How stra(i)nge are your controls? A comparative analysis of metabolic phenotypes in commonly used C57BL/6 substrains.

In recent years, insufficiently characterised controls have been a contributing factor to irreproducibility in biomedical research including neuroscience and metabolism. There is now a growing awareness of phenotypic differences between the C57BL/6 substrains which are commonly used as control animals. We here investigated baseline metabolic characteristics such as glucose regulation, fasted serum insulin levels and hepatic insulin signalling in five different C57BL/6 substrains (N, J, JOla, JRcc) of both sexes, obtained from two commercial vendors, Charles River Laboratories (Crl) and Envigo (Env). Our results indicate systematic and tissue-specific differences between substrains, affected by both vendor and sex, in all parameters investigated, and not necessarily mediated by the presence of the NntC57BL/6J mutation. Not only were there differences between 6J and 6N as expected, all three 6J substrains exhibited different profiles, even from the same breeder. Two distinct metabolic profiles were identified, one in which low insulin levels resulted in impaired glucose clearance (6JCrl; both sexes) and the other, where sustained elevations in fasted basal insulin levels led to glucose intolerance (male 6JRccEnv). Further, 6JRccEnv displayed sex differences in both glucose clearance and hepatic insulin signalling markers. In comparison, the two 6N substrains of either sex, irrespective of vendor, did not exhibit considerable differences, with 6NCrl animals presenting a good choice as a healthy baseline 'control' for many types of experiments. Overall, our data emphasise the importance of selecting and characterising control subjects regarding background, sex, and supplier to ensure proper experimental outcomes in biomedical research.

Sex Differences in Behavior and Molecular Pathology in the 5XFAD Model.

BACKGROUND: The prevalence of Alzheimer's disease (AD) is greater in women compared to men, but the reasons for this remain unknown. This sex difference has been widely neglected in experimental studies using transgenic mouse models of AD. OBJECTIVE: Here, we studied behavior and molecular pathology of 5-month-old 5XFAD mice, which express mutated human amyloid precursor protein and presenilin-1 on a C57BL/6J background, versus their wild-type littermate controls, to compare both sex- and genotype-dependent differences. METHODS: A novel behavioral paradigm was utilized (OF-NO-SI), comprising activity measures (Open Field, OF) arena, followed by Novel Object exploration (NO) and Social Interaction (SI) of a sex-matched conspecific. Each segment consisted of two repeated trials to assess between-trial habituation. Subsequently, brain pathology (amyloid load, stress response and inflammation markers, synaptic integrity, trophic support) was assessed using qPCR and western blotting. RESULTS: Female 5XFAD mice had higher levels of human APP and amyloid-ß and heightened inflammation versus males. These markers correlated with hyperactivity observed in both sexes, yet only female 5XFAD mice presented with subtle deficits in object and social exploration. Male animals had higher expression of stress markers and neurotrophic factors irrespective of genotype, this correlated with cognitive performance. CONCLUSION: The impact of sex on AD-relevant phenotypes is in line with human data and emphasizes the necessity of appropriate study design and reporting. Differential molecular profiles observed in male versus female mice offer insights into possible protective mechanisms, and hence treatment strategies.

PEERS - An Open Science "Platform for the Exchange of Experimental Research Standards" in Biomedicine.

Laboratory workflows and preclinical models have become increasingly diverse and complex. Confronted with the dilemma of a multitude of information with ambiguous relevance for their specific experiments, scientists run the risk of overlooking critical factors that can influence the planning, conduct and results of studies and that should have been considered a priori. To address this problem, we developed "PEERS" (Platform for the Exchange of Experimental Research Standards), an open-access online platform that is built to aid scientists in determining which experimental factors and variables are most likely to affect the outcome of a specific test, model or assay and therefore ought to be considered during the design, execution and reporting stages. The PEERS database is categorized into in vivo and in vitro experiments and provides lists of factors derived from scientific literature that have been deemed critical for experimentation. The platform is based on a structured and transparent system for rating the strength of evidence related to each identified factor and its relevance for a specific method/model. In this context, the rating procedure will not solely be limited to the PEERS working group but will also allow for a community-based grading of evidence. We here describe a working prototype using the Open Field paradigm in rodents and present the selection of factors specific to each experimental setup and the rating system. PEERS not only offers users the possibility to search for information to facilitate experimental rigor, but also draws on the engagement of the scientific community to actively expand the information contained within the platform. Collectively, by helping scientists search for specific factors relevant to their experiments, and to share experimental knowledge in a standardized manner, PEERS will serve as a collaborative exchange and analysis tool to enhance data validity and robustness as well as the reproducibility of preclinical research. PEERS offers a vetted, independent tool by which to judge the quality of information available on a certain test or model, identifies knowledge gaps and provides guidance on the key methodological considerations that should be prioritized to ensure that preclinical research is conducted to the highest standards and best practice.

