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Background 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. New method We asked whether the analysis of spontaneous behavior during the first week in the social homecage system IntelliCage could provide useful prescreening information before specialized and time consuming test batteries are run. To determine how much behavioral variation is captured in this data, we performed principal component analysis on free adaptation data of 1552 mice tested in the IntelliCage during the past years. We then computed individual component scores to characterize and compare groups of mice. Result We found 11 uncorrelated components which accounted for 82% of total variance. They characterize frequency and properties of corner visits and nosepokes, drinking activity, spatial distribution, as well as diurnal time course of activity. Behavioral profiles created using individual component scores were highly characteristic for different inbred strains or different lesion models of the nervous system. They were also remarkably stable across labs and experiments. Comparison with existing methods Monitoring of mutant mice with known deficits in hippocampus-dependent tests produced profiles very similar to those of hippocampally lesioned mice. Conclusions Taken together, our results suggest that already the monitoring of spontaneous behavior during a week of free adaptation in the IntelliCage can contribute significantly to high throughput prescreening of mutant mice. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: The IntelliCage allows fully automated continuous testing of various behaviours in the home cage environment without handling the mice. Here we tested whether conditioned avoidance is retained after a time period delay spent outside the IntelliCage. During the training, nosepokes in one of the four learning corners were punished with an air-puff. After 24h of training, the mice were placed in regular cages for 24h. During the last 18h of this interval, the mice were water deprived and then returned to the IntelliCage for a probe trial where drinking was allowed in all corners. The C57BL/6 mice developed a significant suppression of nosepoking in the punished corner during training, and the avoidance was carried over to the following probe trial. Repetition of the experiment by delivering punishment in a different corner assigned to individual mice revealed a similar performance pattern. Comparison between the different strains revealed a reduced nosepoke suppression in DBA/2 and B6D2F1 mice as compared to C57BL/6 mice in the probe trial, despite similar error rates during the training with short (1-s) air-puffs. However, the performance of the three strains in the probe trial were equalised when the air-puffs were prolonged until the end of the corner visit. Significant extinction of the nosepoke suppression occurred after 6 days. A prolonged interval (7 days) between the training and the probe trial resulted in a loss of suppression in DBA/2 mice, but not in C57BL/6 and B6D2F1 mice. Additional experiments revealed that performance in the probe trial was dependent on a complex set of intramaze cues. Testing of mice with bilateral excitotoxic lesions of the hippocampus or dorso-lateral striatum revealed that learning this task was dependent on an intact hippocampus, but not on an intact striatum. In summary, the conditioned nosepoke suppression test presented here is sensitive to both genetic differences and hippocampal lesions. This test could be applied to the screening of mutant mice with impaired hippocampal functions more efficiently than those of the standard memory tests. [Copyright &y& Elsevier]

Summary: Background: 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. Results: Birds carried both data-loggers for recording GPS position and EEG during flight. First, we classified characteristic EEG frequencies of caged birds and found five main bands: A: 0–3, B: 3–12, C: 12–60, D: 60–130, and E: 130–200 Hz. We analyzed changes in these activity bands when pigeons were released over sea (a featureless environment) and over land. Passing over the coastline and other prominent landmarks produced a pattern of EEG alterations consisting of two phases: activation of EEG in the high-frequency bands (D and/or E), followed by activation of C. Overlaying the EEG activity with GPS tracks allowed us to identify topographical features of interest for the pigeons that were not recognizable by distinct changes of their flight path. Conclusions: We provide evidence that EEG analysis can identify landmarks and objects of interest during homing. Middle-frequency activity (C) reflects visual perception of prominent landmarks, whereas activation of higher frequencies (D and E) is linked with information processing at a higher level. Activation of E bands is likely to reflect an initial process of orientation and is not necessarily linked with processing of visual information. [Copyright &y& Elsevier]

Abstract: The hippocampus plays a central role in various forms of complex learning and memory. Opioid peptides and receptors are abundant in the hippocampus. These peptides are co-released with glutamate from mossy fiber- and lateral perforant path-synapses. In this study, we evaluated the functional relevance of the CA3 Kappa opioid receptors (KOR) by transient pharmacological activation or inactivation using single bilateral intrahippocampal microinjections of a selective agonist (U50,488H, 1 or 2.5nmol), a selective antagonist (nor-binaltorphimine, norBNI 5nmol) or a mixture of both. C57Bl/6J mice were tested in a fear conditioning paradigm (FC) or in a modified version of the water maze task thought to reveal how flexibly animals can learn and manipulate spatial information (WM). In FC, the agonist (2.5nmol) decreased context-induced (but not tone-induced) freezing whereas norBNI had no effect. The impairment caused by the agonist U50,488H was blocked by the injection of norBNI, suggesting that overstimulation of CA3-KOR impairs the acquisition and consolidation of contextual fear-related memory. In the WM task, mice were trained repeatedly each day to find a hidden platform. After having reached this goal, the platform position was changed the next day for a new task. U50,488H injection before the last task abolished the previously acquired ability to find rapidly a new platform location, whereas adding norBNI reversed this impairment. Thus, in the mouse, even partial and topographically restricted activation of CA3-KOR entails impairments in two different hippocampus-dependent tasks, indicating functional relevance of the kappa opioid system. [Copyright &y& Elsevier]

