A brain-enriched circular RNA controls excitatory neurotransmission and restricts sensitivity to aversive stimuli.

Circular RNAs (circRNAs) are a large class of noncoding RNAs. Despite the identification of thousands of circular transcripts, the biological significance of most of them remains unexplored, partly because of the lack of effective methods for generating loss-of-function animal models. In this study, we focused on circTulp4, an abundant circRNA derived from the Tulp4 gene that is enriched in the brain and synaptic compartments. By creating a circTulp4-deficient mouse model, in which we mutated the splice acceptor site responsible for generating circTulp4 without affecting the linear mRNA or protein levels, we were able to conduct a comprehensive phenotypic analysis. Our results demonstrate that circTulp4 is critical in regulating neuronal and brain physiology, modulating the strength of excitatory neurotransmission and sensitivity to aversive stimuli. This study provides evidence that circRNAs can regulate biologically relevant functions in neurons, with modulatory effects at multiple levels of the phenotype, establishing a proof of principle for the regulatory role of circRNAs in neural processes.

Complex Long-term Effects of Radiation on Adult Mouse Behavior.

We have shown previously that a single radiation event (0.063, 0.125 or 0.5 Gy, 0.063 Gy/min) in adult mice (age 10 weeks) can have delayed dose-dependent effects on locomotor behavior 18 months postirradiation. The highest dose (0.5 Gy) reduced, whereas the lowest dose (0.063 Gy) increased locomotor activity at older age independent of sex or genotype. In the current study we investigated whether higher doses administered at a higher dose rate (0.5, 1 or 2 Gy, 0.3 Gy/min) at the same age (10 weeks) cause stronger or earlier effects on a range of behaviors, including locomotion, anxiety, sensorimotor and cognitive behavior. There were clear dose-dependent effects on spontaneous locomotor and exploratory activity, anxiety-related behavior, body weight and affiliative social behavior independent of sex or genotype of wild-type and Ercc2S737P heterozygous mice on a mixed C57BL/6JG and C3HeB/FeJ background. In addition, smaller genotype- and dose-dependent radiation effects on working memory were evident in males, but not in females. The strongest dose-dependent radiation effects were present 4 months postirradiation, but only effects on affiliative social behaviors persisted until 12 months postirradiation. The observed radiation-induced behavioral changes were not related to alterations in the eye lens, as 4 months postirradiation anterior and posterior parts of the lens were still normal. Overall, we did not find any sensitizing effect of the mutation towards radiation effects in vivo.

Post-synaptic scaffold protein TANC2 in psychiatric and somatic disease risk.

Understanding the shared genetic aetiology of psychiatric and medical comorbidity in neurodevelopmental disorders (NDDs) could improve patient diagnosis, stratification and treatment options. Rare tetratricopeptide repeat, ankyrin repeat and coiled-coil containing 2 (TANC2)-disrupting variants were disease causing in NDD patients. The post-synaptic scaffold protein TANC2 is essential for dendrite formation in synaptic plasticity and plays an unclarified but critical role in development. We here report a novel homozygous-viable Tanc2-disrupted function model in which mutant mice were hyperactive and had impaired sensorimotor gating consistent with NDD patient psychiatric endophenotypes. Yet, a multi-systemic analysis revealed the pleiotropic effects of Tanc2 outside the brain, such as growth failure and hepatocellular damage. This was associated with aberrant liver function including altered hepatocellular metabolism. Integrative analysis indicates that these disrupted Tanc2 systemic effects relate to interaction with Hippo developmental signalling pathway proteins and will increase the risk for comorbid somatic disease. This highlights how NDD gene pleiotropy can augment medical comorbidity susceptibility, underscoring the benefit of holistic NDD patient diagnosis and treatment for which large-scale preclinical functional genomics can provide complementary pleiotropic gene function information.

Diabetes type 2 risk gene Dusp8 is associated with altered sucrose reward behavior in mice and humans.

