Cross-disorder risk gene CACNA1C differentially modulates susceptibility to psychiatric disorders during development and adulthood.

Single-nucleotide polymorphisms (SNPs) in CACNA1C, the α1C subunit of the voltage-gated L-type calcium channel Cav1.2, rank among the most consistent and replicable genetics findings in psychiatry and have been associated with schizophrenia, bipolar disorder and major depression. However, genetic variants of complex diseases often only confer a marginal increase in disease risk, which is additionally influenced by the environment. Here we show that embryonic deletion of Cacna1c in forebrain glutamatergic neurons promotes the manifestation of endophenotypes related to psychiatric disorders including cognitive decline, impaired synaptic plasticity, reduced sociability, hyperactivity and increased anxiety. Additional analyses revealed that depletion of Cacna1c during embryonic development also increases the susceptibility to chronic stress, which suggest that Cav1.2 interacts with the environment to shape disease vulnerability. Remarkably, this was not observed when Cacna1c was deleted in glutamatergic neurons during adulthood, where the later deletion even improved cognitive flexibility, strengthened synaptic plasticity and induced stress resilience. In a parallel gene × environment design in humans, we additionally demonstrate that SNPs in CACNA1C significantly interact with adverse life events to alter the risk to develop symptoms of psychiatric disorders. Overall, our results further validate Cacna1c as a cross-disorder risk gene in mice and humans, and additionally suggest a differential role for Cav1.2 during development and adulthood in shaping cognition, sociability, emotional behavior and stress susceptibility. This may prompt the consideration for pharmacological manipulation of Cav1.2 in neuropsychiatric disorders with developmental and/or stress-related origins.

Forebrain glutamatergic, but not GABAergic, neurons mediate anxiogenic effects of the glucocorticoid receptor.

Anxiety disorders constitute a major disease and social burden worldwide; however, many questions concerning the underlying molecular mechanisms still remain open. Besides the involvement of the major excitatory (glutamate) and inhibitory (gamma aminobutyric acid (GABA)) neurotransmitter circuits in anxiety disorders, the stress system has been directly implicated in the pathophysiology of these complex mental illnesses. The glucocorticoid receptor (GR) is the major receptor for the stress hormone cortisol (corticosterone in rodents) and is widely expressed in excitatory and inhibitory neurons, as well as in glial cells. However, currently it is unknown which of these cell populations mediate GR actions that eventually regulate fear- and anxiety-related behaviors. In order to address this question, we generated mice lacking the receptor specifically in forebrain glutamatergic or GABAergic neurons by breeding GRflox/flox mice to Nex-Cre or Dlx5/6-Cre mice, respectively. GR deletion specifically in glutamatergic, but not in GABAergic, neurons induced hypothalamic-pituitary-adrenal axis hyperactivity and reduced fear- and anxiety-related behavior. This was paralleled by reduced GR-dependent electrophysiological responses in the basolateral amygdala (BLA). Importantly, viral-mediated GR deletion additionally showed that fear expression, but not anxiety, is regulated by GRs in glutamatergic neurons of the BLA. This suggests that pathological anxiety likely results from altered GR signaling in glutamatergic circuits of several forebrain regions, while modulation of fear-related behavior can largely be ascribed to GR signaling in glutamatergic neurons of the BLA. Collectively, our results reveal a major contribution of GRs in the brain's key excitatory, but not inhibitory, neurotransmitter system in the regulation of fear and anxiety behaviors, which is crucial to our understanding of the molecular mechanisms underlying anxiety disorders.

CRF receptor type 2 neurons in the posterior bed nucleus of the stria terminalis critically contribute to stress recovery.

