielded sufficient response receive either CBD or placebo preceding 8 exposure sessions in a double-blind fashion. A subsidiary aim is to explore which (combination of) clinical, behavioral and genetic profiles of patients are related to treatment response., Methods/design: This is an 8-week multicenter, randomized, double-blind, placebo-controlled trial. Seventy-two patients with social phobia or panic disorder with agoraphobia with incomplete response to earlier treatment will be included from outpatient clinics in the Netherlands. Patients are randomized to augmentation of exposure therapy with 300 mg CBD or placebo. The study medication is administered orally, 2 h preceding each of the eight 90 min exposure sessions. Measurements will take place at baseline, first administration of medication, every session, mid-treatment, last administration of medication, post-treatment and at 3 and 6 months' follow-up. The primary outcome measure is the score on the Fear Questionnaire (FQ). In addition, determinants of the expected treatment enhancing effect of CBD will be explored., Discussion: This is the first trial to investigate whether the addition of CBD to exposure therapy is effective in reducing phobic symptoms in treatment refractory patients with social phobia or panic disorder with agoraphobia., Trial Registration: Netherlands Trial Register NTR5100 . Registered 13 March 2015. Protocol version: issue date 17 Jan 2018, protocol amendment number 7.

This study evaluated the extent to which a reduction in contextual fear contributes to the anxiolytic effect of benzodiazepines in the fear-potentiated startle response. To this end, chlordiazepoxide, an anxiolytic often used as positive control in preclinical drug studies, and zolpidem, known to have sedative properties and to be devoid of anxiolytic effects, were tested in two contexts: the same context as training had taken place and an alternative context. In addition, the level of muscle relaxation was assessed in a grip strength test. Chlordiazepoxide (2.5-10 mg/kg) decreased the fear-potentiated startle response, confirming its anxiolytic activity. In addition, it dose-dependently decreased the overall startle response in the same, but not the alternative context, and did not affect grip strength, indicating that chlordiazepoxide inhibits contextual fear in the absence of non-specific drug effects. Zolpidem (1.0-10 mg/kg) reduced the overall startle response in both contexts equally and decreased grip strength, indicating that its effects on fear-potentiated startle are due to non-specific drug effects, and not anxiolytic effects. The present findings show that chlordiazepoxide reduces contextual conditioned fear in the absence of non-specific drug effects. In addition, they show that training and testing rats in different contexts makes it possible to distinguish between cued, contextual and non-specific drug effects. As exaggerated contextual fear conditioning contributes to the fear generalization processes implicated in pathological anxiety, focus in screening of anxiolytic effects could be directed more towards the suppression of contextual fear and, therefore, this approach would be a valuable addition to standard preclinical screening., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cognitive impairments, e.g., reward learning, are present in various psychiatric disorders and warrant treatment. Improving reward-related learning could synergistically enhance psychosocial treatments and cognition generally. A critical first step is to understand the mechanisms underlying reward learning. The dopamine system has been implicated in such learning, but less known is how indirect activation of this system may affect reward learning. We determined the role of alpha7 nicotinic acetylcholine receptors (nAChR) on a probabilistic reversal learning task (PRLT) in mice that includes reward and punishment. Male alpha7 knockout (KO), heterozygous (HT), and wildtype (WT) littermate mice (n = 84) were treated with vehicle, 0.03, or 0.3 mg/kg nicotine. Two cohorts of C57BL/6NJ male mice were treated with various alpha7 nAChR ligands, including the full agonists PNU282877 and AR-R-17779, the positive allosteric modulator CCMI, the partial agonist SSR180711, and the antagonist methyllycaconitine. All mice were then tested in the PRLT. Nicotine (0.3 mg/kg) significantly improved initial reward learning in alpha7 WT and HT mice but did not improve learning in KO mice, suggesting an involvement of the alpha7 nAChR in the pro-learning effects of nicotine. Neither alpha7 nAChR treatments (PNU282987, AR-R-17779, CCMI, SSR180711, nor methyllycaconitine) affected mouse PRLT performance however. Nicotine improved reward learning via a mechanism that may include alpha7 nAChRs. This improvement unlikely relied solely on alpha7 nAChRs however, since no alpha7 nAChR ligand improved reward learning in normal mice. Future assessments of the effects of other nAChR subtypes on reward learning are needed., (Copyright © 2018 Elsevier B.V. and European College of Neuropsychopharmacology. All rights reserved.)