BACKGROUND: Dusp8 is the first GWAS-identified gene that is predominantly expressed in the brain and has previously been linked with the development of diabetes type 2 in humans. In this study, we unravel how Dusp8 is involved in the regulation of sucrose reward behavior. METHODS: Female, chow-fed global Dusp8 WT and KO mice were tested in an observer-independent IntelliCage setup for self-administrative sucrose consumption and preference followed by a progressive ratio task with restricted sucrose access to monitor seeking and motivation behavior. Sixty-three human carriers of the major C and minor T allele of DUSP8 SNP rs2334499 were tested for their perception of food cues by collecting a rating score for sweet versus savory high caloric food. RESULTS: Dusp8 KO mice showed a comparable preference for sucrose, but consumed more sucrose compared to WT mice. In a progressive ratio task, Dusp8 KO females switched to a "trial and error" strategy to find sucrose while control Dusp8 WT mice kept their previously established seeking pattern. Nonetheless, the overall motivation to consume sucrose, and the levels of dopaminergic neurons in the brain areas NAcc and VTA were comparable between genotypes. Diabetes-risk allele carriers of DUSP8 SNP rs2334499 preferred sweet high caloric food compared to the major allele carriers, rating scores for savory food remained comparable between groups. CONCLUSION: Our data suggest a novel role for Dusp8 in the perception of sweet high caloric food as well as in the control of sucrose consumption and foraging in mice and humans.

Dusp8 affects hippocampal size and behavior in mice and humans.

Dual-specificity phosphatase 8 (Dusp8) acts as physiological inhibitor for the MAPKs Jnk, Erk and p38 which are involved in regulating multiple CNS processes. While Dusp8 expression levels are high in limbic areas such as the hippocampus, the functional role of Dusp8 in hippocampus morphology, MAPK-signaling, neurogenesis and apoptosis as well as in behavior are still unclear. It is of particular interest whether human carriers of a DUSP8 allelic variant show similar hippocampal alterations to mice. Addressing these questions using Dusp8 WT and KO mouse littermates, we found that KOs suffered from mildly impaired spatial learning, increased locomotor activity and elevated anxiety. Cell proliferation, apoptosis and p38 and Jnk phosphorylation were unaffected, but phospho-Erk levels were higher in hippocampi of the KOs. Consistent with a decreased hippocampus size in Dusp8 KO mice, we found reduced volumes of the hippocampal subregions subiculum and CA4 in humans carrying the DUSP8 allelic variant SNP rs2334499:C > T. Overall, aberrations in morphology and behavior in Dusp8 KO mice and a decrease in hippocampal volume of SNP rs2334499:C > T carriers point to a novel, translationally relevant role of Dusp8 in hippocampus function that warrants further studies on the role of Dusp8 within the limbic network.

Analysis of locomotor behavior in the German Mouse Clinic.

BACKGROUND: Generation and phenotyping of mutant mouse models continues to increase along with the search for the most efficient phenotyping tests. Here we asked if a combination of different locomotor tests is necessary for comprehensive locomotor phenotyping, or if a large data set from an automated gait analysis with the CatWalk system would suffice. NEW METHOD: First we endeavored to meaningfully reduce the large CatWalk data set by Principal Component Analysis (PCA) to decide on the most relevant parameters. We analyzed the influence of sex, body weight, genetic background and age. Then a combination of different locomotor tests was analyzed to investigate the possibility of redundancy between tests. RESULT: The extracted 10 components describe 80% of the total variance in the CatWalk, characterizing different aspects of gait. With these, effects of CatWalk version, sex, body weight, age and genetic background were detected. In addition, the PCA on a combination of locomotor tests suggests that these are independent without significant redundancy in their locomotor measures. COMPARISON WITH EXISTING METHODS: The PCA has permitted the refinement of the highly dimensional CatWalk (and other tests) data set for the extraction of individual component scores and subsequent analysis. CONCLUSION: The outcome of the PCA suggests the possibility to focus on measures of the front and hind paws, and one measure of coordination in future experiments to detect phenotypic differences. Furthermore, although the CatWalk is sensitive for detecting locomotor phenotypes pertaining to gait, it is necessary to include other tests for comprehensive locomotor phenotyping.

Understanding gene functions and disease mechanisms: Phenotyping pipelines in the German Mouse Clinic.

Since decades, model organisms have provided an important approach for understanding the mechanistic basis of human diseases. The German Mouse Clinic (GMC) was the first phenotyping facility that established a collaboration-based platform for phenotype characterization of mouse lines. In order to address individual projects by a tailor-made phenotyping strategy, the GMC advanced in developing a series of pipelines with tests for the analysis of specific disease areas. For a general broad analysis, there is a screening pipeline that covers the key parameters for the most relevant disease areas. For hypothesis-driven phenotypic analyses, there are thirteen additional pipelines with focus on neurological and behavioral disorders, metabolic dysfunction, respiratory system malfunctions, immune-system disorders and imaging techniques. In this article, we give an overview of the pipelines and describe the scientific rationale behind the different test combinations.

