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Microglia, resident myeloid immune cells of the central nervous system (CNS), actively shape the circuitry of the brain, maintain CNS homeostasis during the steady state and orchestrate immune responses upon CNS injury. Both canonical and non-canonical functions of the macroautophagy/autophagy-related protein ATG5 regulate myeloid cell survival and immune responses. Here, we report that loss of ATG5 in postnatal microglia does not perturb CNS tissue integrity, microglial cell survival, or immune activation. Learning task performances were unchanged in mutant mice. Furthermore, lack of ATG5 expression in microglia had no impact on the development of experimental autoimmune encephalomyelitis. These data indicate that, basal autophagy, identified to be essential for the survival and function of neuronal cells, is not required to maintain CNS homeostasis if absent in adult microglia and ATG5 expression is dispensable for the development of autoimmune neuroinflammation. Abbreviations Ag, antigen; APC, antigen presenting cell; ATG/Atg, autophagy-related; CD, cluster of differentiation; CNS, central nervous system; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; fl, floxed; LAP, LC3-associated phagocytosis; LC3, microtubule-associated protein 1 light chain 3; MFI, median fluorescence intensity; MHCII, major histocompatibility complex class II; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between animal facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multicentre study, whereby inbred C57BL/6J mice from a single breeding cohort were allocated to and reared in 5 different animal facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of the rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that environmental variation among animal facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organisation. We detected changes in chromatin organisation in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity, and regulation of behaviour. Our findings demonstrate that common environmental differences between animal facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and thus argue that study designs should take environmental background into account to increase the robustness and replicability of findings.

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions 1–4. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment 5. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between rearing facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multi-center study, where inbred mice from the same breeding stock were reared in five different facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that common variation among rearing facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organization. We detected changes in chromatin organization in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity and regulation of behavior. Our findings demonstrate that common environmental differences between rearing facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. We expect our findings to stimulate further research into the mechanisms and drivers of these epigenetic changes mediated by the laboratory environment. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and a need to account for the animals’ environmental background in study design to produce robust and replicable findings.

Circuit formation in the central nervous system has been historically studied during development, after which cell-autonomous and nonautonomous wiring factors inactivate. In principle, balanced reactivation of such factors could enable further wiring in adults, but their relative contributions may be circuit dependent and are largely unknown. Here, we investigated hippocampal mossy fiber sprouting to gain insight into wiring mechanisms in mature circuits. We found that sole ectopic expression of Id2 in granule cells is capable of driving mossy fiber sprouting in healthy adult mouse and rat. Mice with the new mossy fiber circuit solved spatial problems equally well as controls but appeared to rely on local rather than global spatial cues. Our results demonstrate reprogrammed connectivity in mature neurons by one defined factor and an assembly of a new synaptic circuit in adult brain., Proceedings of the National Academy of Sciences of the United States of America, 118 (40), ISSN:0027-8424, ISSN:1091-6490

Random errors in protein synthesis are prevalent and ubiquitous, yet their effect on organismal health has remained enigmatic for over five decades. Here, we studied whether mice carrying the ribosomal ambiguity (ram) mutation Rps2-A226Y, recently shown to increase the inborn error rate of mammalian translation, if at all viable, present any specific, possibly aging-related, phenotype. We introduced Rps2-A226Y using a Cre/loxP strategy. Resulting transgenic mice were mosaic and showed a muscle-related phenotype with reduced grip strength. Analysis of gene expression in skeletal muscle using RNA-Seq revealed transcriptomic changes occurring in an age-dependent manner, involving an interplay of PGC1α, FOXO3, mTOR, and glucocorticoids as key signaling pathways, and finally resulting in activation of a muscle atrophy program. Our results highlight the relevance of translation accuracy, and show how disturbances thereof may contribute to age-related pathologies., Communications Biology, 4 (1), ISSN:2399-3642

The key role of APP for Alzheimer pathogenesis is well established. However, perinatal lethality of germline knockout mice lacking the entire APP family has so far precluded the analysis of its physiological functions for the developing and adult brain. Here, we generated conditional APP/APLP1/APLP2 triple KO (cTKO) mice lacking the APP family in excitatory forebrain neurons from embryonic day 11.5 onwards. NexCre cTKO mice showed altered brain morphology with agenesis of the corpus callosum and disrupted hippocampal lamination. Further, NexCre cTKOs revealed reduced basal synaptic transmission and drastically reduced long-term potentiation that was associated with reduced dendritic length and reduced spine density of pyramidal cells. With regard to behavior, lack of the APP family leads not only to severe impairments in a panel of tests for learning and memory, but also to an autism-like phenotype including repetitive rearing and climbing, impaired social communication, and deficits in social interaction. Together, our study identifies essential functions of the APP family during development, for normal hippocampal function and circuits important for learning and social behavior., The EMBO Journal, 40 (12), ISSN:0261-4189, ISSN:1460-2075

