Low-Intensity Blast Induces Acute Glutamatergic Hyperexcitability in Mouse Hippocampus Leading to Long-Term Learning Deficits and Altered Expression of Proteins Involved in Synaptic Plasticity and Serine Protease Inhibitors

Neurocognitive consequences of blast-induced traumatic brain injury (bTBI) pose significant concerns for military service members and veterans with the majority of invisible injury. However, the underlying mechanism of such mild bTBI by low-intensity blast (LIB) exposure for long-term cognitive and mental deficits remains elusive. Our previous studies have shown that mice exposed to LIB result in nanoscale ultrastructural abnormalities in the absence of gross or apparent cellular damage in the brain. Here we tested the hypothesis that glutamatergic hyperexcitability may contribute to long-term learning deficits. Using brain slice electrophysiological recordings, we found an increase in averaged frequencies with a burst pattern of miniature excitatory postsynaptic currents (mEPSCs) in hippocampal CA3 neurons in LIB-exposed mice at 1- and 7-days post injury, which was blocked by a specific NMDA receptor antagonist AP5. In addition, cognitive function assessed at 3-months post LIB exposure by automated home-cage monitoring showed deficits in dynamic patterns of discrimination learning and cognitive flexibility in LIB-exposed mice. Collected hippocampal tissue was further processed for quantitative global-proteomic analysis. Advanced data-independent acquisition for quantitative tandem mass spectrometry analysis identified altered expression of proteins involved in synaptic plasticity and serine protease inhibitors in LIB-exposed mice. Some were correlated with the ability of discrimination learning and cognitive flexibility. These findings show that acute glutamatergic hyperexcitability in the hippocampus induced by LIB may contribute to long-term cognitive dysfunction and protein alterations. Studies using this military-relevant mouse model of mild bTBI provide valuable insights into developing a potential therapeutic strategy to ameliorate hyperexcitability-modulated LIB injuries

Systematic assessment of the replicability and generalizability of preclinical findings: Impact of protocol harmonization across laboratory sites.

The influence of protocol standardization between laboratories on their replicability of preclinical results has not been addressed in a systematic way. While standardization is considered good research practice as a means to control for undesired external noise (i.e., highly variable results), some reports suggest that standardized protocols may lead to idiosyncratic results, thus undermining replicability. Through the EQIPD consortium, a multi-lab collaboration between academic and industry partners, we aimed to elucidate parameters that impact the replicability of preclinical animal studies. To this end, 3 experimental protocols were implemented across 7 laboratories. The replicability of results was determined using the distance travelled in an open field after administration of pharmacological compounds known to modulate locomotor activity (MK-801, diazepam, and clozapine) in C57BL/6 mice as a worked example. The goal was to determine whether harmonization of study protocols across laboratories improves the replicability of the results and whether replicability can be further improved by systematic variation (heterogenization) of 2 environmental factors (time of testing and light intensity during testing) within laboratories. Protocols were tested in 3 consecutive stages and differed in the extent of harmonization across laboratories and standardization within laboratories: stage 1, minimally aligned across sites (local protocol); stage 2, fully aligned across sites (harmonized protocol) with and without systematic variation (standardized and heterogenized cohort); and stage 3, fully aligned across sites (standardized protocol) with a different compound. All protocols resulted in consistent treatment effects across laboratories, which were also replicated within laboratories across the different stages. Harmonization of protocols across laboratories reduced between-lab variability substantially compared to each lab using their local protocol. In contrast, the environmental factors chosen to introduce systematic variation within laboratories did not affect the behavioral outcome. Therefore, heterogenization did not reduce between-lab variability further compared to the harmonization of the standardized protocol. Altogether, these findings demonstrate that subtle variations between lab-specific study protocols may introduce variation across independent replicate studies even after protocol harmonization and that systematic heterogenization of environmental factors may not be sufficient to account for such between-lab variation. Differences in replicability of results within and between laboratories highlight the ubiquity of study-specific variation due to between-lab variability, the importance of transparent and fine-grained reporting of methodologies and research protocols, and the importance of independent study replication

Reproducibility via coordinated standardization:A multi-center study in a Shank2 genetic rat model for Autism Spectrum Disorders

Inconsistent findings between laboratories are hampering scientific progress and are of increasing public concern. Differences in laboratory environment is a known factor contributing to poor reproducibility of findings between research sites, and well-controlled multisite efforts are an important next step to identify the relevant factors needed to reduce variation in study outcome between laboratories. Through harmonization of apparatus, test protocol, and aligned and non-aligned environmental variables, the present study shows that behavioral pharmacological responses in Shank2 knockout (KO) rats, a model of synaptic dysfunction relevant to autism spectrum disorders, were highly replicable across three research centers. All three sites reliably observed a hyperactive and repetitive behavioral phenotype in KO rats compared to their wild-type littermates as well as a dose-dependent phenotype attenuation following acute injections of a selective mGluR1 antagonist. These results show that reproducibility in preclinical studies can be obtained and emphasizes the need for high quality and rigorous methodologies in scientific research. Considering the observed external validity, the present study also suggests mGluR1 as potential target for the treatment of autism spectrum disorders

