Introduction to the EQIPD quality system.

While high risk of failure is an inherent part of developing innovative therapies, it can be reduced by adherence to evidence-based rigorous research practices. Supported through the European Union's Innovative Medicines Initiative, the EQIPD consortium has developed a novel preclinical research quality system that can be applied in both public and private sectors and is free for anyone to use. The EQIPD Quality System was designed to be suited to boost innovation by ensuring the generation of robust and reliable preclinical data while being lean, effective and not becoming a burden that could negatively impact the freedom to explore scientific questions. EQIPD defines research quality as the extent to which research data are fit for their intended use. Fitness, in this context, is defined by the stakeholders, who are the scientists directly involved in the research, but also their funders, sponsors, publishers, research tool manufacturers, and collaboration partners such as peers in a multi-site research project. The essence of the EQIPD Quality System is the set of 18 core requirements that can be addressed flexibly, according to user-specific needs and following a user-defined trajectory. The EQIPD Quality System proposes guidance on expectations for quality-related measures, defines criteria for adequate processes (i.e. performance standards) and provides examples of how such measures can be developed and implemented. However, it does not prescribe any pre-determined solutions. EQIPD has also developed tools (for optional use) to support users in implementing the system and assessment services for those research units that successfully implement the quality system and seek formal accreditation. Building upon the feedback from users and continuous improvement, a sustainable EQIPD Quality System will ultimately serve the entire community of scientists conducting non-regulated preclinical research, by helping them generate reliable data that are fit for their intended use.

Pre-Clinical Common Data Elements for Traumatic Brain Injury Research: Progress and Use Cases.

Traumatic brain injury (TBI) is an extremely complex condition due to heterogeneity in injury mechanism, underlying conditions, and secondary injury. Pre-clinical and clinical researchers face challenges with reproducibility that negatively impact translation and therapeutic development for improved TBI patient outcomes. To address this challenge, TBI Pre-clinical Working Groups expanded upon previous efforts and developed common data elements (CDEs) to describe the most frequently used experimental parameters. The working groups created 913 CDEs to describe study metadata, animal characteristics, animal history, injury models, and behavioral tests. Use cases applied a set of commonly used CDEs to address and evaluate the degree of missing data resulting from combining legacy data from different laboratories for two different outcome measures (Morris water maze [MWM]; RotorRod/Rotarod). Data were cleaned and harmonized to Form Structures containing the relevant CDEs and subjected to missing value analysis. For the MWM dataset (358 animals from five studies, 44 CDEs), 50% of the CDEs contained at least one missing value, while for the Rotarod dataset (97 animals from three studies, 48 CDEs), over 60% of CDEs contained at least one missing value. Overall, 35% of values were missing across the MWM dataset, and 33% of values were missing for the Rotarod dataset, demonstrating both the feasibility and the challenge of combining legacy datasets using CDEs. The CDEs and the associated forms created here are available to the broader pre-clinical research community to promote consistent and comprehensive data acquisition, as well as to facilitate data sharing and formation of data repositories. In addition to addressing the challenge of standardization in TBI pre-clinical studies, this effort is intended to bring attention to the discrepancies in assessment and outcome metrics among pre-clinical laboratories and ultimately accelerate translation to clinical research.

Mouse model systems of autism spectrum disorder: Replicability and informatics signature.

Phenotyping mouse model systems of human disease has proven to be a difficult task, with frequent poor inter- and intra-laboratory replicability, particularly in behavioral domains such as social and cognitive function. However, establishing robust animal model systems with strong construct validity is of fundamental importance as they are central tools for understanding disease pathophysiology and developing therapeutics. To complete our studies of mouse model systems relevant to autism spectrum disorder (ASD), we present a replication of the main findings from our two published studies of five genetic mouse model systems of ASD. To assess the intra-laboratory robustness of previous results, we chose the two model systems that showed the greatest phenotypic differences, the Shank3/F and Cntnap2, and repeated assessments of general health, activity and social behavior. We additionally explored all five model systems in the same framework, comparing all results obtained in this three-yearlong effort using informatics techniques to assess commonalities and differences. Our results showed high intra-laboratory replicability of results, even for those with effect sizes that were not particularly large, suggesting that discrepancies in the literature may be dependent on subtle but pivotal differences in testing conditions, housing enrichment, or background strains and less so on the variability of the behavioral phenotypes. The overall informatics analysis suggests that in our behavioral assays we can separate the set of tested mouse model system into two main classes that in some aspects lie on opposite ends of the behavioral spectrum, supporting the view that autism is not a unitary concept.

