Loss of the parkinsonism-associated protein FBXO7 in glutamatergic forebrain neurons in mice leads to abnormal motor behavior and synaptic defects.

Mutations in PARK15, which encodes for the F-box protein FBXO7 have been associated with Parkinsonian Pyramidal syndrome, a rare and complex movement disorder with Parkinsonian symptoms, pyramidal tract signs and juvenile onset. Our previous study showed that systemic loss of Fbxo7 in mice causes motor defects and premature death. We have also demonstrated that FBXO7 has a crucial role in neurons as the specific deletion in tyrosine hydroxylase-positive or glutamatergic forebrain neurons leads to late-onset or early-onset motor dysfunction, respectively. In this study, we examined NEX-Cre;Fbxo7fl/fl mice, in which Fbxo7 was specifically deleted in glutamatergic projection neurons. The effects of FBXO7 deficiency on striatal integrity were investigated with HPLC and histological analyses. NEX-Cre;Fbxo7fl/fl mice revealed an increase in striatal dopamine concentrations, changes in the glutamatergic, GABAergic and dopaminergic pathways, astrogliosis and microgliosis and little or no neuronal loss in the striatum. To determine the effects on the integrity of the synapse, we purified synaptic membranes, subjected them to quantitative mass spectrometry analysis and found alterations in the complement system, endocytosis and exocytosis pathways. These neuropathological changes coincide with alterations in spontaneous home cage behavior. Taken together, our findings suggest that FBXO7 is crucial for corticostriatal projections and the synaptic integrity of the striatum, and consequently for proper motor control.

Isolated catatonia-like executive dysfunction in mice with forebrain-specific loss of myelin integrity.

A key feature of advanced brain aging includes structural defects of intracortical myelin that are associated with secondary neuroinflammation. A similar pathology is seen in specific myelin mutant mice that model 'advanced brain aging' and exhibit a range of behavioral abnormalities. However, the cognitive assessment of these mutants is problematic because myelin-dependent motor-sensory functions are required for quantitative behavioral readouts. To better understand the role of cortical myelin integrity for higher brain functions, we generated mice lacking Plp1, encoding the major integral myelin membrane protein, selectively in ventricular zone stem cells of the mouse forebrain. In contrast to conventional Plp1 null mutants, subtle myelin defects were restricted to the cortex, hippocampus, and underlying callosal tracts. Moreover, forebrain-specific Plp1 mutants exhibited no defects of basic motor-sensory performance at any age tested. Surprisingly, several behavioral alterations reported for conventional Plp1 null mice (Gould et al., 2018) were absent and even social interactions appeared normal. However, with novel behavioral paradigms, we determined catatonia-like symptoms and isolated executive dysfunction in both genders. This suggests that loss of myelin integrity has an impact on cortical connectivity and underlies specific defects of executive function. These observations are likewise relevant for human neuropsychiatric conditions and other myelin-related diseases.

NMDAR1 autoantibodies amplify behavioral phenotypes of genetic white matter inflammation: a mild encephalitis model with neuropsychiatric relevance.

Encephalitis has an estimated prevalence of ≤0.01%. Even with extensive diagnostic work-up, an infectious etiology is identified or suspected in <50% of cases, suggesting a role for etiologically unclear, noninfectious processes. Mild encephalitis runs frequently unnoticed, despite slight neuroinflammation detectable postmortem in many neuropsychiatric illnesses. A widely unexplored field in humans, though clearly documented in rodents, is genetic brain inflammation, particularly that associated with myelin abnormalities, inducing primary white matter encephalitis. We hypothesized that "autoimmune encephalitides" may result from any brain inflammation concurring with the presence of brain antigen-directed autoantibodies, e.g., against N-methyl-D-aspartate-receptor NR1 (NMDAR1-AB), which are not causal of, but may considerably shape the encephalitis phenotype. We therefore immunized young female Cnp-/- mice lacking the structural myelin protein 2'-3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) with a "cocktail" of NMDAR1 peptides. Cnp-/- mice exhibit early low-grade inflammation of white matter tracts and blood-brain barrier disruption. Our novel mental-time-travel test disclosed that Cnp-/- mice are compromised in what-where-when orientation, but this episodic memory readout was not further deteriorated by NMDAR1-AB. In contrast, comparing wild-type and Cnp-/- mice without/with NMDAR1-AB regarding hippocampal learning/memory and motor balance/coordination revealed distinct stair patterns of behavioral pathology. To elucidate a potential contribution of oligodendroglial NMDAR downregulation to NMDAR1-AB effects, we generated conditional NR1 knockout mice. These mice displayed normal Morris water maze and mental-time-travel, but beam balance performance was similar to immunized Cnp-/-. Immunohistochemistry confirmed neuroinflammation/neurodegeneration in Cnp-/- mice, yet without add-on effect of NMDAR1-AB. To conclude, genetic brain inflammation may explain an encephalitic component underlying autoimmune conditions.