Amyloid-β precursor protein (APP) is central to the pathogenesis of Alzheimer’s disease, yet its physiological functions remain incompletely understood. Previous studies had indicated important synaptic functions of APP and the closely related homologue APLP2 in excitatory forebrain neurons for spine density, synaptic plasticity, and behavior. Here, we show that APP is also widely expressed in several interneuron subtypes, both in hippocampus and cortex. To address the functional role of APP in inhibitory neurons, we generated mice with a conditional APP/APLP2 double knockout (cDKO) in GABAergic forebrain neurons using DlxCre mice. These DlxCre cDKO mice exhibit cognitive deficits in hippocampus-dependent spatial learning and memory tasks, as well as impairments in species-typic nesting and burrowing behaviors. Deficits at the behavioral level were associated with altered neuronal morphology and synaptic plasticity Long-Term Potentiation (LTP). Impaired basal synaptic transmission at the Schafer collateral/CA1 pathway, which was associated with altered compound excitatory/inhibitory synaptic currents and reduced action potential firing of CA1 pyramidal cells, points to a disrupted excitation/inhibition balance in DlxCre cDKOs. Together, these impairments may lead to hippocampal dysfunction. Collectively, our data reveal a crucial role of APP family proteins in inhibitory interneurons to maintain functional network activity.

Although most nervous system diseases affect women and men differentially, most behavioral studies using mouse models do not include subjects of both sexes. Many researchers worry that data of female mice may be unreliable due to the estrous cycle. Here, we retrospectively evaluated sex effects on coefficient of variation (CV) in 5,311 mice which had performed the same place navigation protocol in the water-maze and in 4,554 mice tested in the same open field arena. Confidence intervals for Cohen's d as measure of effect size were computed and tested for equivalence with 0.2 as equivalence margin. Despite the large sample size, only few behavioral parameters showed a significant sex effect on CV. Confidence intervals of effect size indicated that CV was either equivalent or showed a small sex difference at most, accounting for less than 2% of total group to group variation of CV. While female mice were potentially slightly more variable in water-maze acquisition and in the open field, males tended to perform less reliably in the water-maze probe trial. In addition to evaluating variability, we also directly compared mean performance of female and male mice and found them to be equivalent in both water-maze place navigation and open field exploration. Our data confirm and extend other large scale studies in demonstrating that including female mice in experiments does not cause a relevant increase of data variability. Our results make a strong case for including mice of both sexes whenever open field or water-maze are used in preclinical research., American Journal of Medical Genetics. Part C, Seminars in Medical Genetics, 175 (3), ISSN:1552-4868, ISSN:1552-4876

Objective Clinical trials targeting β-amyloid peptides (Aβ) for Alzheimer disease (AD) failed for arguable reasons that include selecting the wrong stages of AD pathophysiology or Aβ being the wrong target. Targeting Aβ to prevent cerebral amyloid angiopathy (CAA) has not been rigorously followed, although the causal role of Aβ for CAA and related hemorrhages is undisputed. CAA occurs with normal aging and to various degrees in AD, where its impact and treatment is confounded by the presence of parenchymal Aβ deposition. Methods APPDutch mice develop CAA in the absence of parenchymal amyloid, mimicking hereditary cerebral hemorrhage with amyloidosis Dutch type (HCHWA-D). Mice were treated with a β-site amyloid precursor protein cleaving enzyme 1 (BACE1) inhibitor. We used 3-dimensional ultramicroscopy and immunoassays for visualizing CAA and assessing Aβ in cerebrospinal fluid (CSF) and brain. Results CAA onset in mice was at 22 to 24 months, first in frontal leptomeningeal and superficial cortical vessels followed by vessels penetrating the cortical layers. CSF Aβ increased with aging followed by a decrease of both Aβ40 and Aβ42 upon CAA onset, supporting the idea that combined reduction of CSF Aβ40 and Aβ42 is a specific biomarker for vascular amyloid. BACE1 inhibitor treatment starting at CAA onset and continuing for 4 months revealed a 90% Aβ reduction in CSF and largely prevented CAA progression and associated pathologies. Interpretation This is the first study showing that Aβ reduction at early disease time points largely prevents CAA in the absence of parenchymal amyloid. Our observation provides a preclinical basis for Aβ-reducing treatments in patients at risk of CAA and in presymptomatic HCHWA-D. ANN NEUROL 2019;86:561-571.

Striking differences in the septo-temporal distribution of neurogenesis are found in small rodents. Here, we assessed the association of adult hippocampal neurogenesis with behavioral responses to novelty, temporal and spatial sequence and reversal learning in wild, wild-derived and laboratory rodents using an automated testing apparatus, the IntelliCage. Behaviorally, DBA/2 and wild-derived house mice were quickest to explore a novel environment, wild wood mice and bank voles were slowest, and C57BL/6 intermediate. Rule learning (temporal and spatial) was fastest in wood mice and bank voles, while DBA/2 and house mice performed poorer. C57BL/6 performed similar to the house mice in the temporal task and similar to wild rodents in the spatial task. Using the number of DCX-positive neurons and proliferating, Ki67-positive cells in the septal, intermediate and temporal hippocampus as a proxy, an ANCOVA was used to test for within-group relations between neurogenesis and behavior. We found that higher numbers of DCX-positive cells in the temporal hippocampus were associated with an increased latency and a lower frequency to explore a novel environment. Temporal and spatial sequence learning was not associated with neurogenesis. In the spatial reversal task however, animals with higher septal neurogenesis showed a persevering phenotype and slower re-learning. Our findings provide strong evidence of septo-temporally segregated neurogenesis effects on behavior across five rodent strains and species. While temporal neurogenesis covaries with behavioral responses to novelty, septal neurogenesis relates to perseverance of a successfully learned spatial rule. Importantly, these associations were independent of species or strain and can be found in both wild and domesticated rodents.