Prostaglandin E[sub2] (PGE[sub2]) is a crucial mediator of inflammatory pain sensitization. Here, we demonstrate that inhibition of a specific glycine receptor subtype (GlyR α3) by PCE[sub2]-induced receptor phosphorylation underlies central inflammatory pain sensitization. We show that GlyR α3 is distinctly expressed in superficial layers of the spinal cord dorsal horn. Mice deficient in GlyR α3 not only lack the inhibition of glycinergic neurotransmission by PGE[sub2] seen in wild-type mice but also show a reduction in pain sensitization induced by spinal PGE[sub2] injection or peripheral inflammation. Thus, GlyR α3 may provide a previously unrecognized molecular target in pain therapy. [ABSTRACT FROM AUTHOR]

Several lines of evidence point to the involvement of adenosine in the regulation of important central mechanisms such as cognition, arousal, aggression and anxiety. In order to elucidate the involvement of the adenosine A1 receptor (A1AR) in spatial learning and the control of exploratory behaviour, we assessed A1AR knockout mice (A1AR−/−) and their wild-type littermates (A1AR+/+) in a place navigation task in the water maze and in a battery of forced and free exploration tests. In the water maze, A1AR−/− mice showed normal escape latencies and were indistinguishable from controls with respect to measures of spatial performance during both training and probe trial. But despite normal performance they showed increased wall hugging, most prominently after the relocation of the goal platform for reversal training. Quantitative analysis of strategy choices indicated that wall hugging was increased mainly at the expense of chaining and passive floating, whereas the frequency of trials characterised as direct swims or focal searching was normal in A1AR−/− mice. These results indicate intact spatial cognition, but mildly altered emotional reactions to the water maze environment. In line with this interpretation, A1AR−/− mice showed normal levels and patterns of activity, but a mild increase of some measures of anxiety in our battery of forced and free exploration paradigms. These results are in line with findings published using a genetically similar line, but demonstrate that the magnitude of the changes and the range of affected behavioural measures may vary considerably depending on the environmental conditions during testing. [Copyright &y& Elsevier]

Summary Self-propagating amyloid-β (Aβ) aggregates or seeds possibly drive pathogenesis of Alzheimer's disease (AD). Small molecules targeting such structures might act therapeutically in vivo. Here, a fluorescence polarization assay was established that enables the detection of compound effects on both seeded and spontaneous Aβ42 aggregation. In a focused screen of anti-amyloid compounds, we identified Disperse Orange 1 (DO1) ([4-((4-nitrophenyl)diazenyl)-N-phenylaniline]), a small molecule that potently delays both seeded and non-seeded Aβ42 polymerization at substoichiometric concentrations. Mechanistic studies revealed that DO1 disrupts preformed fibrillar assemblies of synthetic Aβ42 peptides and decreases the seeding activity of Aβ aggregates from brain extracts of AD transgenic mice. DO1 also reduced the size and abundance of diffuse Aβ plaques and decreased neuroinflammation-related gene expression changes in brains of 5xFAD transgenic mice. Finally, improved nesting behavior was observed upon treatment with the compound. Together, our evidence supports targeting of self-propagating Aβ structures with small molecules as a valid therapeutic strategy. Graphical Abstract Highlights • A fluorescence polarization assay recapitulates template-mediated Aβ42 aggregation • The assay identifies compound effects on activity of synthetic and ex vivo Aβ seeds • The small-molecule DO1 delays seeded and non-seeded Aβ42 polymerization • DO1 reduces plaques, gene expression changes, and behavioral dysfunction in AD mice Amyloid seeding may have high relevance for the pathogenesis of neurodegenerative diseases such as Alzheimer's. Boeddrich et al. have established an innovative method to study this phenomenon. It enabled the discovery of a small molecule that potently delays seeding of amyloid-beta and has therapeutic effects in 5xFAD transgenic mice. [ABSTRACT FROM AUTHOR]