Inducing sterile pyramidal neuronal death in mice to model distinct aspects of gray matter encephalitis.

Up to one person in a population of 10,000 is diagnosed once in lifetime with an encephalitis, in 50-70% of unknown origin. Recognized causes amount to 20-50% viral infections. Approximately one third of affected subjects develops moderate and severe subsequent damage. Several neurotropic viruses can directly infect pyramidal neurons and induce neuronal death in cortex and hippocampus. The resulting encephalitic syndromes are frequently associated with cognitive deterioration and dementia, but involve numerous parallel and downstream cellular and molecular events that make the interpretation of direct consequences of sudden pyramidal neuronal loss difficult. This, however, would be pivotal for understanding how neuroinflammatory processes initiate the development of neurodegeneration, and thus for targeted prophylactic and therapeutic interventions. Here we utilized adult male NexCreERT2xRosa26-eGFP-DTA (= 'DTA') mice for the induction of a sterile encephalitis by diphtheria toxin-mediated ablation of cortical and hippocampal pyramidal neurons which also recruits immune cells into gray matter. We report multifaceted aftereffects of this defined process, including the expected pathology of classical hippocampal behaviors, evaluated in Morris water maze, but also of (pre)frontal circuit function, assessed by prepulse inhibition. Importantly, we modelled in encephalitis mice novel translationally relevant sequelae, namely altered social interaction/cognition, accompanied by compromised thermoreaction to social stimuli as convenient readout of parallel autonomic nervous system (dys)function. High resolution magnetic resonance imaging disclosed distinct abnormalities in brain dimensions, including cortical and hippocampal layering, as well as of cerebral blood flow and volume. Fluorescent tracer injection, immunohistochemistry and brain flow cytometry revealed persistent blood-brain-barrier perturbance and chronic brain inflammation. Surprisingly, blood flow cytometry showed no abnormalities in circulating major immune cell subsets and plasma high-mobility group box 1 (HMGB1) as proinflammatory marker remained unchanged. The present experimental work, analyzing multidimensional outcomes of direct pyramidal neuronal loss, will open new avenues for urgently needed encephalitis research.

Autoantibodies against NMDA receptor 1 modify rather than cause encephalitis.

The etiology and pathogenesis of "anti-N-methyl-D-aspartate-receptor (NMDAR) encephalitis" and the role of autoantibodies (AB) in this condition are still obscure. While NMDAR1-AB exert NMDAR-antagonistic properties by receptor internalization, no firm evidence exists to date that NMDAR1-AB by themselves induce brain inflammation/encephalitis. NMDAR1-AB of all immunoglobulin classes are highly frequent across mammals with multiple possible inducers and boosters. We hypothesized that "NMDAR encephalitis" results from any primary brain inflammation coinciding with the presence of NMDAR1-AB, which may shape the encephalitis phenotype. Thus, we tested whether following immunization with a "cocktail" of 4 NMDAR1 peptides, induction of a spatially and temporally defined sterile encephalitis by diphtheria toxin-mediated ablation of pyramidal neurons ("DTA" mice) would modify/aggravate the ensuing phenotype. In addition, we tried to replicate a recent report claiming that immunizing just against the NMDAR1-N368/G369 region induced brain inflammation. Mice after DTA induction revealed a syndrome comprising hyperactivity, hippocampal learning/memory deficits, prefrontal cortical network dysfunction, lasting blood brain-barrier impairment, brain inflammation, mainly in hippocampal and cortical regions with pyramidal neuronal death, microgliosis, astrogliosis, modest immune cell infiltration, regional atrophy, and relative increases in parvalbumin-positive interneurons. The presence of NMDAR1-AB enhanced the hyperactivity (psychosis-like) phenotype, whereas all other readouts were identical to control-immunized DTA mice. Non-DTA mice with or without NMDAR1-AB were free of any encephalitic signs. Replication of the reported NMDAR1-N368/G369-immunizing protocol in two large independent cohorts of wild-type mice completely failed. To conclude, while NMDAR1-AB can contribute to the behavioral phenotype of an underlying encephalitis, induction of an encephalitis by NMDAR1-AB themselves remains to be proven.