Self-propagating amyloid-β (Aβ) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aβ42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aβ42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aβ42 peptides and decreases the seeding activity of Aβ aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aβ plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aβ structures with small molecules as a valid therapeutic strategy.

Mutations in OCRL encoding the inositol polyphosphate 5-phosphatase OCRL (Lowe oculocerebrorenal syndrome protein) disrupt phosphoinositide homeostasis along the endolysosomal pathway causing dysfunction of the cells lining the kidney proximal tubule (PT). The dysfunction can be isolated (Dent disease 2) or associated with congenital cataracts, central hypotonia and intellectual disability (Lowe syndrome). The mechanistic understanding of Dent disease 2/Lowe syndrome remains scarce due to limitations of animal models of OCRL deficiency. Here, we investigate the role of OCRL in Dent disease 2/Lowe syndrome by using OcrlY/− mice, where the lethal deletion of the paralogue Inpp5b was rescued by human INPP5B insertion, and primary culture of proximal tubule cells (mPTCs) derived from OcrlY/− kidneys. The OcrlY/− mice show muscular defects with dysfunctional locomotricity and present massive urinary losses of low-molecular-weight proteins and albumin, caused by selective impairment of receptor-mediated endocytosis in PT cells. The latter was due to accumulation of phosphatidylinositol 4,5–bisphosphate PI(4,5)P2 in endolysosomes, driving local hyper-polymerization of F-actin and impairing trafficking of the endocytic LRP2 receptor, as evidenced in OcrlY/− mPTCs. The OCRL deficiency was also associated with a disruption of the lysosomal dynamic and proteolytic activity. Partial convergence of disease-pathways and renal phenotypes observed in OcrlY/− and Clcn5Y/− mice suggest shared mechanisms in Dent diseases 1 and 2. These studies substantiate the first mouse model of Lowe syndrome and give insights into the role of OCRL in cellular trafficking of multiligand receptors. These insights open new avenues for therapeutic interventions in Lowe syndrome and Dent disease., Human Molecular Genetics, 28 (12), ISSN:0964-6906, ISSN:1460-2083

The 1555 A to G substitution in mitochondrial 12S A‐site rRNA is associated with maternally transmitted deafness of variable penetrance in the absence of otherwise overt disease. Here, we recapitulate the suggested A1555G‐mediated pathomechanism in an experimental model of mitoribosomal mistranslation by directed mutagenesis of mitoribosomal protein MRPS5. We first establish that the ratio of cysteine/methionine incorporation and read‐through of mtDNA‐encoded MT‐CO1 protein constitute reliable measures of mitoribosomal misreading. Next, we demonstrate that human HEK293 cells expressing mutant V336Y MRPS5 show increased mitoribosomal mistranslation. As for immortalized lymphocytes of individuals with the pathogenic A1555G mutation, we find little changes in the transcriptome of mutant V336Y MRPS5 HEK cells, except for a coordinated upregulation of transcripts for cytoplasmic ribosomal proteins. Homozygous knock‐in mutant Mrps5 V338Y mice show impaired mitochondrial function and a phenotype composed of enhanced susceptibility to noise‐induced hearing damage and anxiety‐related behavioral alterations. The experimental data in V338Y mutant mice point to a key role of mitochondrial translation and function in stress‐related behavioral and physiological adaptations.

The increasing resolution of tract-tracing studies has led to the definition of segments along the transverse axis of the hippocampal pyramidal cell layer, which may represent functionally defined elements. This review will summarize evidence for a morphological and functional differentiation of pyramidal cells along the radial (deep to superficial) axis of the cell layer. In many species, deep and superficial sublayers can be identified histologically throughout large parts of the septotemporal extent of the hippocampus. Neurons in these sublayers are generated during different periods of development. During development, deep and superficial cells express genes (Sox5, SatB2) that also specify the phenotypes of superficial and deep cells in the neocortex. Deep and superficial cells differ neurochemically (e.g. calbindin and zinc) and in their adult gene expression patterns. These markers also distinguish sublayers in the septal hippocampus, where they are not readily apparent histologically in rat or mouse. Deep and superficial pyramidal cells differ in septal, striatal, and neocortical efferent connections. Distributions of deep and superficial pyramidal cell dendrites and studies in reeler or sparsely GFP-expressing mice indicate that this also applies to afferent pathways. Histological, neurochemical, and connective differences between deep and superficial neurons may correlate with (patho-) physiological phenomena specific to pyramidal cells at different radial locations. We feel that an appreciation of radial subdivisions in the pyramidal cell layer reminiscent of lamination in other cortical areas may be critical in the interpretation of studies of hippocampal anatomy and function.