Assessing Sociability, Social Memory, and Pup Retrieval in Mice.

Adaptive social behavior is important in mammals, both for the well-being of the individual and for the thriving of the species. Dysfunctions in social behavior occur in many neurodevelopmental and psychiatric diseases, and research into the genetic components of disease-relevant social deficits can open up new avenues for understanding the underlying biological mechanisms and therapeutic interventions. Genetically modified mouse models are particularly useful in this respect, and robust experimental protocols are needed to reliably assess relevant social behavior phenotypes. Here we describe in detail three protocols to quantitatively measure sociability, one of the most frequently investigated social behavior phenotypes in mice, using a three-chamber sociability test. These protocols can be extended to also assess social memory. In addition, we provide a detailed protocol on pup retrieval, which is a particularly robust maternal behavior amenable to various scientific questions. © 2017 by John Wiley & Sons, Inc.

Alterations in neuronal control of body weight and anxiety behavior by glutathione peroxidase 4 deficiency.

Elevated levels of oxidative stress and neuronal inflammation in the hypothalamus or ventral midbrain, respectively, represent common denominators for obesity and Parkinson's Disease (PD). However, little is known about defense mechanisms that protect neurons in these regions from oxidative damage. Here, we aimed to assess whether murine Gpx4, a crucial antioxidant enzyme that protects neurons from membrane damage and ferroptosis, is critical for the protection from neuronal inflammation in two distinct pathophysiologic diseases, namely metabolic dysfunction in diet-induced obesity or PD. Gpx4 was deleted from either AgRP or POMC neurons in the hypothalamus, essential for metabolic homeostasis, or from dopaminergic neurons in the ventral midbrain, governing behaviors such as anxiety or voluntary movement. To induce a pro-inflammatory environment, AgRP and POMC neuron-specific Gpx4 knockout mice were subjected to high-fat high-sucrose (HFHS) diet. To exacerbate oxidative stress in dopaminergic neurons of the ventral midbrain, we systemically co-deleted the PD-related gene DJ-1. Gpx4 was dispensable for the maintenance of cellular health and function of POMC neurons, even in mice exposed to obesogenic conditions. In contrast, HFHS-fed mice with Gpx4 deletion from AgRP neurons displayed increased body adiposity. Gpx4 expression and activity were diminished in the hypothalamus of HFHS-fed mice compared to standard diet-fed controls. Gpx4 deletion from dopaminergic neurons induced anxiety behavior, and diminished spontaneous locomotor activity when DJ-1 was co-deleted. Overall, these data suggest a physiological role for Gpx4 in balancing metabolic control signals and inflammation in AgRP but not POMC neurons. Moreover, Gpx4 appears to constitute an important rheostat against neuronal dysfunction and PD-like symptoms in dopaminergic circuitry within the ventral midbrain.

Serum Response Factor (SRF) Ablation Interferes with Acute Stress-Associated Immediate and Long-Term Coping Mechanisms.

Stress experience modulates behavior, metabolism, and energy expenditure of organisms. One molecular hallmark of an acute stress response is a rapid induction of immediate early genes (IEGs) such as c-Fos and Egr family members. IEG transcription in neurons is mediated by the neuronal activity-driven gene regulator serum response factor (SRF). We show a first role of SRF in immediate and long-lasting acute restraint stress (AS) responses. For this, we employed a standardized mouse phenotyping protocol at the German Mouse Clinic (GMC) including behavioral, metabolic, and cardiologic tests as well as gene expression profiling to analyze the consequences of forebrain-specific SRF deletion in mice exposed to AS. Adult mice with an SRF deletion in glutamatergic neurons (Srf; CaMKIIa-CreERT2 ) showed hyperactivity, decreased anxiety, and impaired working memory. In response to restraint AS, instant stress reactivity including locomotor behavior and corticosterone induction was impaired in Srf mutant mice. Interestingly, even several weeks after previous AS exposure, SRF-deficient mice showed long-lasting AS-associated changes including altered locomotion, metabolism, energy expenditure, and cardiovascular changes. This suggests a requirement of SRF for mediating long-term stress coping mechanisms in wild-type mice. SRF ablation decreased AS-mediated IEG induction and activity of the actin severing protein cofilin. In summary, our data suggest an SRF function in immediate AS reactions and long-term post-stress-associated coping mechanisms.

Conditional Reduction of Adult Born Doublecortin-Positive Neurons Reversibly Impairs Selective Behaviors.

