In animal experiments, animals, husbandry and test procedures are traditionally standardized to maximize test sensitivity and minimize animal use, assuming that this will also guarantee reproducibility. However, by reducing within-experiment variation, standardization may limit inference to the specific experimental conditions. Indeed, we have recently shown in mice that standardization may generate spurious results in behavioral tests, accounting for poor reproducibility, and that this can be avoided by population heterogenization through systematic variation of experimental conditions. Here, we examined whether a simple form of heterogenization effectively improves reproducibility of test results in a multi-laboratory situation. Each of six laboratories independently ordered 64 female mice of two inbred strains (C57BL/6NCrl, DBA/2NCrl) and examined them for strain differences in five commonly used behavioral tests under two different experimental designs. In the standardized design, experimental conditions were standardized as much as possible in each laboratory, while they were systematically varied with respect to the animals' test age and cage enrichment in the heterogenized design. Although heterogenization tended to improve reproducibility by increasing within-experiment variation relative to between-experiment variation, the effect was too weak to account for the large variation between laboratories. However, our findings confirm the potential of systematic heterogenization for improving reproducibility of animal experiments and highlight the need for effective and practicable heterogenization strategies.

Ras-GRF1 is a neuronal specific guanine exchange factor that, once activated by both ionotropic and metabotropic neurotransmitter receptors, can stimulate Ras proteins, leading to long-term phosphorylation of downstream signaling. The two available reports on the behavior of two independently generated Ras-GRF1 deficient mouse lines provide contrasting evidence on the role of Ras-GRF1 in spatial memory and contextual fear conditioning. These discrepancies may be due to the distinct alterations introduced in the mouse genome by gene targeting in the two lines that could differentially affect expression of nearby genes located in the imprinted region containing the Ras-grf1 locus. In order to determine the real contribution of Ras-GRF1 to spatial memory we compared in Morris Water Maze learning Brambilla's mice with a third mouse line (GENA53) in which a non-sense mutation was introduced in the Ras-GRF1 coding region without additional changes in the genome and we found that memory in this task is normal. Also, we measured both contextual and cued fear conditioning, which were previously reported to be affected in Brambilla's mice, and we confirmed that contextual learning but not cued conditioning is impaired in both mouse lines. In addition, we also tested both lines for the first time in conditioned place aversion in the Intellicage, an ecological and remotely controlled behavioral test, and we observed normal learning. Finally, based on previous reports of other mutant lines suggesting that Ras-GRF1 may control body weight, we also measured this non-cognitive phenotype and we confirmed that both Ras-GRF1 deficient mutants are smaller than their control littermates. In conclusion, we demonstrate that Ras-GRF1 has no unique role in spatial memory while its function in contextual fear conditioning is likely to be due not only to its involvement in amygdala functions but possibly to some distinct hippocampal connections specific to contextual learning.

Nerve injury triggers numerous changes in the injured neurons and surrounding nonneuronal cells that ultimately result in successful target reinnervation or cell death. c-Jun is a component of the heterodimeric AP-1 transcription factor, and c-Jun is highly expressed in response to neuronal trauma. Here we have investigated the role of c-jun during axonal regeneration using mice lacking c-jun in the central nervous system. After transection of the facial nerve, the absence of c-Jun caused severe defects in several aspects of the axonal response, including perineuronal sprouting, lymphocyte recruitment, and microglial activation. c-Jun-deficient motorneurons were atrophic, resistant to axotomy-induced cell death, and showed reduced target muscle reinnervation. Expression of CD44, galanin, and alpha7beta1 integrin, molecules known to be involved in regeneration, was greatly impaired, suggesting a mechanism for c-Jun-mediated axonal growth. Taken together, our results identify c-Jun as an important regulator of axonal regeneration in the injured central nervous system.

GPS analysis of flight trajectories of pigeons can reveal that topographic features influence their flight paths. Recording electrical brain activity that reflects attentional processing could indicate objects of interest that do not cause changes in the flight path. Therefore, we investigated whether crossing particular visual landmarks when homing from a familiar release site is associated with changes in EEG.