Many calcium-binding proteins are expressed in a region-and cell-type specific manner in the mammalian hippocampus. Neuronal calcium-binding proteins (NECABs) are also expressed in hippocampal neurons, but few species have been investigated, with partly controversial findings. We here describe NECAB1, NECAB2 and NECAB3 as well as parvalbumin, calbindin, and calretinin in the European mole, and compare staining patterns of these proteins with those in mouse and other species. While subtle differences are present, NECAB staining in the European mole was generally similar to those in mouse. Common to European moles, mice, and other species we investigated, large hilar polymorphic cells, likely to represent mossy cells, were positive for all three NECABs. NECAB1 and 2 are suitable as markers for these cells along the entire septotemporal axis of the hippocampus. In the European mole, parvalbumin, calbindin and calretinin showed traits that have been described in other species before, albeit in a unique combination. In summary, we provide the first description of distribution of these proteins in the hippocampus of the European mole. This subterranean, insectivorous, and solitary living species belongs to the Order of Eulipotyphla. Despite many similarities with other subterranean species from the rodent order in terms of lifestyle, its hippocampus is cytoarchitecturally much more elaborated than in, e.g., mole-rats. It remains an open question if the hippocampal structure of the European mole reflects evolutionary constraints or ecology. Our descriptive study highlights the diversity in hippocampal cytoarchitecture even in small mammalian species., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Maliković, Amrein, Vinciguerra, Wolfer and Slomianka.)

The IntelliCage (IC) permits the assessment of the behavior and learning abilities of mice in a social home cage context. To overcome water deprivation as an aversive driver of learning, we developed protocols in which spatial learning is motivated appetitively by the preference of mice for sweetened over plain water. While plain water is available at all times, only correct task responses give access to sweetened water rewards. Under these conditions, C57BL/6J mice successfully mastered a corner preference task with the reversal and also learned a more difficult time-place task with reversal. However, the rate of responding to sweetened water decreased strongly with increasing task difficulty, indicating that learning challenges and reduced success in obtaining rewards decreased the motivation of the animals to seek sweetened water. While C57BL/6J mice of both sexes showed similar initial taste preferences and learned similarly well in simple learning tasks, the rate of responding to sweetened water and performance dropped more rapidly in male than in female mice in response to increasing learning challenges. Taken together, our data indicate that male mice can have a disadvantage relative to females in mastering difficult, appetitively motivated learning tasks, likely due to sex differences in value-based decision-making., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Nigri, Bramati, Steiner and Wolfer.)

Theoretical and empirical evidence indicates that low external validity due to rigorous standardization of study populations is a cause of poor replicability in animal research. Here we report a multi-laboratory study aimed at investigating whether heterogenization of study populations by using animals from different breeding sites increases the replicability of results from single-laboratory studies. We used male C57BL/6J mice from six different breeding sites to test a standardized against a heterogenized (HET) study design in six independent replicate test laboratories. For the standardized design, each laboratory ordered mice from a single breeding site (each laboratory from a different one), while for the HET design, each laboratory ordered proportionate numbers of mice from the five remaining breeding sites. To test our hypothesis, we assessed 14 outcome variables, including body weight, behavioral measures obtained from a single session on an elevated plus maze, and clinical blood parameters. Both breeding site and test laboratory affected variation in outcome variables, but the effect of test laboratory was more pronounced for most outcome variables. Moreover, heterogenization of study populations by breeding site (HET) did not reduce variation in outcome variables between test laboratories, which was most likely due to the fact that breeding site had only little effect on variation in outcome variables, thereby limiting the scope for HET to reduce between-lab variation. We conclude that heterogenization of study populations by breeding site has limited capacity for improving the replicability of results from single-laboratory animal studies., (© 2023. The Author(s).)

The IntelliCage allows automated testing of cognitive abilities of mice in a social home cage environment without handling by human experimenters. Restricted water access in combination with protocols in which only correct responses give access to water is a reliable learning motivator for hippocampus-dependent tasks assessing spatial memory and executive function. However, water restriction may negatively impact on animal welfare, especially in poor learners. To better comply with the 3R principles, we previously tested protocols in which water was freely available but additional access to sweetened water could be obtained by learning a task rule. While this purely appetitive motivation worked for simple tasks, too many mice lost interest in the sweet reward during more difficult hippocampus-dependent tasks. In the present study, we tested a battery of increasingly difficult spatial tasks in which water was still available without learning the task rule, but rendered less attractive either by adding bitter tasting quinine or by increasing the amount of work to obtain it. As in previous protocols, learning of the task rule provided access to water sweetened with saccharin. The two approaches of dual motivation were tested in two cohorts of female C57BL/6 N mice. Compared to purely appetitive motivation, both novel protocols strongly improved task engagement and increased task performance. Importantly, neither of the added disincentives had an adverse impact on liquid consumption, health status or body weight of the animals. Our results show that it is possible to refine test protocols in the IntelliCage so that they challenge cognitive functions without restricting access to water., Competing Interests: DW was involved in the development of the IntelliCage system. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ma, Schildknecht, Steiner, Amrein, Nigri, Bramati and Wolfer.)