The prevalence of anxiety disorders is higher in women than in men. Yet preclinical studies on anxiety are mostly performed in male subjects. This may have limited our understanding of mechanisms contributing to anxiety disorders. Since fear conditioning is considered an important factor in the etiology of anxiety disorders, the present study aimed to investigate the effect of sex and estrous cycle on conditioned fear and the anxiolytic effect of benzodiazepines in rats. We measured the fear-potentiated startle response in male and female rats during different estrous cycle stages and performed a replication study in a separate cohort. In addition, we assessed the response to diazepam (0-3.0 mg/kg IP) and chlordiazepoxide (0-10 mg/kg IP) in male and female rats in proestrous/estrous and diestrous stage. Our results showed that there were no sex differences in the expression of fear-potentiated startle. The estrous cycle also did not affect the fear-potentiated startle response. In addition, male and female rats did not differ in their fear-potentiated startle response following treatment with either diazepam or chlordiazepoxide. In conclusion, the current study shows that male and female rats do not differ in their conditioned fear response and the responsiveness to benzodiazepines. The results further indicate that conditioned fear-related processes are not affected by gonadal hormone fluctuations in this paradigm. These findings may suggest that the higher prevalence of anxiety disorders in women more likely results from differences in responding to previous experiences or differences in other predisposing factors, rather than differences in conditioned fear per se., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Degeneration of dopamine (DA) neurons in the substantia nigra pars compacta is the pathological hallmark of Parkinson's disease (PD). In PD multiple pathogenic mechanisms initiate and drive this neurodegenerative process, making the development of effective treatments challenging. To date, PD patients are primarily treated with dopaminergic drugs able to temporarily enhance DA levels, therefore relieving motor symptoms. However, the drawbacks of these therapies including the inability to alter disease progression are constantly supporting the search for alternative treatment approaches. Over the past years efforts have been put into the development of new therapeutic strategies based on the delivery of therapeutic genes using viral vectors or transplantation of DA neurons for cell-based DA replacement. Here, past achievements and recent advances in gene- and cell-based therapies for PD are outlined. We discuss how current gene and cell therapy strategies hold great promise for the treatment of PD and how the use of stem cells and recent developments in cellular reprogramming could contribute to open a new avenue in PD therapy., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Patients with bipolar disorder mania exhibit poor cognition, impulsivity, risk-taking, and goal-directed activity that negatively impact their quality of life. To date, existing treatments for bipolar disorder do not adequately remediate cognitive dysfunction. Reducing dopamine transporter expression recreates many bipolar disorder mania-relevant behaviors (i.e. hyperactivity and risk-taking). The current study investigated whether dopamine D 1 -family receptor blockade would attenuate the risk-taking, hypermotivation, and hyperactivity of dopamine transporter knockdown mice., Methods: Dopamine transporter knockdown and wild-type littermate mice were tested in mouse versions of the Iowa Gambling Task (risk-taking), Progressive Ratio Breakpoint Test (effortful motivation), and Behavioral Pattern Monitor (activity). Prior to testing, the mice were treated with the dopamine D 1 -family receptor antagonist SCH 23390 hydrochloride (0.03, 0.1, or 0.3 mg/kg), or vehicle., Results: Dopamine transporter knockdown mice exhibited hyperactivity and hyperexploration, hypermotivation, and risk-taking preference compared with wild-type littermates. SCH 23390 hydrochloride treatment decreased premature responding in dopamine transporter knockdown mice and attenuated their hypermotivation. SCH 23390 hydrochloride flattened the safe/risk preference, while reducing activity and exploratory levels of both genotypes similarly., Conclusions: Dopamine transporter knockdown mice exhibited mania-relevant behavior compared to wild-type mice. Systemic dopamine D 1 -family receptor antagonism attenuated these behaviors in dopamine transporter knockdown, but not all effects were specific to only the knockdown mice. The normalization of behavior via blockade of dopamine D 1 -family receptors supports the hypothesis that D 1 and/or D 5 receptors could contribute to the mania-relevant behaviors of dopamine transporter knockdown mice.

Rationale: Given the large number of patients that does not respond sufficiently to currently available treatment for anxiety disorders, there is a need for improved treatment., Objectives: We evaluated the anxiolytic effects of corticotropin releasing factor (CRF) 1 receptor antagonists and glucocorticoid receptor (GR) antagonists in the separation-induced vocalization test in guinea pigs and transgenic mice with central CRF overexpression. Furthermore, we explored effects of these drugs when given in combination with a suboptimal dose of a selective serotonin re-uptake inhibitor (SSRI)., Methods: In guinea pig pups, the CRF 1 receptor antagonists CP-154,526 and DMP695, and the GR antagonists mifepristone and Org34517 (all at 2.5, 10 and 40mg/kg intraperitoneally (IP)) were tested alone or in combination with 0.63mg/kg paroxetine IP. In CRF overexpressing mouse pups and wild type littermates, effects of CP-154,526 (10, 20 and 40mg/kg subcutaneously (SC)) and mifepristone (5, 15, 45mg/kg SC) were studied alone or in combination with 0.03mg/kg paroxetine SC., Results: CRF 1 but not GR antagonists reduced the number of calls relative to vehicle in guinea pigs and mice, independent of genotype. Treatment of CRF 1 receptor or GR antagonists with paroxetine had no combined effect in guinea pigs, wild type or CRF overexpressing mice., Conclusions: Current results indicate robust anxiolytic properties of CRF 1 receptor antagonists in guinea pigs and mice overexpressing CRF, and lack thereof of GR antagonists. Although no combined treatment effects were observed, it would be interesting to study combined treatment of CRF 1 receptor antagonists with SSRIs following chronic drug administration., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

We recently showed that a genetic polymorphism (rs878886) in the human corticotropin-releasing hormone receptor 1 (CRHR1) is associated with reduced fear-conditioned responses to a threat cue. This is a potentially important finding considering that the failure to acquire fear contingencies can leave an individual in a maladaptive state of more generalized anxiety. Consistent with that idea, the CRHR1-dependent fear acquisition deficit translated into heightened contextual anxiety when taking genetic variability within the serotonin transporter long polymorphic region (5-HTTLPR) into account. To replicate our previous findings, we conducted a replication study in 224 healthy medication-free human subjects using the exact same cue and context virtual reality fear-conditioning procedure as in study by Heitland et al. (2013). In the replication study, consistent with the original findings, CRHR1 rs878886 G-allele carriers showed reduced acquisition of cue-specific fear-conditioned responses compared with C/C homozygotes. Also, in this larger sample the cue acquisition deficit of G-allele carriers translated into heightened contextual anxiety, even independent of 5-HTT gene variation. In contrast to our earlier findings, there was an additional interaction effect of CRHR1 rs878886 and the triallelic 5-HTTLPR/rs25531 variant on cued fear acquisition. In summary, this study replicated the initially reported association of the CRHR1 rs878886 G-allele with cued fear acquisition deficits, albeit with a different pattern of results regarding the interaction with 5-HTT variation. This further supports the notion that the human corticotropin-releasing hormone plays a role in the acquisition of fears., (© 2015 John Wiley & Sons Ltd and International Behavioural and Neural Genetics Society.)

The inability to associate aversive events with relevant cues (i.e. fear learning) may lead to maladaptive anxiety. To further study the role of the serotonin transporter (SERT) in fear learning, classical fear conditioning was studied in SERT knockout rats (SERT(-/-)) using fear potentiation of the startle reflex. Next, fear acquisition and concomitant development of contextual conditioned fear were monitored during training. To differentiate between developmental and direct effects of reduced SERT functioning, effects of acute and chronic SSRI treatment were studied in adult rats. Considering the known interactions between serotonin and corticotropin-releasing factor (CRF), we studied the effect of the CRFR1 antagonist CP154,526 on behavioral changes observed and determined CRF1 receptor levels in SERT(-/-) rats. SERT(-/-) showed blunted fear potentiation and enhanced contextual fear, which resulted from a deficit in fear acquisition. Paroxetine treatment did not affect acquisition or expression of fear-potentiated startle, suggesting that disturbed fear learning in SERT(-/-) results from developmental changes and not from reduced SERT functioning. Although CRF1 receptor levels did not differ significantly between genotypes, CP154,526 treatment normalized both cue- and contextual fear in SERT(-/-) during acquisition, but not expression of fear-potentiated startle. The disrupted fear acquisition and concomitant increase in contextual conditioned fear-potentiated startle fear in SERT(-/-) resembles the associative learning deficit seen in patients with panic disorder and suggests that normal SERT functioning is crucial for the development of an adequate fear neuro-circuitry. Moreover, the normalization of fear acquisition by CP154,526 suggests a role for central CRF signaling in the generalization of fear., (Copyright © 2015 Elsevier B.V. and ECNP. All rights reserved.)