CaMKIIα Expressing Neurons to Report Activity-Related Endogenous Hypoxia upon Motor-Cognitive Challenge.

We previously introduced the brain erythropoietin (EPO) circle as a model to explain the adaptive 'brain hardware upgrade' and enhanced performance. In this fundamental circle, brain cells, challenged by motor-cognitive tasks, experience functional hypoxia, triggering the expression of EPO among other genes. We attested hypoxic cells by a transgenic reporter approach under the ubiquitous CAG promoter, with Hif-1α oxygen-dependent degradation-domain (ODD) fused to CreERT2-recombinase. To specifically focus on the functional hypoxia of excitatory pyramidal neurons, here, we generated CaMKIIα-CreERT2-ODD::R26R-tdTomato mice. Behavioral challenges, light-sheet microscopy, immunohistochemistry, single-cell mRNA-seq, and neuronal cultures under normoxia or hypoxia served to portray these mice. Upon complex running wheel performance as the motor-cognitive task, a distinct increase in functional hypoxic neurons was assessed immunohistochemically and confirmed three-dimensionally. In contrast, fear conditioning as hippocampal stimulus was likely too short-lived to provoke neuronal hypoxia. Transcriptome data of hippocampus under normoxia versus inspiratory hypoxia revealed increases in CA1 CaMKIIα-neurons with an immature signature, characterized by the expression of Dcx, Tbr1, CaMKIIα, Tle4, and Zbtb20, and consistent with accelerated differentiation. The hypoxia reporter response was reproduced in vitro upon neuronal maturation. To conclude, task-associated activity triggers neuronal functional hypoxia as a local and brain-wide reaction mediating adaptive neuroplasticity. Hypoxia-induced genes such as EPO drive neuronal differentiation, brain maturation, and improved performance.

Hippocampal neurons respond to brain activity with functional hypoxia.

Physical activity and cognitive challenge are established non-invasive methods to induce comprehensive brain activation and thereby improve global brain function including mood and emotional well-being in healthy subjects and in patients. However, the mechanisms underlying this experimental and clinical observation and broadly exploited therapeutic tool are still widely obscure. Here we show in the behaving brain that physiological (endogenous) hypoxia is likely a respective lead mechanism, regulating hippocampal plasticity via adaptive gene expression. A refined transgenic approach in mice, utilizing the oxygen-dependent degradation (ODD) domain of HIF-1α fused to CreERT2 recombinase, allows us to demonstrate hypoxic cells in the performing brain under normoxia and motor-cognitive challenge, and spatially map them by light-sheet microscopy, all in comparison to inspiratory hypoxia as strong positive control. We report that a complex motor-cognitive challenge causes hypoxia across essentially all brain areas, with hypoxic neurons particularly abundant in the hippocampus. These data suggest an intriguing model of neuroplasticity, in which a specific task-associated neuronal activity triggers mild hypoxia as a local neuron-specific as well as a brain-wide response, comprising indirectly activated neurons and non-neuronal cells.

Multiple inducers and novel roles of autoantibodies against the obligatory NMDAR subunit NR1: a translational study from chronic life stress to brain injury.