Adult neurogenesis occurs in the adult mammalian subventricular zone (SVZ) along the walls of the lateral ventricles and the subgranular zone (SGZ) of the hippocampal dentate gyrus. While a burgeoning body of research implicates adult neurogenesis in olfactory bulb (OB)- and hippocampal-related behaviors, the precise function continues to elude. To further assess the behavioral importance of adult neurogenesis, we herein generated a novel inducible transgenic mouse model of adult neurogenesis reduction where mice with CreER(T2) under doublecortin (DCX) promoter control were crossed with mice where diphtheria toxin A (DTA) was driven by the Rosa26 promoter. Activation of DTA, through the administration of tamoxifen (TAM), results in a specific reduction of DCX+ immature neurons in both the hippocampal dentate gyrus and OB. We show that the decrease of DCX+ cells causes impaired social discrimination ability in both young adult (from 3 months) and middle aged (from 10 months) mice. Furthermore, these animals showed an age-independent altered coping behavior in the Forced Swim Test without clear changes in anxiety-related behavior. Notably, these behavior changes were reversible on repopulating the neurogenic zones with DCX+ cells on cessation of the TAM treatment, demonstrating the specificity of this effect. Overall, these results support the notion that adult neurogenesis plays a role in social memory and in stress coping but not necessarily in anxiety-related behavior.

Tests for Anxiety-Related Behavior in Mice.

Phenotyping of inbred mouse strains and genetically modified mouse models for characteristics related to neuropsychiatric diseases includes assessing their anxiety-related behavior. A variety of tests have been developed to measure anxiety in laboratory rodents and these tests have been placed under scrutiny over the years concerning their validity. Here we describe the most widely used tests for anxiety in mice. The protocols we present are established methods used in the German Mouse Clinic (GMC), with which alterations in anxiety could successfully be discovered in mouse mutants. Moreover, since baseline anxiety levels in mice are easily influenced by a great variety of disturbances, we carefully outline the critical parameters that need to be considered.

Assessing Cognition in Mice.

Genetically modified mouse models have proven useful to study learning and memory processes and the neurocircuitry and molecular mechanisms involved, as well as to develop therapies for diseases involving cognitive impairment. A variety of tests have been developed to measure cognition in mice, and here we present those established and regularly used in the German Mouse Clinic. The test paradigms have been carefully chosen according to reliability of results and disease relevance of the cognitive functions assessed. Further criteria were time efficiency and ease of application. All tests assess slightly different but also overlapping or interacting aspects of learning and memory so that they can be used to complement each other in a comprehensive assessment of cognitive function. The five protocols described are for spontaneous alternation in the Y-maze, social discrimination, object recognition, automated assessment of learning and memory using the IntelliCage, and olfactory discrimination learning.

A robust and reliable non-invasive test for stress responsivity in mice.

Stress and an altered stress response have been associated with many multifactorial diseases, such as psychiatric disorders or neurodegenerative diseases. As currently mouse mutants for each single gene are generated and phenotyped in a large-scale manner, it seems advisable also to test these mutants for alterations in their stress responses. Here we present the determinants of a robust and reliable non-invasive test for stress-responsivity in mice. Stress is applied through restraining the mice in tubes and recording behavior in the Open Field 20 min after cessation of the stress. Two hours, but not 15 or 50 min of restraint lead to a robust and reproducible increase in distance traveled and number of rearings during the first 5 min in the Open Field in C57BL/6 mice. This behavioral response is blocked by the corticosterone synthesis inhibitor metyrapone, but not by RU486 treatment, indicating that it depends on corticosteroid secretion, but is not mediated via the glucocorticoid receptor type II. We assumed that with a stress duration of 15 min one could detect hyper-responsivity, and with a stress duration of 2 h hypo-responsivity in mutant mouse lines. This was validated with two mutant lines known to show opposing effects on corticosterone secretion after stress exposure, corticotropin-releasing hormone (CRH) over-expressing mice and CRH receptor 1 knockout (KO) mice. Both lines showed the expected phenotype, i.e., increased stress responsivity in the CRH over-expressing mouse line (after 15 min restraint stress) and decreased stress responsivity in the CRHR1-KO mouse line (after 2 h of restraint stress). It is possible to repeat the acute stress test several times without the stressed animal adapting to it, and the behavioral response can be robustly evoked at different ages, in both sexes and in different mouse strains. Thus, locomotor and rearing behavior in the Open Field after an acute stress challenge can be used as reliable, non-invasive indicators of stress responsivity and corticosterone secretion in mice.