Psychopharmacology has had some bad publicity lately. Frankly, there have been some major problems along the way in developing new effective drugs for psychiatric disorders. After a prolonged period of high investments but low success rates, big pharmaceutical companies seem to retract their activities in the psychopharmacology field. Yet, the burden of mental disorders is likely to keep on growing in the next decades. In this position paper, we focus on drug development for depression and anxiety disorders, to narrow the scope of the assay. We describe the current situation of the psychopharmacology field, and analyse some of the methods and paradigms that have brought us here, but which should perhaps change to bring us even further. In addition, some of the factors contributing to the current stagnation in psychopharmacology are discussed. Finally, we suggest a number of changes that could lead to a more rational strategy for central nervous system drug development and which may circumvent some of the pitfalls leading to "me too" approaches. Central to the suggested changes, is the notion that mental disorders do not lead to several symptoms, but a network of causally related symptoms convolutes into a mental disorder. We call upon academia to put these changes in the early phases of drug development into effect., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Stress, through corticotropin-releasing factor (CRF), influences all aspects of cocaine addiction. Earlier studies suggest that individual differences in responsivity to stress affect susceptibility to develop addiction. We have previously found that CRF over-expression alters individual differences in behavioural responses to novelty stress in mice. Therefore, we hypothesised that post-natal, long-term over-expression of brain CRF may alter the rewarding effects of cocaine in a manner that is sensitive to individual differences. In this study we specifically investigated cocaine-induced conditioned place preference (CPP) in transgenic mice over-expressing CRF (CRF-OE) and in wild-type (WT) littermates after determining their individual locomotor and emotional responsivity to inescapable novelty. CRF-OE mice showed decreased overall locomotor activity and increased anxiety-like behaviour in response to novelty compared to WT mice. Low behavioural reactivity to novelty (LR) was associated with heightened anxiety-like behaviour in CRF-OE, but not in WT, mice. WT and CRF-OE mice developed CPP equally to both low (5mg/kg) and high (20mg/kg) doses of cocaine. However, LR CRF-OE mice expressed significantly stronger cocaine CPP than transgenic mice with high locomotor response to novelty (HR). In WT mice, on the other hand, stronger CPP induced by 20mg/kg of cocaine was found in the HR animals. Furthermore, there was a strong negative correlation between locomotor reactivity to novelty and CPP in CRF-OE, but not in WT, mice. Collectively, these results suggest that long-term, post-natal CRF over-expression increases the rewarding effects of cocaine in individuals with high emotional response to stress., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The separation-induced vocalization test in guinea pig pups is one of many that has been used to screen for anxiolytic-like properties of drugs. The test is based on the cross-species phenomenon that infants emit distress calls when placed in social isolation. Here we report a systematic review and meta-analysis of pharmacological intervention in the separation-induced vocalization test in guinea pig pups. Electronic databases were searched for original research articles, yielding 32 studies that met inclusion criteria. We extracted data on pharmacological intervention, animal and methodological characteristics, and study quality indicators. Meta-analysis showed that the different drug classes in clinical use for the treatment of anxiety disorders, have comparable effects on vocalization behaviour, irrespective of their mechanism of action. Of the experimental drugs, nociception (NOP) receptor agonists proved very effective in this test. Analysis further indicated that the commonly used read-outs total number and total duration of vocalizations are equally valid. With regard to methodological characteristics, repeated testing of pups as well as selecting pups with moderate or high levels of vocalization were associated with larger treatment effects. Finally, reporting of study methodology, randomization and blinding was poor and Egger's test for small study effects showed that publication bias likely occurred. This review illustrates the value of systematic reviews and meta-analyses in improving translational value and methodological aspects of animal models. It further shows the urgent need to implement existing publication guidelines to maximize the output and impact of experimental animal studies., (Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.)

Rationale: As enhanced corticotropin-releasing factor (CRF) transmission is associated with induction of sensorimotor gating deficits, CRF₁ receptor antagonists may reverse disrupted prepulse inhibition (PPI), an operational measure of sensorimotor gating., Objectives: To determine the effects of CRF₁ receptor antagonists in pharmacological models of disrupted PPI and to determine if long-term elevated central CRF levels alter sensitivity towards PPI disrupting drugs., Methods: CP154,526 (10-40 mg/kg), SSR125543 (3-30 mg/kg) and DMP695 (40 mg/kg) were tested on PPI disruption provoked by D-amphetamine (2.5, 3 mg/kg), ketamine (5, 30 mg/kg) and MK801 (0.2, 0.5 mg/kg) in Wistar rats, C57Bl/6J and CD1 mice, and on spontaneously low PPI in Iffa Credo rats and DBA/2J mice. PPI-disrupting effects of D-amphetamine (2.5-5 mg/kg) and MK801 (0.3-1 mg/kg) were examined in CRF-overexpressing (CRFtg) mice, which display PPI deficits. Finally, we determined the influence of CP154,526 on D-amphetamine-induced dopamine outflow in nucleus accumbens and prefrontal cortex of CRFtg mice using in vivo microdialysis., Results: No CRF₁-antagonists improved PPI deficits in any test. CRFtg mice showed blunted PPI disruption in response to MK801, but not D-amphetamine. Further, D-amphetamine-induced dopamine release was less pronounced in CRFtg versus wild-type mice, a response normalized by pretreatment with CP154,526., Conclusion: The inability of CRF₁ receptor antagonists to block pharmacological disruption of sensorimotor gating suggests that the involvement of CRF₁ receptors in the modulation of dopaminergic and glutamatergic neurotransmission relevant for sensory gating is limited. Furthermore, the alterations observed in CRFtg mice support the notion that long-term elevated central CRF levels induce changes in these neurotransmitter systems.