Circulating autoantibodies (AB) of different immunoglobulin classes (IgM, IgA, and IgG), directed against the obligatory N-methyl-D-aspartate-receptor subunit NR1 (NMDAR1-AB), belong to the mammalian autoimmune repertoire, and appear with age-dependently high seroprevalence across health and disease. Upon access to the brain, they can exert NMDAR-antagonistic/ketamine-like actions. Still unanswered key questions, addressed here, are conditions of NMDAR1-AB formation/boosting, intraindividual persistence/course in serum over time, and (patho)physiological significance of NMDAR1-AB in modulating neuropsychiatric phenotypes. We demonstrate in a translational fashion from mouse to human that (1) serum NMDAR1-AB fluctuate upon long-term observation, independent of blood-brain barrier (BBB) perturbation; (2) a standardized small brain lesion in juvenile mice leads to increased NMDAR1-AB seroprevalence (IgM + IgG), together with enhanced Ig-class diversity; (3) CTLA4 (immune-checkpoint) genotypes, previously found associated with autoimmune disease, predispose to serum NMDAR1-AB in humans; (4) finally, pursuing our prior findings of an early increase in NMDAR1-AB seroprevalence in human migrants, which implicated chronic life stress as inducer, we independently replicate these results with prospectively recruited refugee minors. Most importantly, we here provide the first experimental evidence in mice of chronic life stress promoting serum NMDAR1-AB (IgA). Strikingly, stress-induced depressive-like behavior in mice and depression/anxiety in humans are reduced in NMDAR1-AB carriers with compromised BBB where NMDAR1-AB can readily reach the brain. To conclude, NMDAR1-AB may have a role as endogenous NMDAR antagonists, formed or boosted under various circumstances, ranging from genetic predisposition to, e.g., tumors, infection, brain injury, and stress, altogether increasing over lifetime, and exerting a spectrum of possible effects, also including beneficial functions.

Vascular response to social cognitive performance measured by infrared thermography: A translational study from mouse to man.

To assess complex social recognition in mice, we previously developed the SocioBox paradigm. Unexpectedly, 4 weeks after performing in the SocioBox, mice displayed robust social avoidance during Y-maze sociability testing. This unique "sociophobia" acquisition could be documented in independent cohorts. We therefore employed infrared thermography as a non-invasive method of stress-monitoring during SocioBox testing (presentation of five other mice) versus empty box. A higher Centralization Index (body/tail temperature) in the SocioBox correlated negatively with social recognition memory and, after 4 weeks, with social preference in the Y-maze. Assuming that social stimuli might be associated with characteristic thermo-responses, we exposed healthy men (N = 103) with a comparably high intelligence level to a standardized test session including two cognitive tests with or without social component (face versus pattern recognition). In some analogy to the Centralization Index (within-subject measure) used in mice, the Reference Index (ratio nose/malar cheek temperature) was introduced to determine the autonomic facial response/flushing during social recognition testing. Whereas cognitive performance and salivary cortisol were comparable across human subjects and tests, the Face Recognition Test was associated with a characteristic Reference Index profile. Infrared thermography may have potential for discriminating disturbed social behaviors.

Genetically induced brain inflammation by Cnp deletion transiently benefits from microglia depletion.

Reduced expression of 2'-3'-cyclic nucleotide 3'-phosphodiesterase (Cnp) in humans and mice causes white matter inflammation and catatonic signs. These consequences are experimentally alleviated by microglia ablation via colony-stimulating factor 1 receptor (CSF1R) inhibition using PLX5622. Here we address for the first time preclinical topics crucial for translation, most importantly 1) the comparison of 2 long-term PLX5622 applications (prevention and treatment) vs. 1 treatment alone, 2) the correlation of catatonic signs and executive dysfunction, 3) the phenotype of leftover microglia evading depletion, and 4) the role of intercellular interactions for efficient CSF1R inhibition. Based on our Cnp-/- mouse model and in vitro time-lapse imaging, we report the unexpected discovery that microglia surviving under PLX5622 display a highly inflammatory phenotype including aggressive premortal phagocytosis of oligodendrocyte precursor cells. Interestingly, ablating microglia in vitro requires mixed glial cultures, whereas cultured pure microglia withstand PLX5622 application. Importantly, 2 extended rounds of CSF1R inhibition are not superior to 1 treatment regarding any readout investigated (magnetic resonance imaging and magnetic resonance spectroscopy, behavior, immunohistochemistry). Catatonia-related executive dysfunction and brain atrophy of Cnp-/- mice fail to improve under PLX5622. To conclude, even though microglia depletion is temporarily beneficial and worth pursuing, complementary treatment strategies are needed for full and lasting recovery.-Fernandez Garcia-Agudo, L., Janova, H., Sendler, L. E., Arinrad, S., Steixner, A. A., Hassouna, I., Balmuth, E., Ronnenberg, A., Schopf, N., van der Flier, F. J., Begemann, M., Martens, H., Weber, M. S., Boretius, S., Nave, K.-A., Ehrenreich, H. Genetically induced brain inflammation by Cnp deletion transiently benefits from microglia depletion.

