Activation of feedforward wiring in adult hippocampal neurons by the basic-helix-loop-helix transcription factor Ascl4.

Although evidence indicates that the adult brain retains a considerable capacity for circuit formation, adult wiring has not been broadly considered and remains poorly understood. In this study, we investigate wiring activation in adult neurons. We show that the basic-helix-loop-helix transcription factor Ascl4 can induce wiring in different types of hippocampal neurons of adult mice. The new axons are mainly feedforward and reconfigure synaptic weights in the circuit. Mice with the Ascl4-induced circuits do not display signs of pathology and solve spatial problems equally well as controls. Our results demonstrate reprogrammed connectivity by a single transcriptional factor and provide insights into the regulation of brain wiring in adults.

Using mice from different breeding sites fails to improve replicability of results from single-laboratory studies.

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.

Bdnf-Nrf-2 crosstalk and emotional behavior are disrupted in a sex-dependent fashion in adolescent mice exposed to maternal stress or maternal obesity.

Maternal obesity has been recognized as a stressor affecting the developing fetal brain, leading to long-term negative outcomes comparable to those resulting from maternal psychological stress, although the mechanisms have not been completely elucidated. In this study, we tested the hypothesis that adverse prenatal conditions as diverse as maternal stress and maternal obesity might affect emotional regulation and stress response in the offspring through common pathways, with a main focus on oxidative stress and neuroplasticity. We contrasted and compared adolescent male and female offspring in two mouse models of maternal psychophysical stress (restraint during pregnancy - PNS) and maternal obesity (high-fat diet before and during gestation - mHFD) by combining behavioral assays, evaluation of the hypothalamic-pituitary-adrenal (HPA) axis reactivity, immunohistochemistry and gene expression analysis of selected markers of neuronal function and neuroinflammation in the hippocampus, a key region involved in stress appraisal. Prenatal administration of the antioxidant N-acetyl-cysteine (NAC) was used as a strategy to protect fetal neurodevelopment from the negative effects of PNS and mHFD. Our findings show that these two stressors produce overlapping effects, reducing brain anti-oxidant defenses (Nrf-2) and leading to sex-dependent impairments of hippocampal Bdnf expression and alterations of the emotional behavior and HPA axis functionality. Prenatal NAC administration, by restoring the redox balance, was able to exert long-term protective effects on brain development, suggesting that the modulation of redox pathways might be an effective strategy to target common shared mechanisms between different adverse prenatal conditions.

Refinement of IntelliCage protocols for complex cognitive tasks through replacement of drinking restrictions by incentive-disincentive paradigms.

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.

Environmental enrichment improves hippocampus-dependent spatial learning in female C57BL/6 mice in novel IntelliCage sweet reward-based behavioral tests.

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.

Bank Voles Show More Impulsivity in IntelliCage Learning Tasks than Wood Mice.

Impulsivity is a personality trait of healthy individuals, but in extreme forms common in mental disorders. Previous behavioral testing of wild-caught bank voles and wood mice suggested impulsiveness in bank voles. Here, we compared behavioral performance of bank voles and wood mice in tests for response control in the IntelliCage. In the reaction time task, a test similar to the five-choice serial-reaction time task (5CSRTT), bank voles made more premature responses. Impulsivity in the reaction time task was associated with smaller medial habenular nucleus in bank voles. Additional tests revealed reduced behavioral flexibility in the self-paced flexibility task in bank voles, but equal spatial and reversal learning in the chaining/reversal task in both species. Expression of immediate early gene Arc after behavioral testing was low in medial prefrontal cortex, but high in hypothalamic supraoptic and paraventricular nucleus in bank voles. Wood mice showed the opposite pattern. Numbers of Arc-positive cells in the dorsal hippocampus were higher in bank voles than wood mice. Due to continuous behavioral testing (24/7), associations between behavioral performance and Arc were rare. Corticosterone measurements at the end of experiments suggested that IntelliCage testing did not elicit a stress response in these wild rodents. In summary, habenular size differences and altered activation of brain areas after testing might indicate differently balanced activations of cortico-limbic and cortico-hypothalamic circuits in bank voles compared to wood mice. Behavioral performance of bank voles suggest that these rodents could be a natural animal model for investigating impulsive and perseverative behaviors.

Lack of APLP1 leads to subtle alterations in neuronal morphology but does not affect learning and memory.

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.