Corticotropin-releasing factor (CRF) is implicated in the pathogenesis of bipolar disorder, an illness associated with deficits in prepulse inhibition (PPI) of the acoustic startle response. Valproate is used in the treatment of bipolar disorder and may alter CRF activity via a GABA(A)-ergic mechanism. This study determined the effect of valproate on CRF-disrupted PPI and examined the role of the hypothalamic-pituitary-adrenal axis and GABA-ergic signaling in the effect of valproate. Valproate (60-240 mg/kg) dose-dependently reversed PPI deficits displayed by transgenic mice overexpressing CRF (CRFtg), and normalized PPI deficits induced by CRF i.c.v. infusion in 129Sv mice. Valproate enhanced corticosterone secretion more effectively in CRFtg than in wild-type mice. The effect of valproate on PPI was not blocked by the GABA(A) receptor antagonist bicuculline, the GABA(B) receptor antagonists phaclofen and SCH 50911 or combined administration of a GABA(A) and GABA(B) receptor antagonist. The beneficial effect of valproate on PPI was not mimicked by the GABA(A) receptor agonist muscimol, the GABA transaminase inhibitor vigabatrin, the histone deacetylase (HDAC) inhibitor sodium butyrate or by the mood stabilizers lithium, carbamazepine, lamotrigine or topiramate. Thus, we showed that valproate improves CRF-induced PPI deficits, albeit via a so far unknown mechanism. These marked beneficial effects of valproate on CRF-induced sensorimotor gating deficits suggest that valproate may be of particular value in specific subgroups of bipolar patients that are characterized by alterations in the CRF system., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The ability to identify predictors of aversive events allows organisms to appropriately respond to these events, and failure to acquire these fear contingencies can lead to maladaptive contextual anxiety. Recently, preclinical studies demonstrated that the corticotropin-releasing factor and serotonin systems are interactively involved in adaptive fear acquisition. Here, 150 healthy medication-free human subjects completed a cue and context fear conditioning procedure in a virtual reality environment. Fear potentiation of the eyeblink startle reflex (FPS) was measured to assess both uninstructed fear acquisition and instructed fear expression. All participants were genotyped for polymorphisms located within regulatory regions of the corticotropin releasing hormone receptor 1 (CRHR1 - rs878886) and the serotonin transporter (5HTTLPR). These polymorphisms have previously been linked to panic disorder and anxious symptomology and personality, respectively. G-allele carriers of CRHR1 (rs878886) showed no acquisition of fear conditioned responses (FPS) to the threat cue in the uninstructed phase, whereas fear acquisition was present in C/C homozygotes. Moreover, carrying the risk alleles of both rs878886 (G-allele) and 5HTTLPR (short allele) was associated with increased FPS to the threat context during this phase. After explicit instructions regarding the threat contingency were given, the cue FPS and context FPS normalized in all genotype groups. The present results indicate that genetic variability in the corticotropin-releasing hormone receptor 1, especially in interaction with the 5HTTLPR, is involved in the acquisition of fear in humans. This translates prior animal findings to the human realm.

Rationale: Repeated activation of corticotropin-releasing factor (CRF) receptors is associated with increased anxiety and enhanced stress responsivity, which may be mediated via limbic GABAergic and glutamatergic transmission., Objective: The present study investigated molecular and functional alterations in GABA(A) receptor (GABA(A)R) and metabotropic glutamate receptor (mGluR) responsivity in transgenic mice that chronically overexpress CRF., Methods: CRF(1) receptor, GABA(A)R, and mGluR sensitivity were determined in CRF-overexpressing mice using the stress-induced hyperthermia (SIH) test. In addition, we measured mRNA expression levels of GABA(A)R α subunits and mGluRs in the amygdala and hypothalamus., Results: CRF-overexpressing mice were less sensitive to the anxiolytic effects of the CRF(1) receptor antagonists CP154,526 and DMP695, the GABA(A)R α(3)-selective agonist TP003 (0-3 mg/kg) and the mGluR(2/3) agonist LY379268 (0-10 mg/kg) in the SIH test. The hypothermic effect of the non-selective GABA(A)R agonist diazepam (0-4 mg/kg) and the α(1)-subunit-selective GABA(A)R agonist zolpidem (0-10 mg/kg) was reduced in CRF-overexpressing mice. No genotype differences were found using the GABA(A)R α(5)-subunit preferential compound SH-053-2'F-R-CH(3) and mGluR(5) antagonists MPEP and MTEP. CRF-overexpressing mice showed decreased expression levels of GABA(A)R α(2) subunit and mGluR(3) mRNA levels in the amygdala, whereas these expression levels were increased in the hypothalamus. CRF-overexpressing mice also showed increased hypothalamic mRNA levels of α(1) and α(5) GABA(A)R subunits., Conclusions: We found that lifelong CRF overproduction is associated with altered gene expression and reduced functional sensitivity of discrete GABA(A) and mGluR receptor subtypes. These findings suggest that sustained over-activation of cerebral CRF receptors may contribute to the development of altered stress-related behavior via modulation of GABAergic and glutamatergic transmission.

Background: Within the GABA(A)-receptor field, two important questions are what molecular mechanisms underlie benzodiazepine tolerance, and whether tolerance can be ascribed to certain GABA(A)-receptor subtypes., Methods: We investigated tolerance to acute anxiolytic, hypothermic and sedative effects of diazepam in mice exposed for 28-days to non-selective/selective GABA(A)-receptor positive allosteric modulators: diazepam (non-selective), bretazenil (partial non-selective), zolpidem (α(1) selective) and TPA023 (α(2/3) selective). In-vivo binding studies with [(3)H]flumazenil confirmed compounds occupied CNS GABA(A) receptors., Results: Chronic diazepam treatment resulted in tolerance to diazepam's acute anxiolytic, hypothermic and sedative effects. In mice treated chronically with bretazenil, tolerance to diazepam's anxiolytic and hypothermic, but not sedative, effects was seen. Chronic zolpidem treatment resulted in tolerance to diazepam's hypothermic effect, but partial anxiolytic tolerance and no sedative tolerance. Chronic TPA023 treatment did not result in tolerance to diazepam's hypothermic, anxiolytic or sedative effects., Conclusions: OUR DATA INDICATE THAT: (i) GABA(A)-α(2)/α(3) subtype selective drugs might not induce tolerance; (ii) in rodents quantitative and temporal variations in tolerance development occur dependent on the endpoint assessed, consistent with clinical experience with benzodiazepines (e.g., differential tolerance to antiepileptic and anxiolytic actions); (iii) tolerance to diazepam's sedative actions needs concomitant activation of GABA(A)-α(1)/GABA(A)-α(5) receptors. Regarding mechanism, in-situ hybridization studies indicated no gross changes in expression levels of GABA(A) α(1), α(2) or α(5) subunit mRNA in hippocampus or cortex. Since selective chronic activation of either GABA(A) α(2), or α(3) receptors does not engender tolerance development, subtype-selective GABA(A) drugs might constitute a promising class of novel drugs.