Uncoupling the widespread occurrence of anti-NMDAR1 autoantibodies from neuropsychiatric disease in a novel autoimmune model.

Autoantibodies of the IgG class against N-methyl-D-aspartate-receptor subunit-NR1 (NMDAR1-AB) were considered pathognomonic for anti-NMDAR encephalitis. This view has been challenged by the age-dependent seroprevalence (up to >20%) of functional NMDAR1-AB of all immunoglobulin classes found in >5000 individuals, healthy or affected by different diseases. These findings question a merely encephalitogenic role of NMDAR1-AB. Here, we show that NMDAR1-AB belong to the normal autoimmune repertoire of dogs, cats, rats, mice, baboons, and rhesus macaques, and are functional in the NMDAR1 internalization assay based on human IPSC-derived cortical neurons. The age dependence of seroprevalence is lost in nonhuman primates in captivity and in human migrants, raising the intriguing possibility that chronic life stress may be related to NMDAR1-AB formation, predominantly of the IgA class. Active immunization of ApoE-/- and ApoE+/+ mice against four peptides of the extracellular NMDAR1 domain or ovalbumin (control) leads to high circulating levels of specific AB. After 4 weeks, the endogenously formed NMDAR1-AB (IgG) induce psychosis-like symptoms upon MK-801 challenge in ApoE-/- mice, characterized by an open blood-brain barrier, but not in their ApoE+/+ littermates, which are indistinguishable from ovalbumin controls. Importantly, NMDAR1-AB do not induce any sign of inflammation in the brain. Immunohistochemical staining for microglial activation markers and T lymphocytes in the hippocampus yields comparable results in ApoE-/- and ApoE+/+ mice, irrespective of immunization against NMDAR1 or ovalbumin. These data suggest that NMDAR1-AB of the IgG class shape behavioral phenotypes upon access to the brain but do not cause brain inflammation on their own.

Cognitive, emotional and social phenotyping of mice in an observer-independent setting.

Based on the intellicage paradigm, we have developed a novel cognitive, emotional and social phenotyping battery that permits comprehensive standardized behavioral characterization of mice in an experimenter-independent social setting. Evaluation of this battery in a large number of male and female C57BL/6 wildtype mice, tested in >20 independent cohorts, revealed high reproducibility of the behavioral readouts and may serve as future reference tool. We noticed robust sex-specific differences in general activity, cognitive and emotional behavior, but not regarding preference for social pheromones. Specifically, female mice revealed higher activity, decreased sucrose preference, impaired reversal and place-time-reward learning. Furthermore, female mice reacted more sensitively than males to reward-withdrawal showing a negative emotional contrast/Crespi-effect. In a series of validation experiments, we tested mice with different pathologies, including neuroligin-3 deficient mice (male Nlgn3y/- and female Nlgn3+/-) for autistic behavior, oligodendrocyte-specific erythropoietin receptor knockout (oEpoR-/-) mice for cognitive impairment, as well as mouse models of renal failure (unilateral ureteral obstruction and 5/6 nephrectomy) and of type 2 diabetes (ApoE-/-) - for delineating potentially confounding effects of motivational factors (thirst, glucose-craving) on learning and memory assessments. As prominent features, we saw in Nlgn3 mutants reduced preference for social pheromones, whereas oEpoR-/- mice showed learning deficits in place or reversal learning tasks. Renal failure led to increased water intake, and diabetic metabolism to enhanced glucose preference, limiting interpretation of hereon based learning and memory performance in these mice. The phenotyping battery presented here may be well-suited as high-throughput multifaceted diagnostic instrument for translational neuropsychiatry and behavioral genetics.

