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Research resources like transgenic animals and antibodies are the workhorses of biomedicine, enabling investigators to relatively easily study specific disease conditions. As key biological resources, transgenic animals and antibodies are often validated, maintained, and distributed from university based stock centers. As these centers heavily rely largely on grant funding, it is critical that they are cited by investigators so that usage can be tracked. However, unlike systems for tracking the impact of papers, the conventions and systems for tracking key resource usage and impact lag behind. Previous studies have shown that about 50% of the resources are not findable, making the studies they are supporting irreproducible, but also makes tracking resources difficult. The RRID project is filling this gap by working with journals and resource providers to improve citation practices and to track the usage of these key resources. Here, we reviewed 10 years of citation practices for five university based stock centers, characterizing each reference into two broad categories: findable (authors could use the RRID, stock number, or full name) and not findable (authors could use a nickname or a common name that is not unique to the resource). The data revealed that when stock centers asked their communities to cite resources by RRID, in addition to helping stock centers more easily track resource usage by increasing the number of RRID papers, authors shifted from citing resources predominantly by nickname (~50% of the time) to citing them by one of the findable categories (~85%) in a matter of several years. In the case of one stock center, the MMRRC, the improvement in findability is also associated with improvements in the adherence to NIH rigor criteria, as determined by a significant increase in the Rigor and Transparency Index for studies using MMRRC mice. From this data, it was not possible to determine whether outreach to authors or changes to stock center websites drove better citation practices, but findability of research resources and rigor adherence was improved.
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BACKGROUND: Improving rigor and transparency measures should lead to improvements in reproducibility across the scientific literature; however, the assessment of measures of transparency tends to be very difficult if performed manually. OBJECTIVE: This study addresses the enhancement of the Rigor and Transparency Index (RTI, version 2.0), which attempts to automatically assess the rigor and transparency of journals, institutions, and countries using manuscripts scored on criteria found in reproducibility guidelines (eg, Materials Design, Analysis, and Reporting checklist criteria). METHODS: The RTI tracks 27 entity types using natural language processing techniques such as Bidirectional Long Short-term Memory Conditional Random Field-based models and regular expressions; this allowed us to assess over 2 million papers accessed through PubMed Central. RESULTS: Between 1997 and 2020 (where data were readily available in our data set), rigor and transparency measures showed general improvement (RTI 2.29 to 4.13), suggesting that authors are taking the need for improved reporting seriously. The top-scoring journals in 2020 were the Journal of Neurochemistry (6.23), British Journal of Pharmacology (6.07), and Nature Neuroscience (5.93). We extracted the institution and country of origin from the author affiliations to expand our analysis beyond journals. Among institutions publishing >1000 papers in 2020 (in the PubMed Central open access set), Capital Medical University (4.75), Yonsei University (4.58), and University of Copenhagen (4.53) were the top performers in terms of RTI. In country-level performance, we found that Ethiopia and Norway consistently topped the RTI charts of countries with 100 or more papers per year. In addition, we tested our assumption that the RTI may serve as a reliable proxy for scientific replicability (ie, a high RTI represents papers containing sufficient information for replication efforts). Using work by the Reproducibility Project: Cancer Biology, we determined that replication papers (RTI 7.61, SD 0.78) scored significantly higher (P<.001) than the original papers (RTI 3.39, SD 1.12), which according to the project required additional information from authors to begin replication efforts. CONCLUSIONS: These results align with our view that RTI may serve as a reliable proxy for scientific replicability. Unfortunately, RTI measures for journals, institutions, and countries fall short of the replicated paper average. If we consider the RTI of these replication studies as a target for future manuscripts, more work will be needed to ensure that the average manuscript contains sufficient information for replication attempts.
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Lista de Verificación , Edición , Humanos , Noruega , Reproducibilidad de los Resultados , Proyectos de InvestigaciónRESUMEN
The reproducibility crisis is a multifaceted problem involving ingrained practices within the scientific community. Fortunately, some causes are addressed by the author's adherence to rigor and reproducibility criteria, implemented via checklists at various journals. We developed an automated tool (SciScore) that evaluates research articles based on their adherence to key rigor criteria, including NIH criteria and RRIDs, at an unprecedented scale. We show that despite steady improvements, less than half of the scoring criteria, such as blinding or power analysis, are routinely addressed by authors; digging deeper, we examined the influence of specific checklists on average scores. The average score for a journal in a given year was named the Rigor and Transparency Index (RTI), a new journal quality metric. We compared the RTI with the Journal Impact Factor and found there was no correlation. The RTI can potentially serve as a proxy for methodological quality.
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BACKGROUND INFORMATION: During development, growth cones of outgrowing neurons express proteins involved in vesicular secretion, such as SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins, Munc13 and Munc18. Vesicles are known to fuse in growth cones prior to synapse formation, which may contribute to outgrowth. RESULTS: We tested this possibility in dissociated cell cultures and organotypic slice cultures of two release-deficient mice (Munc18-1 null and Munc13-1/2 double null). Both types of release-deficient neurons have a decreased outgrowth speed and therefore have a smaller total neurite length during early development [DIV1-4 (day in vitro 1-4)]. In addition, more filopodia per growth cone were observed in Munc18-1 null, but not WT (wild-type) or Munc13-1/2 double null neurons. The smaller total neurite length during early development was no longer observed after synaptogenesis (DIV14-23). CONCLUSION: These data suggest that the inability of vesicle fusion in the growth cone affects outgrowth during the initial phases when outgrowth speed is high, but not during/after synaptogenesis. Overall, the outgrowth speed is probably not rate-limiting during neuronal network formation, at least in vitro. In addition, Munc18, but not Munc13, regulates growth cone filopodia, potentially via its previously observed effect on filamentous actin.