Sampling is a critical step in procedures that generate quantitative morphological data in the neurosciences. Samples need to be representative to allow statistical evaluations, and samples need to deliver a precision that makes statistical evaluations not only possible but also meaningful. Sampling generated variability should, e.g., not be able to hide significant group differences from statistical detection if they are present. Estimators of the coefficient of error (CE) have been developed to provide tentative answers to the question if sampling has been "good enough" to provide meaningful statistical outcomes. We tested the performance of the commonly used Gundersen-Jensen CE estimator, using the layers of the mouse hippocampal dentate gyrus as an example (molecular layer, granule cell layer and hilus). We found that this estimator provided useful estimates of the precision that can be expected from samples of different sizes. For all layers, we found that a smoothness factor (m) of 0 generally provided better estimates than an m of 1. Only for the combined layers, i.e., the entire dentate gyrus, better CE estimates could be obtained using an m of 1. The orientation of the sections impacted on CE sizes. Frontal (coronal) sections are typically most efficient by providing the smallest CEs for a given amount of work. Applying the estimator to 3D-reconstructed layers and using very intense sampling, we observed CE size plots with m = 0 to m = 1 transitions that should also be expected but are not often observed in real section series. The data we present also allows the reader to approximate the sampling intervals in frontal, horizontal or sagittal sections that provide CEs of specified sizes for the layers of the mouse dentate gyrus.

Behavioral phenotyping of mice has received a great deal of attention during the past three decades. However, there is still a pressing need to understand the variability caused by environmental and biological factors, human interference, and poorly standardized experimental protocols. The inconsistency of results is often attributed to the inter-individual difference between the experimenters and environmental conditions. The present work aims to dissect the combined influence of the experimenter and the environment on the detection of behavioral traits in two inbred strains most commonly used in behavioral genetics due to their contrasting phenotypes, the C57BL/6J and DBA/2J mice. To this purpose, the elevated O-maze, the open field with object, the accelerating rotarod and the Barnes maze tests were performed by two experimenters in two diverse laboratory environments. Our findings confirm the well-characterized behavioral differences between these strains in exploratory behavior, motor performance, learning and memory. Moreover, the results demonstrate how the experimenter and the environment influence the behavioral tests with a variable-dependent effect, often with mutually exclusive contributions. In this context, our study highlights how both the experimenter and the environment can have an impact on the strain effect size without altering the direction of the conclusions. Importantly, the general agreement on the results is reached by converging evidence from multiple measures addressing the same trait. In conclusion, the present work elucidates the contribution of both the experimenter and the laboratory environment in the intricate field of reproducibility in mouse behavioral phenotyping.

Age-related neurodegenerative diseases (NDDs) are associated with the aggregation and propagation of specific pathogenic protein species (e.g., Aβ, α-synuclein). However, whether disruption of synaptic homeostasis results from protein misfolding per se rather than accumulation of a specific rogue protein is an unexplored question. Here, we show that error-prone translation, with its frequent outcome of random protein misfolding, is sufficient to recapitulate many early features of NDDs, including perturbed Ca2+ signaling, neuronal hyperexcitability, and mitochondrial dysfunction. Mice expressing the ribosomal ambiguity mutation Rps9 D95N exhibited disrupted synaptic homeostasis resulting in behavioral changes reminiscent of early Alzheimer disease (AD), such as learning and memory deficits, maladaptive emotional responses, epileptiform discharges, suppressed circadian rhythmicity, and sleep fragmentation, accompanied by hippocampal NPY expression and cerebral glucose hypometabolism. Collectively, our findings suggest that random protein misfolding may contribute to the pathogenesis of age-related NDDs, providing an alternative framework for understanding the initiation of AD.

The concept of intentions is often taken for granted in the cognitive and neural sciences, and comparing outcomes with internal goals is seen as critical for our sense of agency. We created an experiment where participants decided which face they preferred, and we either created outcome errors by covertly switching the position of the chosen face or induced motor errors by deviating the mouse cursor, or we did both at the same time. In the final case, participants experienced a motor error, but the outcome ended up correct. The result showed that when they received the right face, but at the wrong place, participants rejected the outcome they actually wanted in a majority of the trials. Thus, contrary to common belief, higher-order outcomes do not always regulate our actions. Instead, motor "wrongness" might sometimes override goal "rightness" and lead us to reject the outcome we actually want.

Persistent neuropathic pain is a frequent consequence of peripheral nerve injuries, particularly in the elderly. Using the IntelliCage we studied if sciatic nerve injury obstructed learning and memory in young and aged mice, each in wild type and progranulin deficient mice, which develop premature signs of brain aging. Both young and aged mice developed long-term nerve injury-evoked hyperalgesia and allodynia. In both genotypes, aged mice with neuropathic pain showed high error rates in place avoidance acquisition tasks. However, once learnt, these aged mice with neuropathic pain showed a significantly stronger maintenance of the aversive memory. Nerve injury did not affect place preference behavior in neither genotype, neither in young nor aged mice. However, nerve injury in progranulin deficient mice impaired the learning of spatial sequences of awarded places, particularly in the aged mice. This task required a discrimination of clockwise and anti-clockwise sequences. The chaining failure occurred only in progranulin deficient mice after nerve injury, but not in sham operated or wildtype mice, suggesting that progranulin was particularly important for compensatory adaptations after nerve injury. In contrast, all aged mice with neuropathic pain, irrespective of the genotype, had a long maintenance of aversive memory suggesting a negative alliance and possibly mutual aggravation of chronic neuropathic pain and aversive memory at old age.