The IntelliCage is an automated home-cage system that allows researchers to investigate the spontaneous behavior and learning abilities of group-housed mice. The IntelliCage enables us to increase the standardization and reproducibility of behavioral outcomes by the omission of experimenter-mouse interactions. Although the IntelliCage provides a less stressful environment for animals, standard IntelliCage protocols use controlled water access as the motivational driver for learning. To overcome possible water restrictions in slow learners, we developed a series of novel protocols based on appetitive learning, in which mice had permanent access to plain water but were additionally rewarded with sweetened water upon solving the task. C57BL/6NCrl female mice were used to assess the efficacy of these sweet reward-based protocols in a series of learning tasks. Compared to control mice tested with standard protocols, mice motivated with a sweet reward did equal to or better in operant performance and place learning tasks. Learning of temporal rules was slower than that in controls. When faced with a combined temporal x spatial working memory task, sweet-rewarded mice learned little and chose plain water. In a second set of experiments, the impact of environmental enrichment on appetitive learning was tested. Mice kept under enriched environment (EE) or standard housing (SH) conditions prior to the IntelliCage experiments performed similarly in the sweet-rewarded place learning task. EE mice performed better in the hippocampus-dependent spatial working memory task. The improved performance of EE mice in the hippocampus-dependent spatial working memory task might be explained by the observed larger volume of their mossy fibers. Our results confirm that environmental enrichment increases complex spatial learning abilities and leads to long-lasting morphological changes in the hippocampus. Furthermore, simple standard IntelliCage protocols could easily be adapted to sweet rewards, which improve animal welfare by removing the possibility of water restriction. However, complex behavioral tasks motivated by sweet reward-based learning need further adjustments to reach the same efficacy as standard protocols., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bramati, Stauffer, Nigri, Wolfer and Amrein.)

The hippocampus of many mammals contains a histoarchitectural region that is not present in laboratory mice and rats-the reflected blade of the CA3 pyramidal cell layer. Pyramidal cells of the reflected blade do not extend dendrites into the hippocampal molecular layer, and recent evidence indicates that they, like the proximal CA3 pyramids in laboratory rats and mice, partially integrate functionally with the dentate circuitry in pattern separation. Quantitative assessments of phylogenetic or disease-related changes in the hippocampal structure and function treat the reflected blade heterogeneously. Depending on the ease with which it can be differentiated, it is either assigned to the dentate hilus or to the remainder of CA3. Here, we investigate the impact that the differential assignment of reflected blade neurons may have on the outcomes of quantitative comparisons. We find it to be massive. If reflected blade neurons are treated as a separate entity or pooled with dentate hilar cells, the quantitative makeup of hippocampal cell populations can differentiate between species in a taxonomically sensible way. Assigning reflected blade neurons to CA3 greatly diminishes the differentiating power of all hippocampal principal cell populations, which may point towards a quantitative hippocampal archetype. A heterogeneous assignment results in a differentiation pattern with little taxonomic semblance. The outcomes point towards the reflected blade as either a major potential player in hippocampal functional and structural differentiation or a region that may have cloaked that hippocampi are more similarly organized across species than generally believed., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Maliković, Amrein, Vinciguerra, Lalošević, Wolfer and Slomianka.)

The amyloid precursor protein APP plays a crucial role in Alzheimer pathogenesis. Its physiological functions, however, are only beginning to be unraveled. APP belongs to a small gene family, including besides APP the closely related amyloid precursor-like proteins APLP1 and APLP2, that all constitute synaptic adhesion proteins. While APP and APLP2 are ubiquitously expressed, APLP1 is specific for the nervous system. Previous genetic studies, including combined knockouts of several family members, pointed towards a unique role for APLP1, as only APP/APLP1 double knockouts were viable. We now examined brain and neuronal morphology in APLP1 single knockout (KO) animals, that have to date not been studied in detail. Here, we report that APLP1-KO mice show normal spine density in hippocampal CA1 pyramidal cells and subtle alterations in dendritic complexity. Extracellular field recordings revealed normal basal synaptic transmission and no alterations in synaptic plasticity (LTP). Further, behavioral studies revealed in APLP1-KO mice a small deficit in motor function and reduced diurnal locomotor activity, while learning and memory were not affected by the loss of APLP1. In summary, our study indicates that APP family members serve both distinct and overlapping functions that need to be considered for therapeutic treatments of Alzheimer's disease., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Erdinger, Amrein, Back, Ludewig, Korte, von Engelhardt, Wolfer and Müller.)