"Ecstasy" [3,4-methylenedioxymetamphetamine (MDMA)] is of considerable interest in light of its prosocial properties and risks associated with widespread recreational use. Recently, it was found to bind trace amine-1 receptors (TA(1)Rs), which modulate dopaminergic transmission. Accordingly, using mice genetically deprived of TA(1)R (TA(1)-KO), we explored their significance to the actions of MDMA, which robustly activated human adenylyl cyclase-coupled TA(1)R transfected into HeLa cells. In wild-type (WT) mice, MDMA elicited a time-, dose-, and ambient temperature-dependent hypothermia and hyperthermia, whereas TA(1)-KO mice displayed hyperthermia only. MDMA-induced increases in dialysate levels of dopamine (DA) in dorsal striatum were amplified in TA(1)-KO mice, despite identical levels of MDMA itself. A similar facilitation of the influence of MDMA upon dopaminergic transmission was acquired in frontal cortex and nucleus accumbens, and induction of locomotion by MDMA was haloperidol-reversibly potentiated in TA(1)-KO versus WT mice. Conversely, genetic deletion of TA(1)R did not affect increases in DA levels evoked by para-chloroamphetamine (PCA), which was inactive at hTA(1) sites. The TA(1)R agonist o-phenyl-3-iodotyramine (o-PIT) blunted the DA-releasing actions of PCA both in vivo (dialysis) and in vitro (synaptosomes) in WT but not TA(1)-KO animals. MDMA-elicited increases in dialysis levels of serotonin (5-HT) were likewise greater in TA(1)-KO versus WT mice, and 5-HT-releasing actions of PCA were blunted in vivo and in vitro by o-PIT in WT mice only. In conclusion, TA(1)Rs exert an inhibitory influence on both dopaminergic and serotonergic transmission, and MDMA auto-inhibits its neurochemical and functional actions by recruitment of TA(1)R. These observations have important implications for the effects of MDMA in humans.

In rodents, administration of a mixture of the psychostimulant d-amphetamine and the benzodiazepine chlordiazepoxide results in supra-additive hyperlocomotion, a phenomenon used to identify mood stabilizers. In an attempt to determine whether the d-amphetamine/chlordiazepoxide assay could extend to other behaviors that are affected in mania, we evaluated the effects of the mixture on prepulse inhibition. In addition, we combined chlordiazepoxide with the selective dopamine reuptake inhibitor GBR 12909 or the noradrenergic stimulant (-) ephedrine, and tested these alternative mixtures in locomotor activity and prepulse inhibition tests. Chlordiazepoxide (3mg/kg) robustly potentiated amphetamine-induced hyperactivity, but did not change the amphetamine-induced disruption of prepulse inhibition. This indicates that the d-amphetamine-chlordiazepoxide-induced hyperlocomotion does not extend to other dopamine-driven behaviors. GBR 12909 (16mg/kg) and (-) ephedrine (50mg/kg) both enhanced locomotor activity and disrupted PPI, but combined treatment of either of these compounds with chlordiazepoxide had no significant additive effect on locomotor activity or prepulse inhibition. These findings suggest that the effect of the d-amphetamine/chlordiazepoxide mixture cannot be accounted for by the dopamine enhancing properties of amphetamine alone. Last, valproic acid (120-240mg/kg) did not reduce the GBR-induced hyperactivity. Therefore, further pharmacological evaluation of GBR 12909-induced hyperactivity is warranted to determine its pharmacological potential to model mania-like behavior. Based on the current results, it is concluded that the utility of the pharmacological d-amphetamine/chlordiazepoxide assay as a tool to study brain mechanisms relevant to mania is limited., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The 5-HT(1B) receptor has been implicated in disorders such as depression, anxiety and obsessive-compulsive disorder. In mice lacking the 5-HT(1B) receptor (5-HT(1B) knockout mice), important changes in physiology and behavior exist. In the absence of presynaptic 5-HT(1B) receptor inhibition, chronic SSRI treatment may differentially affect 5-HT(1A) receptor functionality. The present studies tested the hypothesis that chronically reducing 5-HT transporter (5-HTT) function with selective serotonin reuptake inhibitor (SSRI) treatment would accelerate 5-HT(1A) receptor desensitization in 5-HT(1B) knockout mice. Moreover, as 5-HT(1B) knockout mice have been found to display exaggerated autonomic and locomotor responses to environmental stressors, the effects of chronic SSRI treatment on the hyperreactive phenotype of 5-HT(1B) knockout mice were investigated. The stress-reducing effect of the 5-HT(1A) receptor agonist flesinoxan on increases in body temperature, heart rate and locomotor activity was similar in wild type and 5-HT(1B) knockout mice before and after chronic 21-day treatment with the SSRI fluvoxamine, indicating no apparent alteration of 5-HT(1A) receptor sensitivity in 5-HT(1B) knockout mice. Also, chronic SSRI treatment did not alter the increased stress reactivity to mild environmental stressors in 5-HT(1B) knockout mice. We demonstrate that no apparent differences in 5-HT(1A) receptor sensitivity occur between 5-HT(1B) knockout and wild type mice after chronic fluvoxamine treatment. Also, the hyperreactive phenotype of 5-HT(1B) knockout mice is unresponsive to chronic SSRI treatment. Taken together, these results indicate that constitutive absence of 5-HT(1B) receptors does not result in adaptive changes in 5-HT(1A) receptor functionality and that chronic SSRI treatment does not modify stress reactivity in 5-HT(1B) knockout mice., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We examined juvenile social recognition and discrimination in mice with early post-natal onset, transgenic CRF over-expression (CRF-OE) and in their wild-type littermates (WT). CRF-OE mice showed enhanced social investigation during the first encounter, normal short-term and facilitated long-term social recognition memory, compared to WT. These results suggest that chronically elevated brain CRF tone may contribute in better remembering ethologically relevant and emotionally salient stimuli, such as social interaction., (Copyright © 2010 Elsevier B.V. All rights reserved.)