Increasing evidence suggests that synaptic functions of the amyloid precursor protein (APP), which is key to Alzheimer pathogenesis, may be carried out by its secreted ectodomain (APPs). The specific roles of APPsα and APPsβ fragments, generated by non-amyloidogenic or amyloidogenic APP processing, respectively, remain however unclear. Here, we expressed APPsα or APPsβ in the adult brain of conditional double knockout mice (cDKO) lacking APP and the related APLP2. APPsα efficiently rescued deficits in spine density, synaptic plasticity (LTP and PPF), and spatial reference memory of cDKO mice. In contrast, APPsβ failed to show any detectable effects on synaptic plasticity and spine density. The C-terminal 16 amino acids of APPsα (lacking in APPsβ) proved sufficient to facilitate LTP in a mechanism that depends on functional nicotinic α7-nAChRs. Further, APPsα showed high-affinity, allosteric potentiation of heterologously expressed α7-nAChRs in oocytes. Collectively, we identified α7-nAChRs as a crucial physiological receptor specific for APPsα and show distinct in vivo roles for APPsα versus APPsβ. This implies that reduced levels of APPsα that might occur during Alzheimer pathogenesis cannot be compensated by APPsβ.

To evaluate permanent effects of hippocampal and prefrontal cortex lesion on spatial tasks, lesioned and sham-operated female C57BL/6 mice were exposed to a series of conditioning schemes in IntelliCages housing 8–10 transponder-tagged mice from each treatment group. Sequential testing started at 51–172 days after bilateral lesions and lasted for 154 and 218 days in two batches of mice, respectively. Spontaneous undisturbed behavioral patterns clearly separated the three groups, hippocampals being characterized by more erratic hyperactivity, and strongly impaired circadian synchronization ability. Hippocampal lesions led to deficits in spatial passive avoidance, as well as in spatial reference and working memory tasks. Impairment was minimal in rewarded preference/reversal schemes, but prominent if behavioral responses required precise circadian timing or included punishment of wrong spatial choices. No differences between sham-operated and prefrontally lesioned subjects in conditioning success were discernible. These results corroborate the view that hippocampal dysfunction spares simple spatial learning tasks but impairs the ability to cope with conflicting task-inherent spatial, temporal or emotional cues. Methodologically, the results show that automated testing and data analysis of socially kept mice is a powerful, efficient and animal-friendly tool for dissecting complex features and behavioral profiles of hippocampal dysfunction characterizing many transgenic or pharmacological mouse models., Behavioural Brain Research, 352, ISSN:0166-4328, ISSN:1872-7549

The discovery of adult-born neurons in the hippocampus has triggered a wide range of studies that link the new neurons to various behavioral functions. However, the role of new neurons in behavior is still equivocal. Conflicting results may be due to the difficulty in manipulating neurogenesis without off-target effects as well as the statistical approach used, which fail to account for neurogenesis-independent effects of experimental manipulations on behavior. In this study, we apply a more comprehensive statistical and conceptual approach. Instead of between-group analyses, we consider the within-group relationships between neurogenesis and behavior (ANCOVA and mediation analysis) in a large-scale experiment, in which distinct age- (3 and 5 months) and strain- (DBA and C57) related differences in basal levels of neurogenesis in mice are compared with a large number (∼1,500) of behavioral read outs. The analysis failed to detect any association between anxiety and motor impulsivity with neurogenesis. However, within-group adult hippocampal neurogenesis is associated with the reaction to novelty. Specifically, more neurogenesis is associated with a longer latency to explore and a lower frequency of exploratory actions, overall indicative of a phenotype where animals with more neurogenesis were slower to explore a novel environment. This effect is observed in 5-months-old, but not in 3-months-old mice of both strains. An association between the reaction to novelty and adult neurogenesis can have a major impact on results from previous studies using classical behavioral experiments, in which animals are tested in a--for the animal--novel experimental set-up. The neurogenesis-novelty association found here is also a necessary link in the relation that has been suggested to exist between neurogenesis and psychiatric disorders marked by a failure to cope with novelty.

Phosphatidylinositol-3-kinase (PI3K) signaling influences susceptibility to virus infections, anoxia, obstetric complications, and cancer; which are changed in patients with schizophrenia and their first degree relatives. Therefore PI3K signaling might have impact on the pathophysiology of schizophrenia. PI3K signaling crucially involves phosphoinositide-dependent protein kinase (PDK1). Increased anxiety behavior is observed in PDK1 hypomorphic mice. Here we show enhanced prevalence of schizophrenia in carriers of the PDK1 CC genotype in human beings. Moreover, decreased parietal P300 amplitude, which is a well-studied schizophrenic endophenotype, was observed in PDK1 CC carriers. Glutamate and glutamine concentrations are increased in the frontal lobe of PDK1 dysmorphic mice and human CC individuals. Our results demonstrate that the PDK1 CC genotype is associated with increased risk to develop schizophrenia, a typical endophenotype profile observed in the disease and modified neurotransmitter concentrations in brain regions associated with the disease.

Work on laboratory and wild rodents suggests that domestication may impact on the extent of adult hippocampal neurogenesis and its responsiveness to regulatory factors. There is, however, no model of laboratory rodents and their nondomesticated conspecifics that would allow a controlled comparison of the effect of domestication. Here, we present a controlled within-species comparison of adult hippocampal neurogenesis in farm-bred foxes (Vulpes vulpes) that differ in their genetically determined degree of tameness. Quantitative comparisons of cell proliferation (Ki67) and differentiating cells of neuronal lineage (doublecortin, DCX) in the hippocampus of foxes were performed as a proxy for neurogenesis. Higher neurogenesis was observed in tameness-selected foxes, notably in an extended subgranular zone of the middle and temporal compartments of the hippocampus. Increased neurogenesis is negatively associated with aggressive behavior. Across all animals, strong septotemporal gradients were found, with higher numbers of proliferating cells and young neurons relative to resident granule cells in the temporal than in the septal hippocampus. The opposite gradient was found for the ratio of DCX/Ki67- positive cells. When tameness-selected and unselected foxes are compared with rodents and primates, proliferation is similar, while the number of young neurons is higher. The difference may be mediated by an extended period of differentiation or higher rate of survival. On the background of this species-specific neurogenic pattern, selection of foxes for a single behavioral trait key to domestication, i.e., genetic tameness, is accompanied by global and region-specific increases in neurogenesis.