The phenotype of an organism results from its genotype and the influence of the environment throughout development. Even when using animals of the same genotype, independent studies may test animals of different phenotypes, resulting in poor replicability due to genotype-by-environment interactions. Thus, genetically defined strains of mice may respond differently to experimental treatments depending on their rearing environment. However, the extent of such phenotypic plasticity and its implications for the replicability of research findings have remained unknown. Here, we examined the extent to which common environmental differences between animal facilities modulate the phenotype of genetically homogeneous (inbred) mice. We conducted a comprehensive multicentre study, whereby inbred C57BL/6J mice from a single breeding cohort were allocated to and reared in 5 different animal facilities throughout early life and adolescence, before being transported to a single test laboratory. We found persistent effects of the rearing facility on the composition and heterogeneity of the gut microbial community. These effects were paralleled by persistent differences in body weight and in the behavioural phenotype of the mice. Furthermore, we show that environmental variation among animal facilities is strong enough to influence epigenetic patterns in neurons at the level of chromatin organisation. We detected changes in chromatin organisation in the regulatory regions of genes involved in nucleosome assembly, neuronal differentiation, synaptic plasticity, and regulation of behaviour. Our findings demonstrate that common environmental differences between animal facilities may produce facility-specific phenotypes, from the molecular to the behavioural level. Furthermore, they highlight an important limitation of inferences from single-laboratory studies and thus argue that study designs should take environmental background into account to increase the robustness and replicability of findings., Competing Interests: The authors have declared that no competing interests exist.

Translational research in behavioral neuroscience seeks causes and remedies for human mental health problems in animals, following leads imposed by clinical research in psychiatry. This endeavor faces several problems because scientists must read and interpret animal movements to represent human perceptions, mood, and memory processes. Yet, it is still not known how mammalian brains bundle all these processes into a highly compressed motor output in the brain stem and spinal cord, but without that knowledge, translational research remains aimless. Based on some four decades of experience in the field, the article identifies sources of interpretation problems and illustrates typical translational pitfalls. (1) The sensory world of mice is different. Smell, hearing, and tactile whisker sensations dominate in rodents, while visual input is comparatively small. In humans, the relations are reversed. (2) Mouse and human brains are equated inappropriately: the association cortex makes up a large portion of the human neocortex, while it is relatively small in rodents. The predominant associative cortex in rodents is the hippocampus itself, orchestrating chiefly inputs from secondary sensorimotor areas and generating species-typical motor patterns that are not easily reconciled with putative human hippocampal functions. (3) Translational interpretation of studies of memory or emotionality often neglects the ecology of mice, an extremely small species surviving by freezing or flight reactions that do not need much cognitive processing. (4) Further misinterpretations arise from confounding neuronal properties with system properties, and from rigid mechanistic thinking unaware that many experimentally induced changes in the brain do partially reflect unpredictable compensatory plasticity. (5) Based on observing hippocampal lesion effects in mice indoors and outdoors, the article offers a simplistic general model of hippocampal functions in relation to hypothalamic input and output, placing hypothalamus and the supraspinal motor system at the top of a cerebral hierarchy. (6) Many translational problems could be avoided by inclusion of simple species-typical behaviors as end-points comparable to human cognitive or executive processing, and to rely more on artificial intelligence for recognizing patterns not classifiable by traditional psychological concepts., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lipp and Wolfer.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Nigri, Åhlgren, Wolfer and Voikar.)