Measuring Behavior in the Home Cage : Study Design, Applications, Challenges, and Perspectives

FUNDING This research was supported by NIH grants R00AG056662, P20GM125528, AG057424 to WS and SLPeer reviewedPublisher PD

AHCODA-DB:a data repository with web-based mining tools for the analysis of automated high-content mouse phenomics data

Background: Systematic, standardized and in-depth phenotyping and data analyses of rodent behaviour empowers gene-function studies, drug testing and therapy design. However, no data repositories are currently available for standardized quality control, data analysis and mining at the resolution of individual mice.Description: Here, we present AHCODA-DB, a public data repository with standardized quality control and exclusion criteria aimed to enhance robustness of data, enabled with web-based mining tools for the analysis of individually and group-wise collected mouse phenotypic data. AHCODA-DB allows monitoring in vivo effects of compounds collected from conventional behavioural tests and from automated home-cage experiments assessing spontaneous behaviour, anxiety and cognition without human interference. AHCODA-DB includes such data from mutant mice (transgenics, knock-out, knock-in), (recombinant) inbred strains, and compound effects in wildtype mice and disease models. AHCODA-DB provides real time statistical analyses with single mouse resolution and versatile suite of data presentation tools. On March 9th, 2017 AHCODA-DB contained 650 k data points on 2419 parameters from 1563 mice.Conclusion: AHCODA-DB provides users with tools to systematically explore mouse behavioural data, both with positive and negative outcome, published and unpublished, across time and experiments with single mouse resolution. The standardized (automated) experimental settings and the large current dataset (1563 mice) in AHCODA-DB provide a unique framework for the interpretation of behavioural data and drug effects. The use of common ontologies allows data export to other databases such as the Mouse Phenome Database. Unbiased presentation of positive and negative data obtained under the highly standardized screening conditions increase cost efficiency of publicly funded mouse screening projects and help to reach consensus conclusions on drug responses and mouse behavioural phenotypes. The website is publicly accessible through https://public.sylics.com and can be viewed in every recent version of all commonly used browsers

Glycine receptor subunit-β-deficiency in a mouse model of spasticity results in attenuated physical performance, growth, and muscle strength

Spasticity is the most common neurological disorder associated with increased muscle contraction causing impaired movement and gait. The aim of this study was to characterize the physical performance, skeletal muscle function and phenotype of mice with a hereditary spastic mutation (B6.Cg-Glrbspa/J). Motor function, gait and physical activity of juvenile and adult spastic mice and the morphological, histological and mechanical characteristics of their soleus and gastrocnemius medialis muscles were compared with their wild-type (WT) littermates. Spastic mice showed attenuated growth, impaired motor function and low physical activity. Gait of spastic mice was characterized by a typical hopping pattern. Spastic mice showed lower muscle forces, which were related to the smaller physiological cross-sectional area of spastic muscles. The muscle-tendon complex length-force relationship of adult gastrocnemius medialis was shifted towards shorter lengths, which was explained by attenuated longitudinal tibia growth. Spastic gastrocnemius medialis was more fatigue resistant than WT gastrocnemius medialis. This was largely explained by a higher mitochondrial content in muscle fibers and relatively higher percentage of slow-type muscle fibers. Muscles of juvenile spastic mice showed similar differences compared with WT juvenile mice, but these were less pronounced than between adult mice. This study shows that in spastic mice, disturbed motor function and gait is likely to be the result of hyperactivity of skeletal muscle and impaired skeletal muscle growth, which progress with age

Sheltering behavior and locomotor activity in 11 genetically diverse common inbred mouse strains using home-cage monitoring

Functional genetic analyses in mice rely on efficient and in-depth characterization of the behavioral spectrum. Automated home-cage observation can provide a systematic and efficient screening method to detect unexplored, novel behavioral phenotypes. Here, we analyzed high-throughput automated home-cage data using existing and novel concepts, to detect a plethora of genetic differences in spontaneous behavior in a panel of commonly used inbred strains (129S1/SvImJ, A/J, C3H/HeJ, C57BL/6J, BALB/cJ, DBA/2J, NOD/LtJ, FVB/NJ, WSB/EiJ, PWK/PhJ and CAST/EiJ). Continuous video-tracking observations of sheltering behavior and locomotor activity were segmented into distinguishable behavioral elements, and studied at different time scales, yielding a set of 115 behavioral parameters of which 105 showed highly significant strain differences. This set of 115 parameters was highly dimensional; principal component analysis identified 26 orthogonal components with eigenvalues above one. Especially novel parameters of sheltering behavior and parameters describing aspects of motion of the mouse in the home-cage showed high genetic effect sizes. Multi-day habituation curves and patterns of behavior surrounding dark/light phase transitions showed striking strain differences, albeit with lower genetic effect sizes. This spontaneous home-cage behavior study demonstrates high dimensionality, with a strong genetic contribution to specific sets of behavioral measures. Importantly, spontaneous home-cage behavior analysis detects genetic effects that cannot be studied in conventional behavioral tests, showing that the inclusion of a few days of undisturbed, labor extensive homecage assessment may greatly aid gene function analyses and drug target discovery