Increasing evidence suggests that synaptic functions of the amyloid precursor protein (APP), which is key to Alzheimer pathogenesis, may be carried out by its secreted ectodomain (APPs). The specific roles of APPsα and APPsβ fragments, generated by non‐amyloidogenic or amyloidogenic APP processing, respectively, remain however unclear. Here, we expressed APPsα or APPsβ in the adult brain of conditional double knockout mice (cDKO) lacking APP and the related APLP2. APPsα efficiently rescued deficits in spine density, synaptic plasticity (LTP and PPF), and spatial reference memory of cDKO mice. In contrast, APPsβ failed to show any detectable effects on synaptic plasticity and spine density. The C‐terminal 16 amino acids of APPsα (lacking in APPsβ) proved sufficient to facilitate LTP in a mechanism that depends on functional nicotinic α7‐nAChRs. Further, APPsα showed high‐affinity, allosteric potentiation of heterologously expressed α7‐nAChRs in oocytes. Collectively, we identified α7‐nAChRs as a crucial physiological receptor specific for APPsα and show distinct in vivo roles for APPsα versus APPsβ. This implies that reduced levels of APPsα that might occur during Alzheimer pathogenesis cannot be compensated by APPsβ.

Introduction Objective data on the association of maximal aerobic capacity (VO2max) with work related physical activity are sparse. Thus, it is not clear whether occupational physical activity (OPA) contributes to an increase of VO2max. This study examined the association of VO2max with work and non-work related physical activity in a Swiss working population. Methods In this cross-sectional study, a total of 337 healthy and full-time employed adults were recruited. Demographic data, height, weight and BMI were recorded in all subjects. Participants were classified into nine occupational categories (ISCO-88) and merged into three groups with low, moderate, and high OPA. Physical activity was objectively measured by the SenseWear Mini Armband on seven consecutive days (23 hours per day). Participants were regarded as sufficiently active when accumulating ≥30 min of moderate-to-vigorous physical activity per day. VO2max was evaluated using the multistage 20-meter shuttle run test. Results Data of 303 participants were considered for analysis (63% male, age 33 yrs, SD 12). Multiple linear regression analysis (adjusted R2 = 0.69) revealed significant positive associations of VO2max with leisure-time physical activity (LTPA) at vigorous intensity (β = 0.212) and sufficient moderate-to-vigorous physical activity (β = 0.100) on workdays. Female gender (β = -0.622), age (β = -0.264), BMI (β = -0.220), the ratio of maximum to resting heart rate (β = 0.192), occupational group (low vs. high OPA, β = -0.141), and smoking (β = -0.133) were also identified as independent predictors of VO2max. Conclusions The present results suggest that VO2max is positively associated with LTPA, but not with OPA on workdays. This finding emphasizes the need for employees to engage in sufficient high-intensity physical activity in recreation for maintaining or improving VO2max with regard to health benefits., PLoS ONE, 12 (1), ISSN:1932-6203

Selective serotonin reuptake inhibitors (SSRIs) represent the most common treatment for major depression. However, their efficacy is variable and incomplete. In order to elucidate the cause of such incomplete efficacy, we explored the hypothesis positing that SSRIs may not affect mood per se but, by enhancing neural plasticity, render the individual more susceptible to the influence of the environment. Consequently, SSRI administration in a favorable environment promotes a reduction of symptoms, whereas in a stressful environment leads to a worse prognosis. To test such hypothesis, we exposed C57BL/6 mice to chronic stress in order to induce a depression-like phenotype and, subsequently, to fluoxetine treatment (21 days), while being exposed to either an enriched or a stressful condition. We measured the most commonly investigated molecular, cellular and behavioral endophenotypes of depression and SSRI outcome, including depression-like behavior, neurogenesis, brain-derived neurotrophic factor levels, hypothalamic-pituitary-adrenal axis activity and long-term potentiation. Results showed that, in line with our hypothesis, the endophenotypes investigated were affected by the treatment according to the quality of the living environment. In particular, mice treated with fluoxetine in an enriched condition overall improved their depression-like phenotype compared with controls, whereas those treated in a stressful condition showed a distinct worsening. Our findings suggest that the effects of SSRI on the depression- like phenotype is not determined by the drug per se but is induced by the drug and driven by the environment. These findings may be helpful to explain variable effects of SSRI found in clinical practice and to device strategies aimed at enhancing their efficacy by means of controlling environmental conditions.