The rearing environment persistently modulates mouse phenotypes from the molecular to the behavioural level.

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.

Multimodal anatomy of the human forniceal commissure.

Ambiguity surrounds the existence and morphology of the human forniceal commissure. We combine advanced in-vivo tractography, multidirectional ex-vivo fiber dissection, and multiplanar histological analysis to characterize this structure's anatomy. Across all 178 subjects, in-vivo fiber dissection based on the Human Connectome Project 7 T MRI data identifies no interhemispheric connections between the crura fornicis. Multidirectional ex-vivo fiber dissection under the operating microscope demonstrates the psalterium as a thin soft-tissue membrane spanning between the right and left crus fornicis, but exposes no commissural fibers. Multiplanar histological analysis with myelin and Bielchowsky silver staining, however, visualizes delicate cruciform fibers extending between the crura fornicis, enclosed by connective tissue, the psalterium. The human forniceal commissure is therefore much more delicate than previously described and presented in anatomical textbooks. This finding is consistent with the observed phylogenetic trend of a reduction of the forniceal commissure in non-human primates compared to non-primate eutherian mammals.

Cell numbers in the reflected blade of CA3 and their relation to other hippocampal principal cell populations across seven species.

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.

Recurrent rewiring of the adult hippocampal mossy fiber system by a single transcriptional regulator, Id2.

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.

Microglial-glucocorticoid receptor depletion alters the response of hippocampal microglia and neurons in a chronic unpredictable mild stress paradigm in female mice.

Chronic psychological stress is one of the most important triggers and environmental risk factors for neuropsychiatric disorders. Chronic stress can influence all organs via the secretion of stress hormones, including glucocorticoids by the adrenal glands, which coordinate the stress response across the body. In the brain, glucocorticoid receptors (GR) are expressed by various cell types including microglia, which are its resident immune cells regulating stress-induced inflammatory processes. To study the roles of microglial GR under normal homeostatic conditions and following chronic stress, we generated a mouse model in which the GR gene is depleted in microglia specifically at adulthood to prevent developmental confounds. We first confirmed that microglia were depleted in GR in our model in males and females among the cingulate cortex and the hippocampus, both stress-sensitive brain regions. Then, cohorts of microglial-GR depleted and wild-type (WT) adult female mice were housed for 3 weeks in a standard or stressful condition, using a chronic unpredictable mild stress (CUMS) paradigm. CUMS induced stress-related behavior in both microglial-GR depleted and WT animals as demonstrated by a decrease of both saccharine preference and progressive ratio breakpoint. Nevertheless, the hippocampal microglial and neural mechanisms underlying the adaptation to stress occurred differently between the two genotypes. Upon CUMS exposure, microglial morphology was altered in the WT controls, without any apparent effect in microglial-GR depleted mice. Furthermore, in the standard environment condition, GR depleted-microglia showed increased expression of pro-inflammatory genes, and genes involved in microglial homeostatic functions (such as Trem2, Cx3cr1 and Mertk). On the contrary, in CUMS condition, GR depleted-microglia showed reduced expression levels of pro-inflammatory genes and increased neuroprotective as well as anti-inflammatory genes compared to WT-microglia. Moreover, in microglial-GR depleted mice, but not in WT mice, CUMS led to a significant reduction of CA1 long-term potentiation and paired-pulse ratio. Lastly, differences in adult hippocampal neurogenesis were observed between the genotypes during normal homeostatic conditions, with microglial-GR deficiency increasing the formation of newborn neurons in the dentate gyrus subgranular zone independently from stress exposure. Together, these findings indicate that, although the deletion of microglial GR did not prevent the animal's ability to respond to stress, it contributed to modulating hippocampal functions in both standard and stressful conditions, notably by shaping the microglial response to chronic stress.

Loss of all three APP family members during development impairs synaptic function and plasticity, disrupts learning, and causes an autism-like phenotype.

The key role of APP for Alzheimer pathogenesis is well established. However, perinatal lethality of germline knockout mice lacking the entire APP family has so far precluded the analysis of its physiological functions for the developing and adult brain. Here, we generated conditional APP/APLP1/APLP2 triple KO (cTKO) mice lacking the APP family in excitatory forebrain neurons from embryonic day 11.5 onwards. NexCre cTKO mice showed altered brain morphology with agenesis of the corpus callosum and disrupted hippocampal lamination. Further, NexCre cTKOs revealed reduced basal synaptic transmission and drastically reduced long-term potentiation that was associated with reduced dendritic length and reduced spine density of pyramidal cells. With regard to behavior, lack of the APP family leads not only to severe impairments in a panel of tests for learning and memory, but also to an autism-like phenotype including repetitive rearing and climbing, impaired social communication, and deficits in social interaction. Together, our study identifies essential functions of the APP family during development, for normal hippocampal function and circuits important for learning and social behavior.