The bed nucleus of the stria terminalis (BNST) is critical in mediating states of anxiety, and its dysfunction has been linked to stress-related mental disease. Although the anxiety-related role of distinct subregions of the anterior BNST was recently reported, little is known about the contribution of the posterior BNST (pBNST) to the behavioral and neuroendocrine responses to stress. Previously, we observed abnormal expression of corticotropin-releasing factor receptor type 2 (CRFR2) to be associated with post-traumatic stress disorder (PTSD)-like symptoms. Here, we found that CRFR2-expressing neurons within the pBNST send dense inhibitory projections to other stress-related brain regions (for example, the locus coeruleus, medial amygdala and paraventricular nucleus), implicating a prominent role of these neurons in orchestrating the neuroendocrine, autonomic and behavioral response to stressful situations. Local CRFR2 activation by urocortin 3 depolarized the cells, increased the neuronal input resistance and increased firing of action potentials, indicating an enhanced excitability. Furthermore, we showed that CRFR2-expressing neurons within the pBNST are critically involved in the modulation of the behavioral and neuroendocrine response to stress. Optogenetic activation of CRFR2 neurons in the pBNST decreased anxiety, attenuated the neuroendocrine stress response, ameliorated stress-induced anxiety and impaired the fear memory for the stressful event. Moreover, activation following trauma exposure reduced the susceptibility for PTSD-like symptoms. Optogenetic inhibition of pBNST CRFR2 neurons yielded opposite effects. These data indicate the relevance of pBNST activity for adaptive stress recovery.

Beware of your Cre-Ation: lacZ expression impairs neuronal integrity and hippocampus-dependent memory.

Expression of the lacZ-sequence is a widely used reporter-tool to assess the transgenic and/or transfection efficacy of a target gene in mice. Once activated, lacZ is permanently expressed. However, protein accumulation is one of the hallmarks of neurodegenerative diseases. Furthermore, the protein product of the bacterial lacZ gene is ß-galactosidase, an analog to the mammalian senescence-associated ß-galactosidase, a molecular marker for aging. Therefore we studied the behavioral, structural and molecular consequences of lacZ expression in distinct neuronal sub-populations. lacZ expression in cortical glutamatergic neurons resulted in severe impairments in hippocampus-dependent memory accompanied by marked structural alterations throughout the CNS. In contrast, GFP expression or the expression of the ChR2/YFP fusion product in the same cell populations did not result in either cognitive or structural deficits. GABAergic lacZ expression caused significantly decreased hyper-arousal and mild cognitive deficits. Attenuated structural and behavioral consequences of lacZ expression could also be induced in adulthood, and lacZ transfection in neuronal cell cultures significantly decreased their viability. Our findings provide a strong caveat against the use of lacZ reporter mice for phenotyping studies and point to a particular sensitivity of the hippocampus formation to detrimental consequences of lacZ expression. © 2016 Wiley Periodicals, Inc.

Evidence for the role of corticotropin-releasing factor in major depressive disorder.

Major depressive disorder (MDD) is a devastating disease affecting over 300 million people worldwide, and costing an estimated 380 billion Euros in lost productivity and health care in the European Union alone. Although a wealth of research has been directed toward understanding and treating MDD, still no therapy has proved to be consistently and reliably effective in interrupting the symptoms of this disease. Recent clinical and preclinical studies, using genetic screening and transgenic rodents, respectively, suggest a major role of the CRF1 gene, and the central expression of CRF1 receptor protein in determining an individual's risk of developing MDD. This gene is widely expressed in brain tissue, and regulates an organism's immediate and long-term responses to social and environmental stressors, which are primary contributors to MDD. This review presents the current state of knowledge on CRF physiology, and how it may influence the occurrence of symptoms associated with MDD. Additionally, this review presents findings from multiple laboratories that were presented as part of a symposium on this topic at the annual 2014 meeting of the International Behavioral Neuroscience Society (IBNS). The ideas and data presented in this review demonstrate the great progress that has been made over the past few decades in our understanding of MDD, and provide a pathway forward toward developing novel treatments and detection methods for this disorder.

Enriched environment impacts trimethylthiazoline-induced anxiety-related behavior and immediate early gene expression: critical role of Crhr1.