The gravity vector theory postulates that birds determine their position to set a home course by comparing the memorized gravity vector at the home loft with the local gravity vector at the release site, and that they should adjust their flight course to the gravity anomalies encountered. As gravity anomalies are often intermingled with geomagnetic anomalies, we released experienced pigeons from the center of a strong circular gravity anomaly (25 km diameter) not associated with magnetic anomalies and from a geophysical control site, equidistant from the home loft (91 km). After crossing the border zone of the anomaly – expected to be most critical for pigeon navigation – they dispersed significantly more than control birds, except for those having met a gravity anomaly en route. These data increase the credibility of the gravity vector hypothesis.

SUMMARY Homing pigeons (Columba livia) are believed to adopt a map-and-compass strategy to find their way home. Surprisingly, to date a clear demonstration of the use of a cognitive map in free-flight experiments is missing. In this study, we investigated whether homing pigeons use a mental map in which – at an unknown release site – their own position, the home loft and a food loft are represented simultaneously. In order to test this, homing pigeons were trained to fly to a 25–30 km distant food loft. A total of 131 hungry and satiated pigeons were then released from an unfamiliar site equidistant from the food loft and the home loft. Their vanishing bearings and homing times were assessed conventionally at four sites, and also their flight tracks from one release site by means of GPS loggers. The vanishing bearings of fed and hungry birds differed significantly at all release sites and a highly significant proportion of hungry birds flew to the food loft, while the fed birds headed home. The GPS experiment revealed a number of pigeons flying very precisely to the food loft, others correcting their flight direction after topography-induced detours. This implies that the pigeons knew their geographical position in relation to the targets, and chose a flight direction according to their locally manipulated needs – clearly the essence of a cognitive navigational map.

It has been hypothesized that selective serotonin reuptake inhibitors (SSRIs), the most common treatment for major depression, affect mood through changes in immune function. However, the effects of SSRIs on inflammatory response are contradictory since these act either as anti- or pro-inflammatory drugs. Previous experimental and clinical studies showed that the quality of the living environment moderates the outcome of antidepressant treatment. Therefore, we hypothesized that the interplay between SSRIs and the environment may, at least partially, explain the apparent incongruence regarding the effects of SSRI treatment on the inflammatory response. In order to investigate such interplay, we exposed C57BL/6 mice to chronic stress to induce a depression-like phenotype and, subsequently, to fluoxetine treatment or vehicle (21days) while being exposed to either an enriched or a stressful condition. At the end of treatment, we measured the expression levels of several anti- and pro-inflammatory cytokines and inflammatory mediators in the whole hippocampus and in isolated microglia. We also determined microglial density, distribution, and morphology to investigate their surveillance state. Results show that the effects of fluoxetine treatment on inflammation and microglial function, as compared to vehicle, were dependent on the quality of the living environment. In particular, fluoxetine administered in the enriched condition increased the expression of pro-inflammatory markers compared to vehicle, while treatment in a stressful condition produced anti-inflammatory effects. These findings provide new insights regarding the effects of SSRIs on inflammation, which may be crucial to devise pharmacological strategies aimed at enhancing antidepressant efficacy by means of controlling environmental conditions.

Human Molecular Genetics, 21 (2), ISSN:0964-6906, ISSN:1460-2083

The amyloid precursor protein family (APP/APLPs) has essential roles for neuromuscular synapse development and for the formation and plasticity of synapses within the CNS. Despite this, it has remained unclear whether APP mediates its functions primarily as a cell surface adhesion and signaling molecule or via its numerous proteolytic cleavage products. To address these questions, we followed a genetic approach and used APPΔCT15 knockin mice lacking the last 15 amino acids of APP, including the highly conserved YENPTY protein interaction motif. To circumvent functional compensation by the closely related APLP2, these mice were bred to an APLP2-KO background to generate APPΔCT15-DM double mutants. These APPΔCT15-DM mice were partially viable and displayed defects in neuromuscular synapse morphology and function with impairments in the ability to sustain transmitter release that resulted in muscular weakness. In the CNS, we demonstrate pronounced synaptic deficits including impairments in LTP that were associated with deficits in spatial learning and memory. Thus, the APP-CT15 domain provides essential physiological functions, likely via recruitment of specific interactors. Together with the well-established role of APPsα for synaptic plasticity, this shows that multiple domains of APP, including the conserved C-terminus, mediate signals required for normal PNS and CNS physiology. In addition, we demonstrate that lack of the APP-CT15 domain strongly impairs Aβ generationin vivo, establishing the APP C-terminus as a target for Aβ-lowering strategies.SIGNIFICANCE STATEMENTSynaptic dysfunction and cognitive decline are early hallmark features of Alzheimer′s disease. Thus, it is essential to elucidate thein vivofunction(s) of APP at the synapse. At present, it is unknown whether APP family proteins function as cell surface receptors, or mainly via shedding of their secreted ectodomains, such as neurotrophic APPsα. Here, to dissect APP functional domains, we used APP mutant mice lacking the last 15 amino acids that were crossed onto an APLP2-KO background. These APPΔCT15-DM mice showed defects in neuromuscular morphology and function. Synaptic deficits in the CNS included impairments of synaptic plasticity, spatial learning, and memory. Collectively, this indicates that multiple APP domains, including the C-terminus, are required for normal nervous system function.