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Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas Munc18/fisiología , Proteínas del Tejido Nervioso/fisiología , Neuritas/fisiología , Animales , Conos de Crecimiento/fisiología , Péptidos y Proteínas de Señalización Intracelular/deficiencia , Ratones , Ratones Transgénicos , Proteínas Munc18/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Seudópodos/metabolismoRESUMEN
BACKGROUND: Transgenic mice with mosaic, Golgi-staining-like expression of enhanced green fluorescent protein (EGFP) have been very useful in studying the dynamics of neuronal structure and function. In order to further investigate the molecular events regulating structural plasticity, it would be useful to express multiple proteins in the same sparse neurons, allowing co-expression of functional proteins or co-labeling of subcellular compartments with other fluorescent proteins. However, it has been difficult to obtain reproducible expression in the same subset of neurons for direct comparison of neurons expressing different functional proteins. PRINCIPAL FINDINGS: Here we describe a Cre-transgenic line that allows reproducible expression of transgenic proteins of choice in a small number of neurons of the adult cortex, hippocampus, striatum, olfactory bulb, subiculum, hypothalamus, superior colliculus and amygdala. We show that using these Cre-transgenic mice, multiple Cre-dependent transgenes can be expressed together in the same isolated neurons. We also describe a Cre-dependent transgenic line expressing a membrane associated EGFP (EGFP-F). Crossed with the Cre-transgenic line, EGFP-F expression starts in the adolescent forebrain, is present in dendrites, dendritic protrusions, axons and boutons and is strong enough for acute or chronic in vivo imaging. SIGNIFICANCE: This triple transgenic approach will aid the morphological and functional characterization of neurons in various Cre-dependent transgenic mice.
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Neuronas/fisiología , Prosencéfalo/fisiología , Adulto , Animales , Encéfalo/fisiología , Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Humanos , Integrasas/genética , Integrasas/metabolismo , Ratones , Ratones Transgénicos , MosaicismoRESUMEN
INTRODUCTIONThis protocol describes two transfection methods for expressing GFP-tagged actin in primary neurons. The lipid reagent DOTAP (Roche Diagnostics) method produces actin-GFP-expressing hippocampal neurons that survive well during long periods in culture. The calcium phosphate method can be used to transfect neurons that have already been growing on coverslips in vitro. Transfected cells suitable for imaging can be obtained in cultures up to 15 days in vitro. One to two percent transfected cells is a typical result. A disadvantage of the calcium phosphate method is that hippocampal neurons become "fragile" after treatment.
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Mechanisms of synaptic plasticity in CNS circuits are commonly investigated using in vitro preparations such as brain slices or slice culture. During their preparation, slices are exposed to low temperatures, and electrophysiological measurements are sometimes made below physiological temperature. Because dendritic spines, which occur at the majority of excitatory synapses, are morphologically plastic, we investigated the influence of reduced temperature on their morphology and plasticity using live cell imaging of hippocampal slices from transgenic mice expressing a green fluorescent protein-based neuronal surface marker and electron microscopy of adult brain slices. Our data show that dendritic spines are highly sensitive to reduced temperature with rapid loss of actin-based motility followed at longer times by reversible loss of the entire spine structure. Thus, reduced temperature significantly affects synaptic morphology, which is in turn known to influence several key aspects of synaptic transmission. Evidence that hypothermia potentiates anesthesia and is associated with spine loss in hibernating animals further suggests that spine morphology may have a widespread influence on brain function.
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Artefactos , Frío , Dendritas/ultraestructura , Hipocampo/ultraestructura , Manejo de Especímenes/métodos , Vesículas Sinápticas/ultraestructura , Animales , Genes Reporteros , Proteínas Fluorescentes Verdes/análisis , Ratones , Ratones Transgénicos , Microscopía Confocal , Microscopía Electrónica , Proteínas del Tejido Nervioso/análisis , Técnicas de Cultivo de Órganos , Factores de TiempoRESUMEN
The stability of neuronal networks is thought to depend on synaptic transmission which provides activity-dependent maintenance signals for both synapses and neurons. Here, we tested the relationship between presynaptic secretion and neuronal maintenance using munc18-1-null mutant mice as a model. These mutants have a specific defect in secretion from synaptic and large dense-cored vesicles [Verhage et al. (2000), Science, 287, 864-869; Voets et al. (2001), Neuron, 31, 581-591]. Neuronal networks in these mutants develop normally up to synapse formation but eventually degenerate. The proposed relationship between secretion and neuronal maintenance was tested in low-density and organotypic cultures and, in vivo, by conditional cell-specific inactivation of the munc18-1 gene. Dissociated munc18-1-deficient neurons died within 4 days in vitro (DIV). Application of trophic factors, insulin or BDNF delayed degeneration up to 7 DIV. In organotypic cultures, munc18-1-deficient neurons survived until 9 DIV. On glial feeders, these neurons survived up to 10 DIV and 14 DIV when insulin was applied. Co-culturing dissociated mutant neurons with wild-type neurons did not prolong survival beyond 4 DIV, but coculturing mutant slices with wild-type slices prolonged survival up to 19 DIV. Cell-specific deletion of munc18-1 expression in cerebellar Purkinje cells in vivo resulted in the specific loss of these neurons without affecting connected or surrounding neurons. Together, these data allow three conclusions. First, the lack of synaptic activity cannot explain the degeneration in munc18-1-null mutants. Second, trophic support delays but cannot prevent degeneration. Third, a cell-intrinsic yet unknown function of munc18-1 is essential for prolonged survival.