It has been shown previously (Sotnikov et al., ) that mice selectively inbred for high anxiety-related behavior (HAB) vs. low anxiety-related behavior in the elevated plus maze differentially respond to trimethylthiazoline (TMT), a synthetic fox fecal odor. However, less is known about whether environmental factors can rescue these extreme phenotypes. Here, we found that an enriched environment (EE) provided during early adolescence induced anxiolytic effects in HAB (HAB-EE) mice, rescuing their strong avoidance behavior induced by TMT. In a series of experiments, the contribution of maternal, juvenile and adolescent behavior to the anxiolytic effects elicited by EE was investigated. At the molecular level, using c-fos expression mapping, we found that the activity of the medial and basolateral amygdala was significantly reduced in HAB-EE mice after TMT exposure. We further analysed the expression of Crhr1, as its amount in the amygdala has been reported to be important for the regulation of anxiety-related behavior after EE. Indeed, in situ hybridisation indicated significantly decreased Crhr1 expression in the basolateral and central amygdala of HAB-EE mice. To further test the involvement of Crhr1 in TMT-induced avoidance, we exposed conditional glutamatergic-specific Crhr1-knockout mice to the odor. The behavioral response of Crhr1-knockout mice mimicked that of HAB-EE mice, and c-fos expression in the amygdala after TMT exposure was significantly lower compared with controls, thereby further supporting a critical involvement of Crhr1 in environmentally-induced anxiolysis. Altogether, our results indicate that EE can rescue strong avoidance of TMT by HAB mice with Crhr1 expression in the amygdala being critically involved.

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.

Conditional corticotropin-releasing hormone overexpression in the mouse forebrain enhances rapid eye movement sleep.

Impaired sleep and enhanced stress hormone secretion are the hallmarks of stress-related disorders, including major depression. The central neuropeptide, corticotropin-releasing hormone (CRH), is a key hormone that regulates humoral and behavioral adaptation to stress. Its prolonged hypersecretion is believed to play a key role in the development and course of depressive symptoms, and is associated with sleep impairment. To investigate the specific effects of central CRH overexpression on sleep, we used conditional mouse mutants that overexpress CRH in the entire central nervous system (CRH-COE-Nes) or only in the forebrain, including limbic structures (CRH-COE-Cam). Compared with wild-type or control mice during baseline, both homozygous CRH-COE-Nes and -Cam mice showed constantly increased rapid eye movement (REM) sleep, whereas slightly suppressed non-REM sleep was detected only in CRH-COE-Nes mice during the light period. In response to 6-h sleep deprivation, elevated levels of REM sleep also became evident in heterozygous CRH-COE-Nes and -Cam mice during recovery, which was reversed by treatment with a CRH receptor type 1 (CRHR1) antagonist in heterozygous and homozygous CRH-COE-Nes mice. The peripheral stress hormone levels were not elevated at baseline, and even after sleep deprivation they were indistinguishable across genotypes. As the stress axis was not altered, sleep changes, in particular enhanced REM sleep, occurring in these models are most likely induced by the forebrain CRH through the activation of CRHR1. CRH hypersecretion in the forebrain seems to drive REM sleep, supporting the notion that enhanced REM sleep may serve as biomarker for clinical conditions associated with enhanced CRH secretion.

Amygdaloid pERK1/2 in corticotropin-releasing hormone overexpressing mice under basal and acute stress conditions.