Circuit formation in the central nervous system has been historically studied during development, after which cell-autonomous and nonautonomous wiring factors inactivate. In principle, balanced reactivation of such factors could enable further wiring in adults, but their relative contributions may be circuit dependent and are largely unknown. Here, we investigated hippocampal mossy fiber sprouting to gain insight into wiring mechanisms in mature circuits. We found that sole ectopic expression of Id2 in granule cells is capable of driving mossy fiber sprouting in healthy adult mouse and rat. Mice with the new mossy fiber circuit solved spatial problems equally well as controls but appeared to rely on local rather than global spatial cues. Our results demonstrate reprogrammed connectivity in mature neurons by one defined factor and an assembly of a new synaptic circuit in adult brain., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Amyloid-β precursor protein (APP) is central to the pathogenesis of Alzheimer's disease, yet its physiological functions remain incompletely understood. Previous studies had indicated important synaptic functions of APP and the closely related homologue APLP2 in excitatory forebrain neurons for spine density, synaptic plasticity, and behavior. Here, we show that APP is also widely expressed in several interneuron subtypes, both in hippocampus and cortex. To address the functional role of APP in inhibitory neurons, we generated mice with a conditional APP/APLP2 double knockout (cDKO) in GABAergic forebrain neurons using DlxCre mice. These DlxCre cDKO mice exhibit cognitive deficits in hippocampus-dependent spatial learning and memory tasks, as well as impairments in species-typic nesting and burrowing behaviors. Deficits at the behavioral level were associated with altered neuronal morphology and synaptic plasticity Long-Term Potentiation (LTP). Impaired basal synaptic transmission at the Schafer collateral/CA1 pathway, which was associated with altered compound excitatory/inhibitory synaptic currents and reduced action potential firing of CA1 pyramidal cells, points to a disrupted excitation/inhibition balance in DlxCre cDKOs. Together, these impairments may lead to hippocampal dysfunction. Collectively, our data reveal a crucial role of APP family proteins in inhibitory interneurons to maintain functional network activity., (© The Author(s) 2020. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

The calcium-binding protein parvalbumin (PV) is a recognized marker of short-axon GABA-ergic neurons in the cortex and the hippocampus. However in addition, PV is expressed by excitatory, glutamatergic neurons in various areas of the brain and spinal cord. Depending on the location of these neurons, loading of their synaptic vesicles with glutamate is mediated by either of three vesicular glutamate transporters (VGlut): VGlut1, VGlut2, or VGlut3. Driven by our interest in one of these glutamatergic/PV-expressing cell clusters-the lateral hypothalamic parvafox nucleus-we investigated the functions of this population of neurons by the selective deletion of VGlut2 expression in PV-expressing cells according to the Cre/Lox-approach. PV-Cre;VGlut2-Lox mutant mice are phenotypically characterized by deficits in locomotion and vocalization, by a decreased thermal nociception, and by an increased social dominance. We conducted a search of the Allen Brain Atlas for regions that might co-express the genes encoding PV and VGlut2, and that might thus contribute to the manifestation of the observed phenotypes. Our survey revealed several structures that could contribute to the deficits in locomotion and vocalization, such as the red, the subthalamic and the deep cerebellar nuclei. It also disclosed that a shift in the balance of afferental glutamatergic neurotransmission to the periaqueductal gray matter might be accountable for the decrease in sensitivity to pain and for the increase in social dominance. As a whole, this study broadens the state of knowledge about PV-expressing excitatory neurons.

Hyperpolarization-activated cyclic nucleotide-gated channels (HCNs) in the nervous system are implicated in a variety of neuronal functions including learning and memory, regulation of vigilance states and pain. Dysfunctions or genetic loss of these channels have been shown to cause human diseases such as epilepsy, depression, schizophrenia, and Parkinson's disease. The physiological functions of HCN1 and HCN2 channels in the nervous system have been analyzed using genetic knockout mouse models. By contrast, there are no such genetic studies for HCN3 channels so far. Here, we use a HCN3-deficient (HCN3 -/- ) mouse line, which has been previously generated in our group to examine the expression and function of this channel in the CNS. Specifically, we investigate the role of HCN3 channels for the regulation of circadian rhythm and for the determination of behavior. Contrary to previous suggestions we find that HCN3 -/- mice show normal visual, photic, and non-photic circadian function. In addition, HCN3 -/- mice are impaired in processing contextual information, which is characterized by attenuated long-term extinction of contextual fear and increased fear to a neutral context upon repeated exposure.

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 CE s 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 CE s of specified sizes for the layers of the mouse dentate gyrus.

Although most nervous system diseases affect women and men differentially, most behavioral studies using mouse models do not include subjects of both sexes. Many researchers worry that data of female mice may be unreliable due to the estrous cycle. Here, we retrospectively evaluated sex effects on coefficient of variation (CV) in 5,311 mice which had performed the same place navigation protocol in the water-maze and in 4,554 mice tested in the same open field arena. Confidence intervals for Cohen's d as measure of effect size were computed and tested for equivalence with 0.2 as equivalence margin. Despite the large sample size, only few behavioral parameters showed a significant sex effect on CV. Confidence intervals of effect size indicated that CV was either equivalent or showed a small sex difference at most, accounting for less than 2% of total group to group variation of CV. While female mice were potentially slightly more variable in water-maze acquisition and in the open field, males tended to perform less reliably in the water-maze probe trial. In addition to evaluating variability, we also directly compared mean performance of female and male mice and found them to be equivalent in both water-maze place navigation and open field exploration. Our data confirm and extend other large scale studies in demonstrating that including female mice in experiments does not cause a relevant increase of data variability. Our results make a strong case for including mice of both sexes whenever open field or water-maze are used in preclinical research., (© 2017 The Authors. American Journal of Medical Genetics Part C Published by Wiley Periodicals, Inc.)