Rationale: Early life stress is a risk factor for the development of psychopathology in later life. Consequences of adverse life events, however, may depend on the genetic makeup of an individual. Reduced serotonin(1A) receptor function may predispose to the development of anxiety disorders., Objective: Determine susceptibility of serotonin(1A) receptor knockout (1AKO) mice on different background strains to the effects of maternal separation (MS) by assessing startle plasticity in adulthood., Methods: 1AKO mice on a 129S6 and a Swiss Webster (SW) background were used. MS groups were separated daily from their mother for 180 min/day from postnatal days 2 to 14. Control groups underwent normal animal facility rearing. In adulthood, effects on acoustic startle response, habituation, prepulse inhibition (PPI), and foot shock sensitization were determined., Results: MS increased startle reactivity and reduced PPI in 129S6 mice. These effects of MS were independent of genotype. MS had no effect on the other readouts. In SW mice, MS had no consistent effect on startle reactivity and did not alter startle plasticity in wild type or in 1AKO mice. 1AKO mice did not differ from wild-type mice in startle plasticity., Conclusion: Serotonin(1A) receptor deletion does not enhance vulnerability to the effects of MS on startle plasticity. The life-long increase in startle reactivity and PPI deficit induced by MS are strain-dependent. Further, the use of startle reactivity and plasticity may have added value in translational studies relating to early life stress.

Increased central corticotropin-releasing factor (CRF) signaling has been associated with various psychiatric symptoms, including anxiety, depression and psychosis. CRF signaling in both the basolateral amygdala (BLA) and medial prefrontal cortex (mPFC) has been implicated in anxiety-like behavior. In addition, repeated activation of CRF receptors within the BLA induces a chronic anxious state. Here we studied the effects of local repeated CRF infusion in the BLA and mPFC on different forms of anxiety, as assessed during light-enhanced startle (LES, general anxiety) and acquisition of fear-potentiated startle (FPS, cue-conditioned fear). In addition, as CRF has been implicated in sensorimotor gating, prepulse inhibition (PPI) was assessed to determine if local CRF infusion within the BLA or mPFC would interfere with the processing of sensory information. To this end, canulas were placed bilaterally in either the BLA or mPFC of Wistar rats. After recovery, animals were infused with h/rCRF (200 ng/side) or vehicle for five consecutive days. Long term effects of local CRF infusion on LES and acquisition of FPS were measured 4 and 10 days post-treatment, respectively. In addition, the acute (day 1), sub-chronic (day 5) and long-term (7 days post treatment) effects on PPI were measured in the same animals. A clear regional differentiation was found on the long lasting effect of CRF on anxiety-like behavior: infusion into the BLA only enhanced acquisition of FPS, whereas infusion into the mPFC only enhanced LES. Sub-chronic CRF infusion into the BLA, but not the mPFC, disrupted PPI. This disturbed PPI was normalized 7 days post-treatment. Together, the current study shows that local repeated CRF receptor activation in the BLA and mPFC is differentially involved in anxiety- and fear-related behavior. In addition, the BLA may be involved in CRF-induced sensorimotor gating deficits. The absence of a long-term effect on these PPI deficits suggests that lasting activation of CRF receptors is a prerequisite for CRF-mediated effects on sensorimotor gating. The long-term effects of repeated CRF infusion on LES and acquisition of FPS on the other hand, show that in case of anxiety-related processes repeated CRF infusion may have lasting effects., (© 2011 IBRO. Published by Elsevier Ltd. All rights reserved.)

Background: Stress results in hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis, characterized by increased central CRF activity, elevated circulating glucocorticoid levels, impaired glucocorticoid-mediated negative feedback and abnormal hippocampal functions, possibly contributing to the development of behavioral pathologies, such as depression. The hippocampus is critically involved in the control of the HPA axis as well as in explicit memory, contextual aspects of fear, organization of the behavioral response to environmental novelty and in habituation. We have previously shown that mice that over-express CRF in the brain exhibit impaired novelty detection and altered psychophysiological and behavioral habituation, functions linked to dopamine receptor-dependent hippocampal plasticity., Objective and Methods: Therefore, the aim of the present study was to measure D1 and D2 dopamine receptor expression and related signaling, such as CREB and ERK protein levels and phosphorylation, in the hippocampus and other brain regions of mice with post-natal CRF over-expression (CRF-OE mice)., Results: We found a region-specific down-regulation of both D1 and D2 protein expression, without altered CRF receptor protein expression, in the hippocampus in CRF-OE mice. This was accompanied by an impaired phosphorylation of hippocampal CREB, but not ERK1 and ERK2, in the same animals., Conclusions: These results suggest that post-natal onset CRF over-expression results in an impairment of dopamine signaling in the hippocampus, which may underlie cognitive and motivational aspects of stress-related, CRF-driven mood disorders.

In order to evaluate the influence of changes in affective state on light-enhanced startle, the effects of positive affect, induced by acute cocaine administration, and the effect of negative affect, induced by spontaneous cocaine withdrawal-induced anxiety, were studied. Acute cocaine administration decreased LES, whereas withdrawal from chronic cocaine administration exacerbated LES 24h after withdrawal, an effect indicative of increased anxiety. This exacerbated LES was reduced, but not back to normal, 4 days after withdrawal. The finding that both cocaine-induced positive and negative affect can be detected in LES, suggests that this may be a valuable tool in studying affect regulation in rodents., ((c) 2010 Elsevier B.V. All rights reserved.)