Hypersocial behavior and biological redundancy in mice with reduced expression of PSD95 or PSD93.

The postsynaptic density proteins 95 (PSD95) and 93 (PSD93) belong to a family of scaffolding proteins, the membrane-associated guanylate kinases (MAGUKs), which are highly enriched in synapses and responsible for organizing the numerous protein complexes required for synaptic development and plasticity. Genetic studies have associated MAGUKs with diseases like autism and schizophrenia, but knockout mice show severe, complex defects with difficult-to-interpret behavioral abnormalities due to major motor dysfunction which is atypical for psychiatric phenotypes. Therefore, rather than studying loss-of-function mutants, we comprehensively investigated the behavioral consequences of reduced PSD95 expression, using heterozygous PSD95 knockout mice (PSD95+/-). Specifically, we asked whether heterozygous PSD95 deficient mice would exhibit alterations in the processing of social stimuli and social behavior. Additionally, we investigated whether PSD95 and PSD93 would reveal any indication of functional or biological redundancy. Therefore, homozygous and heterozygous PSD93 deficient mice were examined in a similar behavioral battery as PSD95 mutants. We found robust hypersocial behavior in the dyadic interaction test in both PSD95+/- males and females. Additionally, male PSD95+/- mice exhibited higher levels of aggression and territoriality, while female PSD95+/- mice showed increased vocalization upon exposure to an anesthetized female mouse. Both male and female PSD95+/- mice revealed mild hypoactivity in the open field but no obvious motor deficit. Regarding PSD93 mutants, homozygous (but not heterozygous) knockout mice displayed prominent hypersocial behavior comparable to that observed in PSD95+/- mice, despite a more severe motor phenotype, which precluded several behavioral tests or their interpretation. Considering that PSD95 and PSD93 reduction provoke strikingly similar behavioral consequences, we explored a potential substitution effect and found increased PSD93 protein expression in hippocampal synaptic enrichment preparations of PSD95+/- mice. These data suggest that both PSD95 and PSD93 are involved in processing of social stimuli and control of social behavior. This important role may be partly assured by functional/behavioral and biological/biochemical redundancy.

Ablation of BAF170 in Developing and Postnatal Dentate Gyrus Affects Neural Stem Cell Proliferation, Differentiation, and Learning.

The BAF chromatin remodeling complex plays an essential role in brain development. However its function in postnatal neurogenesis in hippocampus is still unknown. Here, we show that in postnatal dentate gyrus (DG), the BAF170 subunit of the complex is expressed in radial glial-like (RGL) progenitors and in cell types involved in subsequent steps of adult neurogenesis including mature astrocytes. Conditional deletion of BAF170 during cortical late neurogenesis as well as during adult brain neurogenesis depletes the pool of RGL cells in DG, and promotes terminal astrocyte differentiation. These derangements are accompanied by distinct behavioral deficits, as reflected by an impaired accuracy of place responding in the Morris water maze test, during both hidden platform as well as reversal learning. Inducible deletion of BAF170 in DG during adult brain neurogenesis resulted in mild spatial learning deficits, having a more pronounced effect on spatial learning during the reversal test. These findings demonstrate involvement of BAF170-dependent chromatin remodeling in hippocampal neurogenesis and cognition and suggest a specific role of adult neurogenesis in DG in adaptive behavior.

The SocioBox: A Novel Paradigm to Assess Complex Social Recognition in Male Mice.

Impairments in social skills are central to mental disease, and developing tools for their assessment in mouse models is essential. Here we present the SocioBox, a new behavioral paradigm to measure social recognition. Using this paradigm, we show that male wildtype mice of different strains can readily identify an unfamiliar mouse among 5 newly acquainted animals. In contrast, female mice exhibit lower locomotor activity during social exploration in the SocioBox compared to males and do not seem to discriminate between acquainted and unfamiliar mice, likely reflecting inherent differences in gender-specific territorial tasks. In addition to a simple quantification of social interaction time of mice grounded on predefined spatial zones (zone-based method), we developed a set of unbiased, data-driven analysis tools based on heat map representations and characterized by greater sensitivity. First proof-of-principle that the SocioBox allows diagnosis of social recognition deficits is provided using male PSD-95 heterozygous knockout mice, a mouse model related to psychiatric pathophysiology.