The NONO protein has been characterized as an important transcriptional regulator in diverse cellular contexts. Here we show that loss of NONO function is a likely cause of human intellectual disability and that NONO-deficient mice have cognitive and affective deficits. Correspondingly, we find specific defects at inhibitory synapses, where NONO regulates synaptic transcription and gephyrin scaffold structure. Our data identify NONO as a possible neurodevelopmental disease gene and highlight the key role of the DBHS protein family in functional organization of GABAergic synapses.

There has been a long-standing need to develop efficient and standardized behavioral test methods for evaluating higher-order brain functions in mice. Here, we developed and validated a behavioral flexibility test in mice using IntelliCage, a fully automated behavioral analysis system for mice in a group-housed environment. We first developed a "behavioral sequencing task" in the IntelliCage that enables us to assess the learning ability of place discrimination and behavioral sequence for reward acquisition. In the serial reversal learning using the task, the discriminated spatial patterns of the rewarded and never-rewarded places were serially reversed, and thus, mice were accordingly expected to realign the previously acquired behavioral sequence. In general, the tested mice showed rapid acquisition of the behavioral sequencing task and behavioral flexibility in the subsequent serial reversal stages both in intra- and inter-session analyses. It was found that essentially the same results were obtained among three different laboratories, which confirm the high stability of the present test protocol in different strains of mice (C57BL/6, DBA/2, and ICR). In particular, the most trained cohort of C57BL/6 mice achieved a markedly rapid adaptation to the reversal task in the final phase of the long-term serial reversal test, which possibly indicated that the mice adapted to the "reversal rule" itself. In conclusion, the newly developed behavioral test was shown to be a valid assay of behavioral flexibility in mice, and is expected to be utilized in tests of mouse models of cognitive deficits.

Despite its key role in Alzheimer pathogenesis, the physiological function(s) of the amyloid precursor protein (APP) and its proteolytic fragments are still poorly understood. Previously, we generated APPsα knock-in (KI) mice expressing solely the secreted ectodomain APPsα. Here, we generated double mutants (APPsα-DM) by crossing APPsα-KI mice onto an APLP2-deficient background and show that APPsα rescues the postnatal lethality of the majority of APP/APLP2 double knockout mice. Surviving APPsα-DM mice exhibited impaired neuromuscular transmission, with reductions in quantal content, readily releasable pool, and ability to sustain vesicle release that resulted in muscular weakness. We show that these defects may be due to loss of an APP/Mint2/Munc18 complex. Moreover, APPsα-DM muscle showed fragmented post-synaptic specializations, suggesting impaired postnatal synaptic maturation and/or maintenance. Despite normal CNS morphology and unaltered basal synaptic transmission, young APPsα-DM mice already showed pronounced hippocampal dysfunction, impaired spatial learning and a deficit in LTP that could be rescued by GABAA receptor inhibition. Collectively, our data show that APLP2 and APP are synergistically required to mediate neuromuscular transmission, spatial learning and synaptic plasticity.

Tissue inhibitors of metalloproteinases (TIMPs) are a family of closely related proteins that inhibit matrix metalloproteinases (MMPs). In the central nervous system (CNS), TIMPs 2, 3, and 4 are constitutively expressed at high levels, whereas TIMP1 can be induced by various stimuli. Here, we studied the effects of constitutive expression of TIMP1 in the CNS in transgenic mice. Transgene expression started prenatally and persisted throughout lifetime at high levels. Since MMP activity has been implicated in CNS development, in proper function of the adult CNS, and in inflammatory disorders, we investigated Timp1-induced CNS alterations. Despite sufficient MMP inhibition, high expressor transgenic mice had a normal phenotype. The absence of compensatory up-regulation of MMP genes in the CNS of Timp1 transgenic mice indicates that development, learning, and memory functions do not require the entire MMP arsenal. To elucidate the effects of strong Timp1 expression in CNS inflammation, we induced experimental allergic encephalomyelitis. We observed a Timp1 dose-dependent mitigation of both experimental allergic encephalomyelitis symptoms and histological lesions in the CNS of transgenic mice. All in all, our data demonstrate that (1) long-term CNS expression of TIMP1 with complete suppression of gelatinolytic activity does not interfere with physiological brain function and (2) TIMP1 might constitute a promising candidate for long-term therapeutic treatment of inflammatory CNS diseases such as multiple sclerosis.

Recent investigations of Nogo-A, a well characterized protein inhibitor of neurite outgrowth in the brain, have revealed additional functions including a role in neuropsychiatric disorders such as schizophrenia. Here we examined Nogo-A functions in mouse CA3 hippocampal circuitry. Patch clamp recordings showed that the absence of Nogo-A results in a hyperactive network. In addition, mGlu3 metabotropic glutamate receptors, which exhibit mutations in certain forms of schizophrenia, were downregulated specifically in the CA3 area. Furthermore, Nogo-A-/- mice showed disordered theta oscillations with decreased incidence and frequency, similar to those observed in mGlu3-/- mice. As disruptions in theta rhythmicity are associated with impaired spatial navigation, we tested mice using modified Morris water maze tasks. Mice lacking Nogo-A exhibited altered search strategies, displaying greater dependence on global as opposed to local reference frames. This link between Nogo-A and mGlu3 receptors may provide new insights into mechanisms underlying schizophrenia., PLoS ONE, 13 (7), ISSN:1932-6203