Adolescent cannabis exposure increases heroin reinforcement in rats genetically vulnerable to addiction.

On the basis of epidemiological studies it has been proposed that cannabis use plays a causal role in the abuse of highly addictive drugs (Gateway Hypothesis). However, epidemiological studies are intrinsically unable to provide evidence of causality. Experimental studies can provide this evidence but they are feasible only in animal models and to date such evidence is lacking. In view of the importance of genetic factors in drug abuse, we investigated the influence of adolescent cannabis exposure on adult heroin reinforcement in two inbred rat strains differentially vulnerable to drugs of abuse, addiction prone Lewis (LEW) and addiction resistant Fischer 344 (F344) strains. Male LEW and F344 rats aged six weeks were exposed to increasing Δ9-tetrahydrocannabinol (THC) doses, twice a day for 3 days (2, 4, 8 mg/kg, i.p.). At adulthood they were allowed to self-administer heroin (0.025 mg/kg) under both Fixed- (FR) and Progressive- (PR) ratio schedules of responding. Following extinction, responding was reinstated by drug-cues and/or by heroin priming. THC pre-exposure increased responding for heroin and heroin intake under FR-3 and FR-5 as well as PR protocols and increased breaking point in PR schedules in LEW but not F344 rats. Drug cues and heroin priming reinstated responding in LEW and F344, but THC pre-exposure increased reinstatement by priming in LEW rats and by cues in F344 rats. These observations show that in genetically predisposed individuals, adolescent cannabis exposure increases heroin reinforcing properties, thus providing a mechanism for a causal role of adolescent cannabis use in heroin abuse.

Lights and shadows of schizophrenia therapy research: Lessons from oral risperidone and olanzapine.

BACKGROUND: Recently, patents of several atypical antipsychotics have reached their expiration date. AIMS: The purpose of the study was to highlight whether modifications of economic/scientific factors may be associated with possible changes in ongoing clinical research on antipsychotic drugs. METHODS: A large systematic analysis was used to depict the time-dependent distribution of published research articles addressing the clinical properties of oral risperidone and olanzapine conventional tablets, two largely prescribed atypical antipsychotics for which the patents have already expired in most of the countries. RESULTS: The systematic analysis indicated that the time-dependent distribution of the selected research articles followed a wave-shape pattern. A dramatic decline of primary and secondary analyses investigating the clinical effects of oral risperidone and olanzapine has occurred in the last decade, complemented by an expected strong reduction in the numbers of industrial-supported clinical studies and a smaller, but significant, decline in the amount of independent research articles. CONCLUSIONS: To date, greater involvement of independent research seems to be the only realistic chance to properly continue the investigation on the clinical properties of oral risperidone and olanzapine conventional tablets, as well as those of other off-patent antipsychotic drugs. However, the limits and potentialities of independent research in accomplishing such a demanding and enduring scientific effort should be addressed.

Systematic review of guidelines for internal validity in the design, conduct and analysis of preclinical biomedical experiments involving laboratory animals.

Over the last two decades, awareness of the negative repercussions of flaws in the planning, conduct and reporting of preclinical research involving experimental animals has been growing. Several initiatives have set out to increase transparency and internal validity of preclinical studies, mostly publishing expert consensus and experience. While many of the points raised in these various guidelines are identical or similar, they differ in detail and rigour. Most of them focus on reporting, only few of them cover the planning and conduct of studies. The aim of this systematic review is to identify existing experimental design, conduct, analysis and reporting guidelines relating to preclinical animal research. A systematic search in PubMed, Embase and Web of Science retrieved 13 863 unique results. After screening these on title and abstract, 613 papers entered the full-text assessment stage, from which 60 papers were retained. From these, we extracted unique 58 recommendations on the planning, conduct and reporting of preclinical animal studies. Sample size calculations, adequate statistical methods, concealed and randomised allocation of animals to treatment, blinded outcome assessment and recording of animal flow through the experiment were recommended in more than half of the publications. While we consider these recommendations to be valuable, there is a striking lack of experimental evidence on their importance and relative effect on experiments and effect sizes.