Selective expression of a constitutively active erythropoietin receptor in GABAergic neurons alters hippocampal network properties without affecting cognition.

We have previously shown that treatment with erythropoietin (EPO) improves cognition in patients with neuropsychiatric disorders as well as in healthy mice, and that transgenic expression of a constitutively active form of the EPO receptor (cEPOR) in glutamatergic neurons boosts higher cognitive functions in mice. In the present work, we examined whether selective activation of EPOR signaling in GABAergic neurons would also modulate cognitive performance. We generated transgenic mice that express cEPOR under the control of the vesicular inhibitory amino acid transporter (Viaat) promoter and subjected them to comprehensive behavioral, cognitive, and electrophysiological analyses. We demonstrate that transgenic expression of cEPOR in GABAergic neurons alters hippocampal gamma-oscillations and enhances long-term potentiation but neither impairs nor improves cognition. To conclude, constitutively active EPOR in GABAergic neurons changes hippocampal network properties without affecting cognition, which suggests that the effect of EPO on cognition is dominated by its effect on the glutamatergic system. Treatment with EPO improves cognitive performance. We previously demonstrated that this effect is replicated by constitutive autoactivation of cEPOR in glutamatergic neurons. By contrast, cEPOR in GABAergic neurons changes hippocampal network properties but neither impairs nor enhances cognition. Thus, EPO modulates neuronal plasticity, and the cognitive benefits may be mainly attributable to its effect on the glutamatergic system.

Gpm6b deficiency impairs sensorimotor gating and modulates the behavioral response to a 5-HT2A/C receptor agonist.

The neuronal tetraspan proteins, M6A (Gpm6a) and M6B (Gpm6b), belong to the family of proteolipids that are widely expressed in the brain. We recently reported Gpm6a deficiency as a monogenetic cause of claustrophobia in mice. Its homolog proteolipid, Gpm6b, is ubiquitously expressed in neurons and oligodendrocytes. Gpm6b is involved in neuronal differentiation and myelination. It interacts with the N-terminal domain of the serotonin transporter (SERT) and decreases cell-surface expression of SERT. In the present study, we employed Gpm6b null mutant mice (Gpm6b(-/-)) to search for behavioral functions of Gpm6b. We studied male and female Gpm6b(-/-) mice and their wild-type (WT, Gpm6b(+/+)) littermates in an extensive behavioral test battery. Additionally, we investigated whether Gpm6b(-/-) mice exhibit changes in the behavioral response to a 5-HT2A/C receptor agonist. We found that Gpm6b(-/-) mice display completely normal sensory and motor functions, cognition, as well as social and emotionality-like (anxiety, depression) behaviors. On top of this inconspicuous behavioral profile, Gpm6b(-/-) mice of both genders exhibit a selective impairment in prepulse inhibition of the acoustic startle response. Furthermore, in contrast to WT mice that show the typical locomotion suppression and increase in grooming activity after intraperitoneal administration of DOI [(±)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane hydrochloride], Gpm6b(-/-) mice demonstrate a blunted behavioral response to this 5-HT2A/C receptor agonist. To conclude, Gpm6b deficiency impairs sensorimotor gating and modulates the behavioral response to a serotonergic challenge.

St8sia2 deficiency plus juvenile cannabis exposure in mice synergistically affect higher cognition in adulthood.