ATG5 in microglia does not contribute vitally to autoimmune neuroinflammation in mice.

Microglia, resident myeloid immune cells of the central nervous system (CNS), actively shape the circuitry of the brain, maintain CNS homeostasis during the steady state and orchestrate immune responses upon CNS injury. Both canonical and non-canonical functions of the macroautophagy/autophagy-related protein ATG5 regulate myeloid cell survival and immune responses. Here, we report that loss of ATG5 in postnatal microglia does not perturb CNS tissue integrity, microglial cell survival, or immune activation. Learning task performances were unchanged in mutant mice. Furthermore, lack of ATG5 expression in microglia had no impact on the development of experimental autoimmune encephalomyelitis. These data indicate that, basal autophagy, identified to be essential for the survival and function of neuronal cells, is not required to maintain CNS homeostasis if absent in adult microglia and ATG5 expression is dispensable for the development of autoimmune neuroinflammation.Abbreviations Ag, antigen; APC, antigen presenting cell; ATG/Atg, autophagy-related; CD, cluster of differentiation; CNS, central nervous system; DC, dendritic cell; EAE, experimental autoimmune encephalomyelitis; fl, floxed; LAP, LC3-associated phagocytosis; LC3, microtubule-associated protein 1 light chain 3; MFI, median fluorescence intensity; MHCII, major histocompatibility complex class II; MOG, myelin oligodendrocyte glycoprotein; MS, multiple sclerosis.

Phylogenetic variation in cortical layer II immature neuron reservoir of mammals.

The adult mammalian brain is mainly composed of mature neurons. A limited amount of stem cell-driven neurogenesis persists in postnatal life and is reduced in large-brained species. Another source of immature neurons in adult brains is cortical layer II. These cortical immature neurons (cINs) retain developmentally undifferentiated states in adulthood, though they are generated before birth. Here, the occurrence, distribution and cellular features of cINs were systematically studied in 12 diverse mammalian species spanning from small-lissencephalic to large-gyrencephalic brains. In spite of well-preserved morphological and molecular features, the distribution of cINs was highly heterogeneous, particularly in neocortex. While virtually absent in rodents, they are present in the entire neocortex of many other species and their linear density in cortical layer II generally increased with brain size. These findings suggest an evolutionary developmental mechanism for plasticity that varies among mammalian species, granting a reservoir of young cells for the cerebral cortex.

To acquire new skills or recover after injuries, the mammalian brain relies on plasticity, the ability for the brain to change its architecture and its connections during the lifetime of an animal. Creating new nerve cells is one way to achieve plasticity, but this process is rarer in humans than it is in mammals with smaller brains. In particular, it is absent in the human cortex: this region is enlarged in species with large brains, where it carries out complex tasks such as learning and memory. Producing new cells in the cortex would threaten the stability of the structures that retain long-term memories. Another route to plasticity is to reshape the connections between existing, mature nerve cells. This process takes place in the human brain during childhood and adolescence, as some connections are strengthened and others pruned away. An alternative mechanism relies on keeping some nerve cells in an immature, 'adolescent' state. When needed, these nerve cells emerge from their state of arrested development and 'grow up', connecting with the appropriate brain circuits. This mechanism does not involve producing new nerve cells, and so it would be suitable to maintain plasticity in the cortex. Consistent with this idea, in mice some dormant nerve cells are present in a small, primitive part of the cortex. La Rosa et al. therefore wanted to determine if the location and number of immature cells in the cortex differed between mammals, and if so, whether these differences depended on brain size. The study spanned 12 mammal species, from small-brained species like mice to larger-brained animals including sheep and non-human primates. Microscopy imaging was used to identify immature nerve cells in brain samples, which revealed that the cortex in larger-brained species contained more adolescent cells than its mouse counterpart. The difference was greatest in a region called the neocortex, which has evolved most recently. This area is most pronounced in primates ­ especially humans ­ where it carries out high-level cognitive tasks. These results identify immature nerve cells as a potential mechanism for plasticity in the cortex. La Rosa et al. hope that the work will inspire searches for similar reservoirs of young cells in humans, which could perhaps lead to new treatments for brain disorders like dementia.