The key role of APP in the pathogenesis of Alzheimer disease is well established. However, postnatal lethality of double knockout mice has so far precluded the analysis of the physiological functions of APP and the APLPs in the brain. Previously, APP family proteins have been implicated in synaptic adhesion, and analysis of the neuromuscular junction of constitutive APP/APLP2 mutant mice showed deficits in synaptic morphology and neuromuscular transmission. Here, we generated animals with a conditional APP/APLP2 double knockout (cDKO) in excitatory forebrain neurons using NexCre mice. Electrophysiological recordings of adult NexCre cDKOs indicated a strong synaptic phenotype with pronounced deficits in the induction and maintenance of hippocampal LTP and impairments in paired pulse facilitation, indicating a possible presynaptic deficit. These deficits were also reflected in impairments in nesting behavior and hippocampus-dependent learning and memory tasks, including deficits in Morris water maze and radial maze performance. Moreover, while no gross alterations of brain morphology were detectable in NexCre cDKO mice, quantitative analysis of adult hippocampal CA1 neurons revealed prominent reductions in total neurite length, dendritic branching, reduced spine density and reduced spine head volume. Strikingly, the impairment of LTP could be selectively rescued by acute application of exogenous recombinant APPsα, but not APPsβ, indicating a crucial role for APPsα to support synaptic plasticity of mature hippocampal synapses on a rapid time scale. Collectively, our analysis reveals an essential role of APP family proteins in excitatory principal neurons for mediating normal dendritic architecture, spine density and morphology, synaptic plasticity and cognition.

The habenular complex linking forebrain and midbrain structures is subdivided into the medial (mHb) and the lateral nuclei (lHb). The mHb is characterized by the expression of specific nicotinic acetylcholine receptor isoforms and the release of acetylcholine to the interpeduncular nucleus (IPN), the sole output region of the mHb. The specific function of this circuit, however, is poorly understood. Here we generated transgenic mice in which mHb cells were selectively ablated postnatally. These lesions led to large reductions in acetylcholine levels within the IPN. The mutant mice exhibited abnormalities in a wide range of behavioral domains. They tended to be hyperactive during the early night period and were maladapted when repeatedly exposed to new environments. Mutant mice also showed a high rate of premature responses in the 5-choice serial reaction time task (5-CSRTT), indicating impulsive and compulsive behavior. Additionally, mice also exhibited delay and effort aversion in a decision-making test, deficits in spatial memory, a subtle increase in anxiety levels, and attenuated sensorimotor gating. IntelliCage studies under social housing conditions confirmed hyperactivity, environmental maladaptation, and impulsive/compulsive behavior, delay discounting, deficits in long-term spatial memory, and reduced flexibility in complex learning paradigms. In 5-CSRTT and adaptation tasks, systemic administration of nicotine slowed down nose-poke reaction and enhanced adaptation in control but not mutant mice. These findings demonstrate that the mHb-IPN pathway plays a crucial role in inhibitory control and cognition-dependent executive functions.

The IntelliMaze allows automated behavioral analysis of group housed laboratory mice while individually assigned protocols can be applied concomitantly for different operant conditioning components. Here we evaluate the effect of additional component availability (enrichment) on behavioral and cognitive performance of mice in the IntelliCage, by focusing on aspects that had previously been found to consistently differ between three strains, in four European laboratories. Enrichment decreased the activity level in the IntelliCages and enhanced spatial learning performance. However, it did not alter strain differences, except for activity during the initial experimental phase. Our results from non-enriched IntelliCages proved consistent between laboratories, but overall laboratory-consistency for data collected using different IntelliCage set-ups, did not hold for activity levels during the initial adaptation phase. Our results suggest that the multiple conditioning in spatially and cognitively enriched environments are feasible without affecting external validity for a specific task, provided animals have adapted to such an IntelliMaze.

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.