The between-laboratory effects on behavioral phenotypes and spatial learning performance of three strains of laboratory mice known for divergent behavioral phenotypes were evaluated in a fully balanced and synchronized study using a completely automated behavioral phenotyping device (IntelliCage). Activity pattern and spatial conditioning performance differed consistently between strains, i.e. exhibited no interaction with the between-laboratory factor, whereas the gross laboratory effect showed up significantly in the majority of measures. It is argued that overall differences between laboratories may not realistically be preventable, as subtle differences in animal housing and treatment will not be controllable, in practice. However, consistency of strain (or treatment) effects appears to be far more important in behavioral and brain sciences than the absolute overall level of such measures. In this respect, basic behavioral and learning measures proved to be highly consistent in the IntelliCage, therefore providing a valid basis for meaningful research hypothesis testing. Also, potential heterogeneity of behavioral status because of environmental and social enrichment has no detectable negative effect on the consistency of strain effects. We suggest that the absence of human interference during behavioral testing is the most prominent advantage of the IntelliCage and suspect that this is likely responsible for the between-laboratory consistency of findings, although we are aware that this ultimately needs direct testing.

Institute of HumanMovement Sciences and Sport, ETH Zurich, Zurich, Switzerland*Corresponding author: W. E. Crusio, Centre de NeurosciencesInte´gratives et Cognitives, Universite´ de Bordeaux, CNRS UMR5228, Bat B2 - Avenue des Faculte´s, 33405 Talence, France.E-mail: wim_crusio@yahoo.comSince Genes, Brain and Behavior (G2B) started publication in2002, the journal has become one of the favored outlets for,among many other subjects, mouse mutant studies. Unfor-tunately, a significant proportion of mutant studies sufferfrom serious methodological problems, which are the mostfrequent reason of rejection without review for manuscriptssubmitted forpublication to G2B. Most of theseproblems arecurrently not always appreciated in the field and authorssometimesprotestsuchsummaryrejectdecisionsbyarguingthat they followed commonly used procedures that manyother journals publish without any problem. Of course, therealissueiswhetherornotthesecommonlyusedproceduresare scientifically valid. The problems vary from improperbreeding procedures to invalid choices of control animals.Examples of such studies can be found in virtually everyscientific journal, even the most prestigious ones. Because ofthepaucity ofmethodological informationthatmanyso-calledhigh-impact journals provide nowadays, it is often evenimpossible to verify what genetic background was employedand whether proper procedures were followed and one justhas to hope for the best. Many of these problems werepointed out by Gerlai (1996) and it is very disappointing andrather distressing, to say the least, to see that more thanadecadeafterGerlai’soriginalcommentaryappeared,articlescontinue to be published that violate some of the most basicrequirements for the proper conduct of mutant studies.Clearly this situation is highly unsatisfactory and the currentsituation is not only frustrating to authors, reviewers andeditors alike, but to the extent that these design faults areobvious fatal flaws, this also constitutes a waste of time,effort and research resources, not to mention the ethicalimplications of unnecessarily using live animals for flawedstudies. The following editorial will therefore outline theminimum standards a mutant study needs to comply within order to be considered for publication in G2B (Table 1).Manuscripts that do not adhere to these minimum require-ments will be rejected for publication without review,whereas manuscripts that do not provide sufficient experi-mental detail will be returned to their authors for completionbefore being sent out for review. It is hoped that otherjournals publishing mutant work will follow our lead, resultingin a critical improvement of the designs of mutant studies.(Sometimes authors refer to an earlier publication for ‘moredetails’. Although this may be acceptable to avoid repeatingmore arcane details of some experimental procedure, anymanuscript should always contain all experimental detailsnecessary for reviewers (and readers!) to understand whathad been done.)In what follows we will briefly discuss the most commonproblems with the design of mutant studies and brieflyexplain why they are not acceptable. For more detailed dis-cussions and acceptable alternative approaches, the reader isreferred to Crusio (2004), Gerlai (1996, 2000, 2001) andWolfer et al. (2002). It should be noted that basically all ofthe problems discussed are common to any mutant study,regardlessofwhetherthemutation wasinducedinatargetedway by means of transgenesis or homologous recombination(‘knock-out’), or was induced randomly following chemicaltreatmentorradiation,oroccurredspontaneously.Inaddition,most of the recommendations given obviously also apply toother species than rodents.

The effects of aggregation in navigating animals have generated growing interest in field and theoretical studies. The few studies on the effects of group flying on the performance of homing pigeons, Columba livia, have led to controversial conclusions, chiefly because of the lack of appropriate technology to follow pigeons during their entire homeward flight. Therefore, we used GPS data loggers on six highly trained pigeons from a familiar release site first by releasing them six times individually, then six times as a group from the same site, and, finally, again six times individually. Flight data showed that the homing performance of the birds flying as a flock was significantly better than that of the birds released individually. When flying in a flock, pigeons showed no resting episodes, shorter homing times, higher speed, and almost no circling around the start zone in comparison to individual flights. Moreover, flock-flying pigeons took a nearly direct, ‘beeline’ route to the loft, whereas individually flying birds preferred to follow roads and other longitudinal landmarks leading towards the loft, even when it caused a detour. Our results show that group cohesion facilitates a shift towards more efficient homing strategies: individuals prefer navigating by familiar landmarks, while flocks show a compass orientation.