Guidelines and Initiatives for Good Research Practice.

This chapter explores existing data reproducibility and robustness initiatives from a cross-section of large funding organizations, granting agencies, policy makers, journals, and publishers with the goal of understanding areas of overlap and potential gaps in recommendations and requirements. Indeed, vigorous stakeholder efforts to identify and address irreproducibility have resulted in the development of a multitude of guidelines but with little harmonization. This likely results in confusion for the scientific community and may pose a barrier to strengthening quality standards instead of being used as a resource that can be meaningfully implemented. Guidelines are also often framed by funding bodies and publishers as recommendations instead of requirements in order to accommodate scientific freedom, creativity, and innovation. However, without enforcement, this may contribute to uneven implementation. The text concludes with an analysis to provide recommendations for future guidelines and policies to enhance reproducibility and to align on a consistent strategy moving forward.

Repeated recall and PKMζ maintain fear memories in juvenile rats.

We examined the neural substrates of fear memory formation and maintenance when repeated recall was used to prevent forgetting in young animals. In contrast to adult rats, juveniles failed to show contextual fear responses at 4 d post-fear conditioning. Reconsolidation sessions 3 and 6 d after conditioning restored contextual fear responses in juveniles 7 d after initial training. In juveniles that received reconsolidation sessions, protein kinase M zeta (PKMζ) increased in the amygdala, but not in the hippocampus. These data suggest that repeated reminders and increased PKMζ maintain fear responses in juvenile animals that otherwise would not exhibit this behavior.

Comprehensive Analysis of the 16p11.2 Deletion and Null Cntnap2 Mouse Models of Autism Spectrum Disorder.

Autism spectrum disorder comprises several neurodevelopmental conditions presenting symptoms in social communication and restricted, repetitive behaviors. A major roadblock for drug development for autism is the lack of robust behavioral signatures predictive of clinical efficacy. To address this issue, we further characterized, in a uniform and rigorous way, mouse models of autism that are of interest because of their construct validity and wide availability to the scientific community. We implemented a broad behavioral battery that included but was not restricted to core autism domains, with the goal of identifying robust, reliable phenotypes amenable for further testing. Here we describe comprehensive findings from two known mouse models of autism, obtained at different developmental stages, using a systematic behavioral test battery combining standard tests as well as novel, quantitative, computer-vision based systems. The first mouse model recapitulates a deletion in human chromosome 16p11.2, found in 1% of individuals with autism. The second mouse model harbors homozygous null mutations in Cntnap2, associated with autism and Pitt-Hopkins-like syndrome. Consistent with previous results, 16p11.2 heterozygous null mice, also known as Del(7Slx1b-Sept1)4Aam weighed less than wild type littermates displayed hyperactivity and no social deficits. Cntnap2 homozygous null mice were also hyperactive, froze less during testing, showed a mild gait phenotype and deficits in the three-chamber social preference test, although less robust than previously published. In the open field test with exposure to urine of an estrous female, however, the Cntnap2 null mice showed reduced vocalizations. In addition, Cntnap2 null mice performed slightly better in a cognitive procedural learning test. Although finding and replicating robust behavioral phenotypes in animal models is a challenging task, such functional readouts remain important in the development of therapeutics and we anticipate both our positive and negative findings will be utilized as a resource for the broader scientific community.

Medial prefrontal cortex processes threatening stimuli in juvenile rats.

To survive, all mammalian species must recognize and respond appropriately to threatening stimuli. In adults, the prelimbic medial prefrontal cortex (mPFC) appears to be involved in fear expression, whereas the infralimbic mPFC mediates fear extinction. In juvenile rats (PN26), the mPFC receives information on potential predators but does not act on it. To test whether the prefrontal cortex is capable of fear regulation in the young organism, we exposed juvenile rats to a threatening or nonthreatening stimulus and assessed fear and brain Fos activation of the mPFC subdivisions, amygdala and periaqueductal gray (PAG). In response to the threat, juveniles froze more, spent more time far from the threat, and had elevated numbers of Fos-positive cells in the prelimbic mPFC, the medial amygdala, and ventral PAG. To test the hypothesis that the mPFC has a dual role in modulating the amygdala and PAG in juveniles, we pharmacologically disinhibited each of the two subdivisions of the mPFC and assessed freezing and downstream activation to the threat. Juvenile rats infused with picrotoxin into the prelimbic mPFC and exposed to a threatening stimulus froze less, spent less time far from the threat, and increased Fos expression. Infusion of picrotoxin into the infralimbic mPFC also reduced fear responding to the threatening stimulus but had no effect on Fos expression. In sum, it appears that the mPFC can process threatening stimuli in juveniles at this age, even though it is normally not involved in the fear responses.