Consistent within-group covariance of septal and temporal hippocampal neurogenesis with behavioral phenotypes for exploration and memory retention across wild and laboratory small rodents.

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.

OCRL deficiency impairs endolysosomal function in a humanized mouse model for Lowe syndrome and Dent disease.

Mutations in OCRL encoding the inositol polyphosphate 5-phosphatase OCRL (Lowe oculocerebrorenal syndrome protein) disrupt phosphoinositide homeostasis along the endolysosomal pathway causing dysfunction of the cells lining the kidney proximal tubule (PT). The dysfunction can be isolated (Dent disease 2) or associated with congenital cataracts, central hypotonia and intellectual disability (Lowe syndrome). The mechanistic understanding of Dent disease 2/Lowe syndrome remains scarce due to limitations of animal models of OCRL deficiency. Here, we investigate the role of OCRL in Dent disease 2/Lowe syndrome by using OcrlY/- mice, where the lethal deletion of the paralogue Inpp5b was rescued by human INPP5B insertion, and primary culture of proximal tubule cells (mPTCs) derived from OcrlY/- kidneys. The OcrlY/- mice show muscular defects with dysfunctional locomotricity and present massive urinary losses of low-molecular-weight proteins and albumin, caused by selective impairment of receptor-mediated endocytosis in PT cells. The latter was due to accumulation of phosphatidylinositol 4,5-bisphosphate PI(4,5)P2 in endolysosomes, driving local hyper-polymerization of F-actin and impairing trafficking of the endocytic LRP2 receptor, as evidenced in OcrlY/- mPTCs. The OCRL deficiency was also associated with a disruption of the lysosomal dynamic and proteolytic activity. Partial convergence of disease-pathways and renal phenotypes observed in OcrlY/- and Clcn5Y/- mice suggest shared mechanisms in Dent diseases 1 and 2. These studies substantiate the first mouse model of Lowe syndrome and give insights into the role of OCRL in cellular trafficking of multiligand receptors. These insights open new avenues for therapeutic interventions in Lowe syndrome and Dent disease.

Similar reliability and equivalent performance of female and male mice in the open field and water-maze place navigation task.

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.

Effects of Strain and Species on the Septo-Temporal Distribution of Adult Neurogenesis in Rodents.

The functional septo-temporal (dorso-ventral) differentiation of the hippocampus is accompanied by gradients of adult hippocampal neurogenesis (AHN) in laboratory rodents. An extensive septal AHN in laboratory mice suggests an emphasis on a relation of AHN to tasks that also depend on the septal hippocampus. Domestication experiments indicate that AHN dynamics along the longitudinal axis are subject to selective pressure, questioning if the septal emphasis of AHN in laboratory mice is a rule applying to rodents in general. In this study, we used C57BL/6 and DBA2/Crl mice, wild-derived F1 house mice and wild-captured wood mice and bank voles to look for evidence of strain and species specific septo-temporal differences in AHN. We confirmed the septal > temporal gradient in C57BL/6 mice, but in the wild species, AHN was low septally and high temporally. Emphasis on the temporal hippocampus was particularly strong for doublecortin positive (DCX+) young neurons and more pronounced in bank voles than in wood mice. The temporal shift was stronger in female wood mice than in males, while we did not see sex differences in bank voles. AHN was overall low in DBA and F1 house mice, but they exhibited the same inversed gradient as wood mice and bank voles. DCX+ young neurons were usually confined to the subgranular zone and deep granule cell layer. This pattern was seen in all animals in the septal and intermediate dentate gyrus. In bank voles and wood mice however, the majority of temporal DCX+ cells were radially dispersed throughout the granule cell layer. Some but not all of the septo-temporal differences were accompanied by changes in the DCX+/Ki67+ cell ratios, suggesting that new neuron numbers can be regulated by both proliferation or the time course of maturation and survival of young neurons. Some of the septo-temporal differences we observe have also been found in laboratory rodents after the experimental manipulation of the molecular mechanisms that control AHN. Adaptations of AHN under natural conditions may operate on these or similar mechanisms, adjusting neurogenesis to the requirements of hippocampal function.