Rationale: Stress-related disorders are associated with dysfunction of both serotonergic and GABAergic pathways, and clinically effective anxiolytics act via both neurotransmitter systems. As there is evidence that the GABA(A) and the serotonin receptor system interact, a serotonergic component in the anxiolytic actions of benzodiazepines could be present., Objectives: The main aim of the present study was to investigate whether the anxiolytic effects of (non-)selective alpha subunit GABA(A) receptor agonists could be reversed with 5-HT(1A) receptor blockade using the stress-induced hyperthermia (SIH) paradigm., Results: The 5-HT(1A) receptor antagonist WAY-100635 (0.1-1 mg/kg) reversed the SIH-reducing effects of the non-alpha-subunit selective GABA(A) receptor agonist diazepam (1-4 mg/kg) and the GABA(A) receptor alpha(3)-subunit selective agonist TP003 (1 mg/kg), whereas WAY-100635 alone was without effect on the SIH response or basal body temperature. At the same time, co-administration of WAY-100635 with diazepam or TP003 reduced basal body temperature. WAY-100635 did not affect the SIH response when combined with the preferential alpha(1)-subunit GABA(A) receptor agonist zolpidem (10 mg/kg), although zolpidem markedly reduced basal body temperature., Conclusions: The present study suggests an interaction between GABA(A) receptor alpha-subunits and 5-HT(1A) receptor activation in the SIH response. Specifically, our data indicate that benzodiazepines affect serotonergic signaling via GABA(A) receptor alpha(3)-subunits. Further understanding of the interactions between the GABA(A) and serotonin system in reaction to stress may be valuable in the search for novel anxiolytic drugs.

Rationale and Objectives: The light-enhanced startle paradigm (LES) is suggested to model anxiety, because of the non-specific cue and the long-term effect. In contrast, the fear-potentiated startle (FPS) is suggested to model conditioned fear. However, the pharmacological profiles of these two paradigms are very similar. The present study investigated the effects of putative anxiogenic drugs on LES and FPS and aimed at determining the sensitivity of LES for anxiogenic drugs and to potentially showing a pharmacological differentiation between these two paradigms., Methods: Male Wistar rats received each dose of the alpha(2)-adrenoceptor antagonist yohimbine (0.25-1.0mg/kg), the 5-HT(2C) receptor agonist m-chlorophenylpiperazine (mCPP, 0.5-2.0mg/kg) or the GABA(A) inverse receptor agonist pentylenetetrazole (PTZ, 3-30mg/kg) and were subsequently tested in either LES or FPS., Results: None of the drugs enhanced LES, whereas mCPP increased percentage FPS and yohimbine increased absolute FPS values. Furthermore, yohimbine increased baseline startle amplitude in the LES, while mCPP suppressed baseline startle in both the LES and FPS and PTZ suppressed baseline startle in the FPS., Conclusions: In contrast to findings in the FPS paradigm, none of the drugs were able to exacerbate the LES response. Thus, a clear pharmacological differentiation was found between LES and FPS., (Copyright 2010 Elsevier Inc. All rights reserved.)

The first line of antidepressant treatment nowadays are selective serotonin reuptake inhibitors. Although they are relatively safe to use, selective serotonin reuptake inhibitors (SSRIs) can induce severe side effects. New promising antidepressants may be the triple monoamine reuptake inhibitors, which not only enhance serotonin and norepinephrine neurotransmission, but also increase brain dopamine levels. Recently it has been shown that one of the triple reuptake inhibitors, DOV 216,303 has antidepressant-like effects in the olfactory bulbectomy (OBX) model of depression, but the alterations in monoaminergic neurotransmission in these animals are still unknown. In the present study we investigated not only the effect of acute, but also chronic treatment of DOV 216,303 in OBX rats on monoamine and metabolite levels. The main results are decreased baseline dopamine levels in the prefrontal cortex one day after OBX, while 38days after OBX no difference could be observed in monoamine levels after vehicle treatment. Treatment with DOV 216,303 leads to increased extracellular levels of serotonin and norepinephrine neurotransmission, but also increased dopamine levels in OBX animals as well as their controls. This increase could be observed after one single administration, but also after chronic treatment. However, a DOV 216,303 challenge in chronically treated animals resulted in lower monoamine concentrations than the same challenge in untreated animals. More research is needed to investigate this seemingly hyporesponsivity to chronic DOV 216,303 treatment., (Copyright 2010 Elsevier B.V. All rights reserved.)

Affective startle modulation is used to study emotional reactivity in humans, and blunted affective startle modulation has been reported in depressed patients. To determine whether blunted affective startle modulation is also a common feature in animal models for affective disorders, light-enhanced startle was studied in three models: inescapable foot shock (IFS), repeated restraint stress (RRS) and olfactory bulbectomy (OBX). In addition, prepulse inhibition was studied in these models. Light-enhanced startle was blunted following IFS and OBX and RRS decreased overall startle responding. Prepulse inhibition, however, was unaffected. These findings indicate that induction models for affective disorders may be associated with long term effects on affective startle modulation. The lack of changes in sensory motor gating suggests that these changes can be ascribed to alterations in emotional reactivity. In conclusion, our results indicate that the blunted affective startle modulation seen in animal models for affective disorders may be used to examine the mechanisms underlying altered emotional reactivity., (Copyright 2009 Elsevier B.V. All rights reserved.)

Background: The amygdala is involved in the coordination of stress but is also an important gatekeeper involved in the regulation of vigilance. The amygdala is structurally complex, consisting of several nuclei with specific functions in the affective response to environmental stimuli. There are indications that the medial amygdaloid nucleus may be a pivotal player in acute responses to emotional environmental stimuli., Methods: The present study therefore aimed to study the effects of bilateral electrolytic lesions of the medial amygdala on unconditioned anxiety-related behavior as well as a sensorimotor gating parameter (prepulse inhibition, PPI) in rats. Anxiety-related behavior was assessed with the use of stress-induced hyperthermia (SIH), light-enhanced startle (LES) and open field behavior., Results: Bilateral electrolytic lesions of the medial amygdala decreased the SIH response and anxiety-related open field behavior. In contrast, lesioned animals displayed augmented LES and disrupted PPI. No changes in basal locomotor activity, body temperature and acoustic startle were found between lesioned and sham animals., Conclusions: The present study suggests that the medial amygdala is an important player in response to acute environmental stimuli. Decreased unconditioned psychological stress responses were found, whereas LES was enhanced and sensorimotor processing was disrupted. However, considering the existing data on basolateral amygdala involvement in PPI and bed nucleus of the stria terminalis involvement in LES, local infusion studies into the MeA should be performed to further substantiate these findings., (Copyright (c) 2010 Elsevier Inc. All rights reserved.)