There has been a long-standing need to develop efficient and standardized behavioral test methods for evaluating higher-order brain functions in mice. Here, we developed and validated a behavioral flexibility test in mice using IntelliCage, a fully automated behavioral analysis system for mice in a group-housed environment. We first developed a "behavioral sequencing task" in the IntelliCage that enables us to assess the learning ability of place discrimination and behavioral sequence for reward acquisition. In the serial reversal learning using the task, the discriminated spatial patterns of the rewarded and never-rewarded places were serially reversed, and thus, mice were accordingly expected to realign the previously acquired behavioral sequence. In general, the tested mice showed rapid acquisition of the behavioral sequencing task and behavioral flexibility in the subsequent serial reversal stages both in intra- and inter-session analyses. It was found that essentially the same results were obtained among three different laboratories, which confirm the high stability of the present test protocol in different strains of mice (C57BL/6, DBA/2, and ICR). In particular, the most trained cohort of C57BL/6 mice achieved a markedly rapid adaptation to the reversal task in the final phase of the long-term serial reversal test, which possibly indicated that the mice adapted to the "reversal rule" itself. In conclusion, the newly developed behavioral test was shown to be a valid assay of behavioral flexibility in mice, and is expected to be utilized in tests of mouse models of cognitive deficits.

Modern molecular genetics create a rapidly growing number of mutant mouse lines, many of which need to be phenotyped behaviorally. Poor reliability and low efficiency of traditional behavioral tests have prompted the development of new approaches to behavioral phenotyping, such as fully automated analysis of behavior in the homecage.

Mitochondrial dysfunction and oxidative stress have been implicated in Parkinson disease (PD). In addition, genetic evidence points to an important role of protein misfolding, aggregation, and failure in the proteasomal degradation of specific neuronal proteins in the pathogenesis of PD. The chaperone heat-shock protein 70 (Hsp70) reduces protein misfolding and aggregation and protects cells against a variety of adverse conditions, including oxidative stress. Moreover, Hsp70 exerts antiapoptotic activity by blocking the function of several key proapoptotic factors. Recently, Hsp70 was shown to inhibit alpha-synuclein toxicity in a Drosophila model of inherited PD. Here we tested the potential of Hsp70 (approved gene symbol HSPA1A) for gene therapy in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of idiopathic PD. We show that Hsp70 gene transfer to dopamine neurons by a recombinant adeno-associated virus significantly protects the mouse dopaminergic system against MPTP-induced dopamine neuron loss and the associated decline in striatal dopamine levels and tyrosine hydroxylase-positive fibers. Hsp70 reduced MPTP-induced apoptosis in the substantia nigra, and unilateral protection of the dopaminergic system by Hsp70 was associated with increased amphetamine-induced turning toward the uninjected side. Collectively, these results suggest that increasing chaperone activity may be beneficial for the treatment of idiopathic PD.

It has been hypothesized that selective serotonin reuptake inhibitors (SSRIs), the most common treatment for major depression, affect mood through changes in immune function. However, the effects of SSRIs on inflammatory response are contradictory since these act either as anti- or pro-inflammatory drugs. Previous experimental and clinical studies showed that the quality of the living environment moderates the outcome of antidepressant treatment. Therefore, we hypothesized that the interplay between SSRIs and the environment may, at least partially, explain the apparent incongruence regarding the effects of SSRI treatment on the inflammatory response. In order to investigate such interplay, we exposed C57BL/6 mice to chronic stress to induce a depression-like phenotype and, subsequently, to fluoxetine treatment or vehicle (21days) while being exposed to either an enriched or a stressful condition. At the end of treatment, we measured the expression levels of several anti- and pro-inflammatory cytokines and inflammatory mediators in the whole hippocampus and in isolated microglia. We also determined microglial density, distribution, and morphology to investigate their surveillance state. Results show that the effects of fluoxetine treatment on inflammation and microglial function, as compared to vehicle, were dependent on the quality of the living environment. In particular, fluoxetine administered in the enriched condition increased the expression of pro-inflammatory markers compared to vehicle, while treatment in a stressful condition produced anti-inflammatory effects. These findings provide new insights regarding the effects of SSRIs on inflammation, which may be crucial to devise pharmacological strategies aimed at enhancing antidepressant efficacy by means of controlling environmental conditions.