Within-strain variation in behavior differs consistently between common inbred strains of mice

Genetic and environmental factors interact throughout life and give rise to individual differences, i.e., individuality. The diversifying effect of environmental factors is counteracted by genetic mechanisms to yield persistence of specific features (robustness). Here, we compared robustness between cohorts of isogenic mice of eight different commonly used strains by analyzing to what extent environmental variation contributed to individuality in each of the eight genotypes, using a previously published dataset. Behavior was assessed in the home-cage, providing control over environmental factors, to reveal within-strain variability in numerous spontaneous behaviors. Indeed, despite standardization and in line with previous studies, substantial variability among mice of the same inbred strain was observed. Strikingly, across a multidimensional set of 115 behavioral parameters, several strains consistently ranked high in within-strain variability (DBA/2J, 129S1/Sv A/J and NOD/LtJ), whereas other strains ranked low (C57BL/6J and BALB/c). Strain rankings of within-strain variability in behavior were confirmed in an independent, previously published behavioral dataset using conventional behavioral tests administered to different mice from the same breeding colonies. Together, these show that genetically inbred mouse strains consistently differ in phenotypic robustness against environmental variation, suggesting that genetic factors contribute to variation in robustness

A 1-night operant learning task without food-restriction differentiates among mouse strains in an automated home-cage environment

Individuals are able to change their behavior based on its consequences, a process involving instrumental learning. Studying instrumental learning in mice can provide new insights in this elementary aspect of cognition. Conventional appetitive operant learning tasks that facilitate the study of this form of learning in mice, as well as more complex operant paradigms, require labor-intensive handling and food deprivation to motivate the animals. Here, we describe a 1-night operant learning protocol that exploits the advantages of automated home-cage testing and circumvents the interfering effects of food restriction. The task builds on behavior that is part of the spontaneous exploratory repertoire during the days before the task. We compared the behavior of C57BL/6J, BALB/cJ and DBA/2J mice and found various differences in behavior during this task, but no differences in learning curves. BALB/cJ mice showed the largest instrumental learning response, providing a superior dynamic range and statistical power to study instrumental learning by using this protocol. Insights gained with this home-cage-based learning protocol without food restriction will be valuable for the development of other, more complex, cognitive tasks in automated home-cages

The systemic inhibition of the terminal complement system reduces neuroinflammation but does not improve motor function in mouse models of CMT1A with overexpressed PMP22

Charcot-Marie-Tooth disease type 1A (CMT1A) is the most prevalent hereditary demyelinating neuropathy. This autosomal, dominantly inherited disease is caused by a duplication on chromosome 17p which includes the peripheral myelin protein 22 (PMP22) gene. There is clinical evidence that the disability in CMT1A is to a large extend due to axonal damage rather than demyelination. Over-expression of PMP22 is recently thought to impede cholesterol trafficking causing a total shutdown of local cholesterol and lipid synthesis in the Schwann cells, thus disturbing their ability to remyelinate. But there is a large variety in disease burden between CMT1A patients with the same genetic defect, indicating the presence of modifying factors that affect disease severity. One of these potential factors is the immune system. Several reports have described patients with co-occurrence of CMT1A with chronic inflammatory demyelinating disease or Guillain-Barré syndrome. We have previously shown in multiple animal models that the innate immune system and specifically the terminal complement system is a driver of inflammatory demyelination. To test the contribution of the terminal complement system to neuroinflammation and disease progression in CMT1A, we inhibited systemic complement C6 in two transgenic mouse models for CMT1A, the C3-PMP22 and C3-PMP22 c-JunP0Cre models. Both models over-express human PMP22, and one (C3-PMP22 c-JunP0Cre) also has a Schwann cell-specific knockout of c-Jun, a crucial regulator of myelination controlling autophagy. We found that systemic inhibition of C6 using antisense oligonucleotides affects the neuroinflammation, Rho GTPase and ERK/MAPK signalling pathways in the CMT1A mouse models. The cholesterol synthesis pathway remained unaffected. Analysis of motor function during treatment with C6 antisense oligonucleotides did not reveal any significant improvement in the CMT1A mouse models. This study shows that the contribution of the terminal complement system to progressive loss of motor function in the CMT1A mouse models tested is limited