Background: Early-life stress may affect 5-HT(1A) receptor circuitry, which could result in increased anxiety in later life. An increased anxiety phenotype in 5-HT(1A) receptor KO mice (1AKO) mice has been ascribed to 5-HT(1A) receptor absence during the early postnatal period. Thus, subtle and transient serotonergic changes during the early postnatal period may lead to an increased risk for developing stress-related disorders during adulthood., Methods: Wildtype and 1AKO mice on a Swiss-Webster (SW) background were treated during the early postnatal period with vehicle or the 5-HT(1A) receptor antagonist WAY-100,635., Results: Pharmacologic 5-HT(1A) receptor blockade during the early postnatal period induced long-lasting effects on anxiety and benzodiazepine sensitivity in adolescent and adult mice on a Swiss-Webster background and resembles the SW 1AKO phenotype. Furthermore, WAY-100,635-treated mice had increased cortical gamma-aminobutyric acid-A receptor (GABA(A)R) alpha(1) and alpha(3) subunit levels and increased hippocampal GABA(A)R alpha(2) subunit levels., Conclusions: Absence of 5-HT(1A)R signaling during early stages of brain maturation predisposes an organism to affective dysfunction later in life. Because early-life treatment with WAY-100,635 in Swiss-Webster mice reduced diazepam sensitivity and increased GABA(A)R alpha subunit levels in the prefrontal cortex and hippocampus, our data suggest a putative link between early-life disruption of the serotonergic system and the emergence of increased anxiety and decreased benzodiazepine responsivity at adult age. Moreover, early-life 5-HT(1A) receptor functionality appears to be essential for the development of normal GABA(A)R functionality. This study may have clinical implications for psychoactive drug use during pregnancy and for the pharmacogenetic background of benzodiazepine sensitivity., (2010 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

It has been suggested that different brain areas are involved in the modulation and expression of fear and anxiety. In the present study we investigated these potential differences by using the fear-potentiated-startle (FPS) and light-enhanced-startle (LES) paradigms to differentiate between fear and anxiety, respectively. Male Wistar rats were tested in the FPS and LES paradigm and perfused 1 h after the test session. Fos immunoreactivity (IR) was quantified in 21 brain areas and compared between FPS, LES and four control conditions. Both FPS and LES procedures significantly enhanced the acoustic startle response. A principal component analysis of Fos-IR-data showed that 70% of the changes in Fos-IR could be explained by three independent components: an arousal-component, identifying brain areas known to be activated under conditions of vigilance, arousal and stress, a LES- and an FPS-component. The LES component comprised the septohippocampal system and functionally interrelated areas including nucleus accumbens, anterior cingulate cortex, lateral habenula and supramammillary areas, but not the dorsolateral part of the bed nucleus of the stria terminalis. The central amygdaloid nucleus and the dorsolateral part of the bed nucleus of the stria terminalis loaded exclusively on the FPS component. Analysis of the separate brain areas revealed significantly higher Fos-IR in LES relative to FPS in the anterior cingulate cortex, nucleus accumbens shell, lateral septum, lateral habenula and area postrema. We conclude that the neural circuitry activated during FPS and LES shows clear differences. In anxiety as induced by LES, activation of the septohippocampal system and related areas seems to play a major role. In fear as induced by FPS, the central amygdaloid nucleus and the dorsolateral part of the bed nucleus of the stria terminalis loaded on the same component, but Fos-IR observed in these brain regions did not differentiate between anxiety and fear. Furthermore, principal-component analysis appears a useful tool in detecting and describing correlated changes in patterns of neuronal activity.

Background: Stress-induced hyperthermia (SIH) is the transient rise in body temperature after encountering a stressor. The SIH response can be blocked by administration of various anxiolytic drugs prior to inducing stress. However, a drug injection involves handling and injection stress and therefore induces a SIH response itself. In the standard SIH test, drugs are therefore injected 60 min before stress induction to allow injection-induced hyperthermia to decline. This makes it difficult to study putative anxiolytic compounds with a short half-life. The present study therefore aimed to compare the effects of standard (stressful) and stress-free anxiolytic drug administration on the subsequent SIH response with a 10-minute injection-stressor interval., Methods: Anxiolytic drugs with short half-lives (midazolam, 8-OH-DPAT, nicotine) were injected subcutaneously in rats using either a stressful (manual injection) or stress-free injection (subcutaneous cannula) method 10 min before novel cage stress. Body temperature and locomotor activity were measured using telemetric transmitters., Results: Stressful and stress-free drug administration resulted in comparable drug effects on the stress-induced hyperthermia and locomotor responses in rats., Conclusion: The present study shows that both stressful and stress-free drug injection shortly before a stressor results in reproducible attenuation of the SIH response in rats. In rats, a short injection-stressor interval can therefore be applied using the SIH model, enabling the study of putative anxiolytic drugs with short half-lives.

Treatment with pramipexole, a dopamine D(3)/D(2) receptor agonist, reduces depressive symptoms in patients suffering from Parkinson's disease. To test the putative antidepressant quality of pramipexole, its effects were assessed in one of the most attractive animal models of depression, the olfactory bulbectomized (OBX) rat. Two experiments studied the effects of pramipexole on bulbectomy-induced hyperactivity. In experiment I, pramipexole was tested at 0.3 and 1.0 mg/kg together with the reference dopamine D(3) receptor agonist 7-OH-DPAT (0.1 mg/kg) and the tri-cyclic antidepressant imipramine (10 mg/kg). In experiment II, pramipexole was tested at lower doses: 0.03 and 0.1 mg/kg, with the same reference compounds. All animals were tested in the open field on days one (acute), seven (sub-chronic) and fourteen (chronic) of administration, as well as one week after cessation of treatment. Pramipexole, in a U-shaped dose response, reduced bulbectomy-induced hyperactivity after (sub) chronic but not acute administration (like imipramine and 7-OH-DPAT). The highest dose of pramipexole (1.0 mg/kg) did not reduce OBX hyperactivity during treatment. However, one week after cessation of treatment, all pramipexole (including the 1.0 mg/kg dose), 7-OH-DPAT and imipramine groups showed a reduction in OBX-induced hyperactivity. Pramipexole and 7-OH-DPAT exert an antidepressant profile in the OBX-rat model in normalizing bulbectomy-induced hyperactivity during (sub) chronic treatment. Moreover, treatment with both these compounds induced long-lasting changes in the bulbectomized brain similar to established antidepressants, strongly predicting antidepressant activity in major depression.