Striking differences in the septo-temporal distribution of neurogenesis are found in small rodents. Here, we assessed the association of adult hippocampal neurogenesis with behavioral responses to novelty, temporal and spatial sequence and reversal learning in wild, wild-derived and laboratory rodents using an automated testing apparatus, the IntelliCage. Behaviorally, DBA/2 and wild-derived house mice were quickest to explore a novel environment, wild wood mice and bank voles were slowest, and C57BL/6 intermediate. Rule learning (temporal and spatial) was fastest in wood mice and bank voles, while DBA/2 and house mice performed poorer. C57BL/6 performed similar to the house mice in the temporal task and similar to wild rodents in the spatial task. Using the number of DCX-positive neurons and proliferating, Ki67-positive cells in the septal, intermediate and temporal hippocampus as a proxy, an ANCOVA was used to test for within-group relations between neurogenesis and behavior. We found that higher numbers of DCX-positive cells in the temporal hippocampus were associated with an increased latency and a lower frequency to explore a novel environment. Temporal and spatial sequence learning was not associated with neurogenesis. In the spatial reversal task however, animals with higher septal neurogenesis showed a persevering phenotype and slower re-learning. Our findings provide strong evidence of septo-temporally segregated neurogenesis effects on behavior across five rodent strains and species. While temporal neurogenesis covaries with behavioral responses to novelty, septal neurogenesis relates to perseverance of a successfully learned spatial rule. Importantly, these associations were independent of species or strain and can be found in both wild and domesticated rodents.

To evaluate permanent effects of hippocampal and prefrontal cortex lesion on spatial tasks, lesioned and sham-operated female C57BL/6 mice were exposed to a series of conditioning schemes in IntelliCages housing 8-10 transponder-tagged mice from each treatment group. Sequential testing started at 51-172days after bilateral lesions and lasted for 154 and 218days in two batches of mice, respectively. Spontaneous undisturbed behavioral patterns clearly separated the three groups, hippocampals being characterized by more erratic hyperactivity, and strongly impaired circadian synchronization ability. Hippocampal lesions led to deficits in spatial passive avoidance, as well as in spatial reference and working memory tasks. Impairment was minimal in rewarded preference/reversal schemes, but prominent if behavioral responses required precise circadian timing or included punishment of wrong spatial choices. No differences between sham-operated and prefrontally lesioned subjects in conditioning success were discernible. These results corroborate the view that hippocampal dysfunction spares simple spatial learning tasks but impairs the ability to cope with conflicting task-inherent spatial, temporal or emotional cues. Methodologically, the results show that automated testing and data analysis of socially kept mice is a powerful, efficient and animal-friendly tool for dissecting complex features and behavioral profiles of hippocampal dysfunction characterizing many transgenic or pharmacological mouse models.

We have recently identified point mutation V336Y in mitoribosomal protein Mrps5 (uS5m) as a mitoribosomal ram (ribosomal ambiguity) mutation conferring error-prone mitochondrial protein synthesis. In vivo in transgenic knock-in animals, homologous mutation V338Y was associated with a discrete phenotype including impaired mitochondrial function, anxiety-related behavioral alterations, enhanced susceptibility to noise-induced hearing damage, and accelerated metabolic aging in muscle. To challenge the postulated link between Mrps5 V338Y-mediated misreading and the in vivo phenotype, we introduced mutation G315R into the mouse Mrps5 gene as Mrps5 G315R is homologous to the established bacterial ram mutation RpsE (uS5) G104R. However, in contrast to bacterial translation, the homologous G → R mutation in mitoribosomal Mrps5 did not affect the accuracy of mitochondrial protein synthesis. Importantly, in the absence of mitochondrial misreading, homozygous mutant MrpS5G315R/G315R mice did not show a phenotype distinct from wild-type animals.

Two million non-emergency surgeries are being cancelled globally every week due to the COVID-19 pandemic, which will have a major impact on patients and healthcare systems.

To systematically evaluate the spectrum of models providing dedicated resources for emergency urological patients (EUPs).

This study aims to characterize the material properties of ascending thoracic aortic aneurysmal tissue, using regional biomechanical assessment of both tensile and dissection propagation peel strength.

APC mutations (APC-mt) occur in ∼70% of colorectal cancers (CRCs), but their relationship to prognosis is unclear.