Corticotropin-releasing hormone (CRH) coordinates neuroendocrine and behavioral adaptations to stress. Acute CRH administration in vivo activates extracellular signal-regulated kinase 1/2 (ERK1/2) in limbic brain areas, acting through the CRH receptor type 1 (CRH-R1). In the present study, we used CRH-COE-Cam mice that overexpress CRH in limbic-restricted areas, to analyze the effect of chronic CRH overexpression on ERK1/2 activation. By immunohistochemistry and confocal microscopy analysis we found that pERK1/2 levels in the basolateral amygdala (BLA) were similar in control and CRH overexpressing mice under basal conditions. Acute stress caused comparably increased levels of corticosterone in both control (CRH-COEcon-Cam) and CRH overexpressing (CRH-COEhom-Cam) animals. CRH-COEhom-Cam mice after stress showed reduced pERK1/2 immunoreactivity in the BLA compared to CRH-COEhom-Cam animals under basal conditions. Radioligand binding and in situ hybridization revealed higher density of CRH-R1 in the amygdala of CRH-COEhom mice under basal conditions compared to control littermates. A significant reduction of the receptor levels was observed in this area after acute stress, suggesting that stress may trigger CRH-R1 internalization/downregulation in these CRH overexpressing mice. Chronic CRH overexpression leads to reduced ERK1/2 activation in response to acute stress in the BLA.

Endocannabinoids mediate acute fear adaptation via glutamatergic neurons independently of corticotropin-releasing hormone signaling.

Recent evidence showed that the endocannabinoid system plays an important role in the behavioral adaptation of stress and fear responses. In this study, we chose a behavioral paradigm that includes criteria of both fear and stress responses to assess whether the involvement of endocannabinoids in these two processes rely on common mechanisms. To this end, we delivered a footshock and measured the fear response to a subsequently presented novel tone stimulus. First, we exposed different groups of cannabinoid receptor type 1 (CB(1))-deficient mice (CB(1) (-/-)) and their wild-type littermates (CB(1) (+/+)) to footshocks of different intensities. Only application of an intense footshock resulted in a sustained fear response to the tone in CB(1) (-/-). Using the intense protocol, we next investigated whether endocannabinoids mediate their effects via an interplay with corticotropin-releasing hormone (CRH) signaling. Pharmacological blockade of CB(1) receptors by rimonabant in mice deficient for the CRH receptor type 1 (CRHR1(-/-)) or type 2 (CRHR2(-/-)), and in respective wild-type littermates, resulted in a sustained fear response in all genotypes. This suggests that CRH is not involved in the fear-alleviating effects of CB(1). As CRHR1(-/-) are known to be severely impaired in stress-induced corticosterone secretion, our observation also implicates that corticosterone is dispensable for CB(1)-mediated acute fear adaptation. Instead, conditional mutants with a specific deletion of CB(1) in principal neurons of the forebrain (CaMK-CB(1) (-/-)), or in cortical glutamatergic neurons (Glu-CB(1) (-/-)), showed a similar phenotype as CB(1) (-/-), thus indicating that endocannabinoid-controlled glutamatergic transmission plays an essential role in acute fear adaptation.

Transgenic overexpression of corticotropin releasing hormone provides partial protection against neurodegeneration in an in vivo model of acute excitotoxic stress.

Corticotropin releasing hormone (CRH) is the central modulator of the mammalian hypothalamic-pituitary-adrenal (HPA) axis. In addition, CRH affects other processes in the brain including learning, memory, and synaptic plasticity. Moreover, CRH has been shown to play a role in nerve cell survival under apoptotic conditions and to serve as an endogenous neuroprotectant in vitro. Employing mice overexpressing murine CRH in the CNS, we observed a differential response of CRH-overexpressing mice (CRH-COEhom-Nes) to acute excitotoxic stress induced by kainate compared with controls (CRH-COEcon-Nes). Interestingly, CRH-overexpression reduced the duration of epileptic seizures and prevented kainate-induced neurodegeneration and neuroinflammation in the hippocampus. Our findings highlight a neuroprotective action of CRH in vivo. This neuroprotective effect was accompanied by increased levels of brain-derived neurotrophic factor (BDNF) in CRH-COEhom-Nes mice, suggesting a potential role for BDNF in mediating CRH-induced neuroprotective actions against acute excitotoxicity in vivo.

Genetic mouse models for behavioral analysis through transgenic RNAi technology.