Childhood disintegrative disorder and autism spectrum disorder: a systematic review.

AIM: In an attempt to clarify the debate surrounding the diagnostic validity of childhood disintegrative disorder (CDD), we systematically reviewed its characteristics and compared it with autism spectrum disorder (ASD). METHOD: Four databases were searched (PubMed, PsycINFO, Embase, and Web of Science). Included articles had participants with CDD, as defined by symptoms present in the criteria of the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition, Text Revision and the International Classification of Diseases, 10th Revision. Comparison groups were those with ASD and ASD with regression. Case studies were excluded. RESULTS: Twenty articles, comprising 96 participants with CDD (80 males, 16 females), were included. Most studies were cross-sectional. The prevalence of CDD was 1.1 to 9.2 per 100 000, with a mean age at regression of 3 years 2 months (SD 1y 1mo), with a range of 2 years to 7 years. In addition to core CDD symptoms, most had intellectual impairment, anxiety, challenging behaviours, and regressed in toileting skills. Participants with CDD and ASD shared core diagnostic and extra-diagnostic features. However, participants with CDD seemed to have more severe symptoms and a different symptom profile, including apparently typical development before regression, faster regression, more affective symptoms, and more global developmental deficit. Possible genetic and autoimmune neurobiological mechanisms were identified. INTERPRETATION: There is limited high-quality evidence describing the aetiology and outcomes of CDD. However, given the qualitative and prognostic differences between ASD and CDD, we recommend that future diagnostic criteria should distinguish late-onset regression.

TRASTORNO DESINTEGRATIVO INFANTIL Y TRASTORNO DEL ESPECTRO AUTISTA: UNA REVISIÓN SISTEMÁTICA: OBJETIVO: En un intento de aclarar el debate que rodea la validez diagnóstica del trastorno desintegrativo infantil (TDI), revisamos sistemáticamente sus características y lo comparamos con el trastorno del espectro autista (TEA). MÉTODO: Se realizaron búsquedas en cuatro bases de datos (PubMed, PsycINFO, Embase y Web of Science). Los artículos incluidos tenían participantes con TDI, según lo definido por los síntomas presentes en los criterios del Manual diagnóstico y estadístico de trastornos mentales, Cuarta edición, Revisión de texto y Clasificación internacional de enfermedades, Décima revisión. Los grupos de comparación fueron aquellos con TEA y TEA con regresión. Se excluyeron los estudios de caso. RESULTADOS: Se incluyeron 20 artículos, con 96 participantes con TDI (80 varones y 16 mujeres). La mayoría de los estudios fueron de corte transversal. La prevalencia de TDI fue de 1,1 a 9,2 por 100.000, con una edad media de regresión de 3 años a 2 meses (DS 1 años 1 mes), con un rango de 2 años a 7 años. Además de los síntomas centrales de la TDI, la mayoría tenía deterioro intelectual, ansiedad, comportamientos desafiantes y regresión en las habilidades para ir al baño. Los participantes con TDI y TEA compartieron funciones básicas comunes de diagnóstico y de diagnóstico adicional. Sin embargo, los participantes con TDI parecían tener síntomas más graves y un perfil de síntomas diferente, incluido un desarrollo aparentemente típico antes de la regresión, una regresión más rápida, síntomas más afectivos y un déficit de desarrollo más global. Se identificaron posibles mecanismos genéticos y autoinmunes neurobiológicos. INTERPRETACIÓN: Existe una evidencia limitada de alta calidad que describe la etiología y los resultados de la TDI. Sin embargo, dadas las diferencias cualitativas y pronósticas entre la TEA y la TDI, recomendamos que los criterios diagnósticos futuros distingan la regresión de inicio tardío.