The increasing resolution of tract-tracing studies has led to the definition of segments along the transverse axis of the hippocampal pyramidal cell layer, which may represent functionally defined elements. This review will summarize evidence for a morphological and functional differentiation of pyramidal cells along the radial (deep to superficial) axis of the cell layer. In many species, deep and superficial sublayers can be identified histologically throughout large parts of the septotemporal extent of the hippocampus. Neurons in these sublayers are generated during different periods of development. During development, deep and superficial cells express genes (Sox5, SatB2) that also specify the phenotypes of superficial and deep cells in the neocortex. Deep and superficial cells differ neurochemically (e.g. calbindin and zinc) and in their adult gene expression patterns. These markers also distinguish sublayers in the septal hippocampus, where they are not readily apparent histologically in rat or mouse. Deep and superficial pyramidal cells differ in septal, striatal, and neocortical efferent connections. Distributions of deep and superficial pyramidal cell dendrites and studies in reeler or sparsely GFP-expressing mice indicate that this also applies to afferent pathways. Histological, neurochemical, and connective differences between deep and superficial neurons may correlate with (patho-) physiological phenomena specific to pyramidal cells at different radial locations. We feel that an appreciation of radial subdivisions in the pyramidal cell layer reminiscent of lamination in other cortical areas may be critical in the interpretation of studies of hippocampal anatomy and function., (© The Author(s) 2011. This article is published with open access at Springerlink.com)

Alzheimer's disease, responsible for the vast majority of dementia cases in the elderly population, is caused by accumulation of toxic levels of amyloid beta peptide (A beta) in the brain. Neprilysin is a major enzyme responsible for the degradation of A beta in vivo. We have previously shown that elevation of neprilysin levels in the brain delays the deposition of A beta-plaques in a mouse model of amyloidosis and that lack of neprilysin leads to increased A beta generation and to signs of incipient neurodegeneration in mouse brains. This study was designed to test whether low brain levels of neprilysin affect the amyloid pathology or perturb the learning and memory performance of mice. Double-mutated mice carrying a targeted depletion of one allele of Mme, the gene encoding neprilysin, and over-expressing human amyloid precursor protein (APP), exhibited a reinforced amyloid pathology in comparison with their APP transgenic littermates. Moreover, in contrast to their parental lines, these mice were impaired in the Morris water maze learning and memory paradigm and showed facilitated extinction in the conditioned taste aversion test. These data suggest that even a partial neprilysin deficiency, as is found during aging, exacerbates amyloid pathology and may impair cognitive functions.

It is well established that the proteolytic processing of the beta-amyloid precursor protein (APP) generates beta-amyloid (Abeta), which plays a central role in the pathogenesis of Alzheimer's disease (AD). In contrast, the physiological role of APP and of its numerous proteolytic fragments and the question of whether a loss of these functions contributes to AD are still unknown. To address this question, we replaced the endogenous APP locus by gene-targeted alleles and generated two lines of knock-in mice that exclusively express APP deletion variants corresponding either to the secreted APP ectodomain (APPs alpha) or to a C-terminal (CT) truncation lacking the YENPTY interaction motif (APPdeltaCT15). Interestingly, the deltaCT15 deletion resulted in reduced turnover of holoAPP, increased cell surface expression, and strongly reduced Abeta levels in brain, likely because of reduced processing in the endocytic pathway. Most importantly, we demonstrate that in both APP knock-in lines the expression of APP N-terminal domains either grossly attenuated or completely rescued the prominent deficits of APP knock-out mice, such as reductions in brain and body weight, grip strength deficits, alterations in circadian locomotor activity, exploratory activity, and the impairment in spatial learning and long-term potentiation. Together, our data suggest that the APP C terminus is dispensable and that APPs alpha is sufficient to mediate the physiological functions of APP assessed by these tests.

Prostaglandin E2 (PGE2) is a crucial mediator of inflammatory pain sensitization. Here, we demonstrate that inhibition of a specific glycine receptor subtype (GlyR alpha3) by PGE2-induced receptor phosphorylation underlies central inflammatory pain sensitization. We show that GlyR alpha3 is distinctly expressed in superficial layers of the spinal cord dorsal horn. Mice deficient in GlyR alpha3 not only lack the inhibition of glycinergic neurotransmission by PGE2 seen in wild-type mice but also show a reduction in pain sensitization induced by spinal PGE2 injection or peripheral inflammation. Thus, GlyR alpha3 may provide a previously unrecognized molecular target in pain therapy.

We generated defined neuronal loss in hippocampus of genetically identical mice by pilocarpine injections and studied the impact of these seizures on the performance of mice in spatial learning and memory. The numbers of TUNEL-positive degenerating cells paralleled the severity of the seizures. When compared to the numbers found for not-seizured control mice, mild, moderate, and severe seizures produced significant increases in TUNEL-positive neurons in CA1 and CA3 regions by 19, 25, and 63%, respectively. Water maze learning was abolished after the severe seizures. However, spatial learning was normal after mild or moderate seizures. Therefore, there was no linear correlation between the impairment of learning and memory performance with the number of degenerating neurons in hippocampus. Our data suggest that normal spatial learning and memory can be achieved without the full number of hippocampal pyramidal neurons in partially lesioned hippocampus.