The neural cell adhesion molecule (NCAM) and its functionally linked polysialyltransferases, ST8SIA2 and ST8SIA4, are crucial for synaptic plasticity. Variations in encoding genes have been associated with mental illness. Since cannabinoids can alter NCAM polysialylation, we hypothesized that delta-9-tetrahydrocannabinol (Δ9-THC) might act as environmental 'second hit' regarding cognition of St8sia2(-/-) mice. These mice show per se minor behavioral abnormalities, consisting of reduced anxiety and mild cognitive deficits. Chronic Δ9-THC treatment of juvenile male wildtype mice (St8sia2(+/+)) (7mg/kg every other day over 3 weeks) did not appreciably affect cognition. St8sia2(-/-) mice, however, displayed a synergistic negative consequence of Δ9-THC on learning/memory, accompanied by polysialic acid-free NCAM-180 reduction in hippocampus and polysialic acid increase in dentate outer molecular layer. These synergistic effects became obvious only months after the last Δ9-THC. We conclude that juvenile cannabis exposure may cause delayed but lasting damage on cognition in subjects genetically predisposed to altered NCAM polysialylation.

Genetic markers of a Munc13 protein family member, BAIAP3, are gender specifically associated with anxiety and benzodiazepine abuse in mice and humans.

Anxiety disorders and substance abuse, including benzodiazepine use disorder, frequently occur together. Unfortunately, treatment of anxiety disorders still includes benzodiazepines, and patients with an existing comorbid benzodiazepine use disorder or a genetic susceptibility for benzodiazepine use disorder may be at risk of adverse treatment outcomes. The identification of genetic predictors for anxiety disorders, and especially for benzodiazepine use disorder, could aid the selection of the best treatment option and improve clinical outcomes. The brain-specific angiogenesis inhibitor I-associated protein 3 (Baiap3) is a member of the mammalian uncoordinated 13 (Munc13) protein family of synaptic regulators of neurotransmitter exocytosis, with a striking expression pattern in amygdalae, hypothalamus and periaqueductal gray. Deletion of Baiap3 in mice leads to enhanced seizure propensity and increased anxiety, with the latter being more pronounced in female than in male animals. We hypothesized that genetic variation in human BAIAP3 may also be associated with anxiety. By using a phenotype-based genetic association study, we identified two human BAIAP3 single-nucleotide polymorphism risk genotypes (AA for rs2235632, TT for rs1132358) that show a significant association with anxiety in women and, surprisingly, with benzodiazepine abuse in men. Returning to mice, we found that male, but not female, Baiap3 knockout (KO) mice develop tolerance to diazepam more quickly than control animals. Analysis of cultured Baiap3 KO hypothalamus slices revealed an increase in basal network activity and an altered response to diazepam withdrawal. Thus, Baiap3/BAIAP3 is gender specifically associated with anxiety and benzodiazepine use disorder, and the analysis of Baiap3/BAIAP3-related functions may help elucidate mechanisms underlying the development of both disorders.

Development of an autism severity score for mice using Nlgn4 null mutants as a construct-valid model of heritable monogenic autism.

Autism is the short name of a complex and heterogeneous group of disorders (autism spectrum disorders, ASD) with several lead symptoms required for classification, including compromised social interaction, reduced verbal communication and stereotyped repetitive behaviors/restricted interests. The etiology of ASD is still unknown in most cases but monogenic heritable forms exist that have provided insights into ASD pathogenesis and have led to the notion of autism as a 'synapse disorder'. Among the most frequent monogenic causes of autism are loss-of-function mutations of the NLGN4X gene which encodes the synaptic cell adhesion protein neuroligin-4X (NLGN4X). We previously described autism-like behaviors in male Nlgn4 null mutant mice, including reduced social interaction and ultrasonic communication. Here, we extend the phenotypical characterization of Nlgn4 null mutant mice to both genders and add a series of additional autism-relevant behavioral readouts. We now report similar social interaction and ultrasonic communication deficits in females as in males. Furthermore, aggression, nest-building parameters, as well as self-grooming and circling as indicators of repetitive behaviors/stereotypies were explored in both genders. The construction of a gender-specific autism severity composite score for Nlgn4 mutant mice markedly diminishes population/sample heterogeneity typically obtained for single tests, resulting in p values of <0.00001 and a genotype predictability of 100% for male and of >83% for female mice. Taken together, these data underscore the similarity of phenotypical consequences of Nlgn4/NLGN4X loss-of-function in mouse and man, and emphasize the high relevance of Nlgn4 null mutant mice as an ASD model with both construct and face validity.