The role of the medial prefrontal cortex in innate fear regulation in infants, juveniles, and adolescents.

In adult animals, the medial prefrontal cortex (mPFC) plays a significant role in regulating emotions and projects to the amygdala and periaqueductal gray (PAG) to modulate emotional responses. However, little is known about the development of this neural circuit and its relevance to unlearned fear in pre-adulthood. To address these issues, we examined the mPFC of 14-d-old (infants), 26-d-old (juveniles), and 38- to 42-d-old (adolescents) rats to represent different developmental and social milestones. The expression patterns of the neuronal marker FOS were used to assess neurological activity. Muscimol, a GABA agonist, was used to inactivate the prelimbic and infralimbic mPFC subdivisions (400 ng in 200 nl). Animals were exposed to either a threatening or nonthreatening stimulus that was ecologically relevant and age specific. Freezing was measured as an indicator of innate fear behavior. The data indicated that the mPFC is neither active nor responsive to innate fear in infant rats. In juveniles, the prelimbic mPFC became responsive in processing aversive sensory stimulation but did not regulate freezing behavior. Finally, during adolescence, inactivation of the prelimbic mPFC significantly attenuated freezing and decreased FOS expression in the ventral PAG. Surprisingly, across all ages, there were no significant differences in FOS levels in the medial and basolateral/lateral amygdala when either mPFC subdivision was inactivated. Together, unlearned fear has a unique developmental course with different brain areas involved in unlearned fear in the immature animal than the adult. In particular, the mPFC neural circuitry is different in young animals and progressively develops more capacities as the animal matures.

Norepinephrine mediates contextual fear learning and hippocampal pCREB in juvenile rats exposed to predator odor.

Predator odors induce unconditioned fear in the young animal and provide the opportunity to study the mechanisms underlying unlearned and learned fear. In the current study, cat odor produced unlearned, innate fear in infant (postnatal age 14; PN14) and juvenile (PN26) rats, but contextual fear learning occurred only in juveniles. It was hypothesized that contextual fear learning in juveniles is mediated by norepinephrine. Consistent with this hypothesis, pre-training injection of the ß-adrenergic antagonist propranolol reduced the unlearned fear response while post-training injection inhibited contextual fear learning in juvenile rats exposed to cat odor. We suggest that NE mediates the formation of contextual fear memories by activation of the transcription factor CREB in the hippocampus in juveniles but not in infants. Levels of phosphorylated CREB (pCREB) were increased in the dorsal and ventral hippocampi of juvenile rats exposed to cat odor. These levels were not increased in infants or juveniles exposed to a control odor. Further, propranolol blocked these increases in pCREB. In conclusion, although innate fear occurs within the neonatal period, contextual fear learning is a relatively late-occurring event, is hippocampal dependent, and mediated by norepinephrine.

Effects of the stimulus and chamber size on unlearned fear across development.

Predator odors have been found to induce unconditioned fear in adult animals and provide the opportunity to study the mechanisms underlying unlearned and learned fear. Predator threats change across an animal's lifetime, as do abilities that enable the animal to learn or engage in different defensive behaviors. Thus, the objective of this study was to determine the combination of factors that successfully induce unlearned fear to predator odor across development. Infant, juvenile, adolescent, and adult rats were exposed to one of the three odor stimuli (control odor, cat urine, or cat fur) in either a small or large chamber. Though all ages displayed fear-related behavior to cat odors, differences were reflected only in freezing behavior and not, as expected, risk-assessment. Infant and juvenile animals also increased freezing to cat urine compared to the control odor, possibly because these age groups possess limited defensive options to cope with threat and so may respond with freezing to all predator stimuli. Unexpectedly, chamber size had no effect on either freezing or risk-assessment in this study. Once the parameters of unconditioned fear are understood, they can be exploited to develop a learning paradigm to predator odors that could be used in early life.