TRANSTORNO DESINTEGRATIVO DA INFÂNCIA E TRANSTORNO DO ESPECTRO AUTISTA: UMA REVISÃO SISTEMÁTICA: OBJETIVO: Na tentativa de esclarecer o debate em torno da validade diagnóstica do transtorno desintegrativo da infância (TDI), nós revisamos sistematicamente suas características e as comparamos com o transtorno do espectro autista (TEA). MÉTODO: Quatro bases de dados foram pesquisadas (PubMed, PsycINFO, Embase, e Web of Science). Os artigos incluídos tinham participantes com TDI, como definido pelos sintomas presentes nos critérios do Manual Diagnóstico e Estatístico de Transtornos Mentais, quarta edição, com revisão do texto, e na Classificação Internacional de Doenças, 10a edição. Grupos de comparação foram aqueles com TEA e TEA com regressão. Estudos de caso foram excluídos. RESULTADOS: Vinte artigos, incluindo 96 participantes com TDI (80 do sexo masculino, 16 do sexo feminino), foram incluídos. A maior parte dos estudos era transversal. A prevalência de TDI foi de 1,1 a 9,2 por 100.000, com idade média de regressão de 3 anos e 2 meses (DP 1a 1m), com variação de 2 anos a 7 anos. Além dos sintomas centrais de TDI, a maioria tinha deficiência intelectual, ansiedade, comportamentos desafiadores, e regressão na habilidade de usar o banheiro. Participantes com TDI e TEA compartilham aspectos diagósticos e extra-diagnósticos centrais. No entanto, os participantes com TDI pareceram ter sintomas mais severos e um perfil diferente de sintomas, incluindo desenvolvimento aparentemente típico antes da regressão, regressão mais rápida, mais sintomas afetivos, e maior déficit global do desenvolvimento. Possíveis mecanismos neurobiológicos genéticos e autoimunes foram identificados. INTERPRETAÇÃO: Há evidência limitada de alta qualidade descrevendo a etiologia dos resultados do TDI. No entanto, dadas as diferenças qualitativas e prognósticas entre TEA e TDI, recomendamos que futuros critérios diagnósticos distinguam a regressão de início tardio.

Widespread reduction of dopamine cell bodies and terminals in adult rats exposed to a low dose regimen of MDMA during adolescence.

Although MDMA (3,4-methylendioxymethamphetamine, ecstasy) neurotoxicity in serotonin neurons is largely recognized in a wide variety of species including man, neurotoxicity in dopamine (DA) neurons is thought to be species-specific. MDMA is mainly consumed by adolescents, often in conjunction with caffeine (Energy Drinks) and this association has been reported to exacerbate MDMA toxic effects. In order to model these aspects of MDMA use, vis-à-vis their impact on DA neurons, we investigated the effects of adolescent exposure to low doses of MDMA (5 mg/kg for 10 days), alone or in combination with caffeine (10 mg/kg) on neuronal and functional DA indices and on recognition memory in adult rats. MDMA reduced density of tyrosine hydroxylase (TH) positive neurons in the ventral tegmental area and in the substantia nigra pars compacta, and immunoreactivity of TH and DA transporter in the nucleus accumbens (NAc) shell and core, and caudate-putamen. This same treatment caused a reduction of basal dialysate DA in the NAc core. MDMA-pretreated rats also showed behavioral sensitization to a MDMA challenge at adulthood and potentiation of MDMA-induced increase of dialysate DA in the NAc core, but not in the NAc shell. In addition, MDMA-treated rats displayed a deficit in recognition memory. Caffeine co-administration did not affect the above outcomes. Our results show that adolescent exposure of rats to low doses of MDMA induces long-lasting and widespread reduction of DA neurons indicative of a neurotoxic effect on DA neurons and suggestive of a degeneration of the same neurons.

Use of the human hepcidin gene to build a positive-selection vector for periplasmic expression in Escherichia coli.

Recombinant proteins are often produced in the periplasm of Escherichia coli because this facilitates the purification process. The oxidizing environment favors the formation of disulfide bridges. We showed that the periplasmic expression of the human hormone hepcidin 25 (Hep25) fused to the maltose-binding protein (MBP) resulted in cell death. This toxicity was not observed when MBP-Hep25 accumulated in the bacterial cytoplasm, or when Hep25 was addressed to the periplasm without the MBP tag. We then modified the periplasmic expression vector pMALp2E to create pMALp2EH, a positive-selection vector with Hep25 as counterselection gene.