Pharmacological inhibitors and knockout mice have developed into routine tools to analyze the role of specific genes in behavior. Both strategies have limitations like the availability of inhibitors for only a subset of proteins and the large efforts required to construct specific mouse mutants. The recent emergence of RNA interference (RNAi)-mediated gene silencing provides a fast alternative that can be applied to any coding gene. We established an approach for the efficient generation of transgenic knockdown mice by targeted insertion of short hairpin (sh) RNA vectors into a defined genomic locus and studied the efficiency of gene silencing in the adult brain and the utility of such mice for behavioral analysis. We generated shRNA knockdown mice for the corticotropin-releasing hormone receptor type 1 (Crhr1), the leucine-rich repeat kinase 2 (Lrkk2) and the purinergic receptor P2X ligand-gated ion channel 7 (P2rx7) genes and show the ubiquitous expression of shRNA and efficient suppression of the target mRNA and protein in the brain of young and 11-month-old knockdown mice. Knockdown mice for the Crhr1 gene exhibited decreased anxiety-related behavior, an impaired stress response, and thereby recapitulate the phenotype of CRHR1 knockout mice. Our results show the feasibility of gene silencing in the adult brain and validate knockdown mice as new genetic models suitable for behavioral analysis.

Conditional mouse mutants highlight mechanisms of corticotropin-releasing hormone effects on stress-coping behavior.

Hypersecretion of central corticotropin-releasing hormone (CRH) has been implicated in the pathophysiology of affective disorders. Both, basic and clinical studies suggested that disrupting CRH signaling through CRH type 1 receptors (CRH-R1) can ameliorate stress-related clinical conditions. To study the effects of CRH-R1 blockade upon CRH-elicited behavioral and neurochemical changes we created different mouse lines overexpressing CRH in distinct spatially restricted patterns. CRH overexpression in the entire central nervous system, but not when overexpressed in specific forebrain regions, resulted in stress-induced hypersecretion of stress hormones and increased active stress-coping behavior reflected by reduced immobility in the forced swim test and tail suspension test. These changes were related to acute effects of overexpressed CRH as they were normalized by CRH-R1 antagonist treatment and recapitulated the effect of stress-induced activation of the endogenous CRH system. Moreover, we identified enhanced noradrenergic activity as potential molecular mechanism underlying increased active stress-coping behavior observed in these animals. Thus, these transgenic mouse lines may serve as animal models for stress-elicited pathologies and treatments that target the central CRH system.

Differential disinhibition of the neonatal hypothalamic- pituitary-adrenal axis in brain-specific CRH receptor 1-knockout mice.

In the adult, corticotropin-releasing hormone (CRH) is the key mediator for the behavioural and neuroendocrine response to stress. It has also been hypothesized that, during postnatal development of the stress system, CRH controls the activity of the HPA axis and mediates the effects of early disturbances, e.g. 24 h of maternal deprivation. In the current study we investigated the function of specific brain corticotropin-releasing hormone receptor type 1 (CRHR1) subpopulations in the control of the HPA axis during postnatal development under basal conditions as well as after 24 h of maternal deprivation. We used two conditional CRHR1-deficient mouse lines which lack this receptor, either specifically in forebrain and limbic structures (Cam-CRHR1) or in all neurons (Nes-CRHR1). Basal circulating corticosterone was increased in Nes-CRHR1 mice compared to controls. Corticosterone response to maternal deprivation was significantly increased in both CRHR1-deficient lines. In the paraventricular nucleus, Cam-CRHR1 animals displayed enhanced CRH and decreased vasopressin expression levels. In contrast, gene expression in Nes-CRHR1 pups was strikingly similar to that in maternally deprived control pups. Furthermore, maternal deprivation resulted in an enhanced response of Cam-CRHR1 pups in the brain, while expression levels in Nes-CRHR1 mouse pups were mostly unchanged. Our results demonstrate that brainstem and/or hypothalamic CRHR1 contribute to the suppression of basal corticosterone secretion in the neonate, while limbic and/or forebrain CRHR1 dampen the activation of the neonatal HPA axis induced by maternal deprivation.