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Advances in gene therapy, especially for brain diseases, have created new imaging demands for noninvasive monitoring of gene expression. While reporter gene imaging using co-expression of fluorescent protein-encoding gene has been widely developed, these conventional methods face significant limitations in longitudinal in vivo applications. Magnetic resonance imaging (MRI), specifically chemical exchange saturation transfer (CEST) MRI, provides a robust noninvasive alternative that offers unlimited depth penetration, reliable spatial resolution, and specificity toward particular molecules. In this study, we explore the potential of CEST-MRI for monitoring gene expression in neurons. We designed a CEST polypeptide reporter expressing 150 arginine residues and evaluated its expression in the living brain after viral vector delivery. A longitudinal study performed at one and 2 months postinjection showed that specific CEST signal was observable. In particular, the CEST contrast exhibited distinct peaks at 0.75 and 1.75 ppm, consistent with the expected hydroxyl and guanidyl protons resonance frequencies. Histological study confirmed the specific neuronal expression of the transgene evidenced by the fluorescence signal from the td-Tomato fluorophore fused to the polypeptide. The ability to image noninvasively a neuron-specific CEST-MRI reporter gene could offer valuable insights for further developments of gene therapy for neurological disorders., (© 2024 The Author(s). NMR in Biomedicine published by John Wiley & Sons Ltd.)

Astrocytes control brain activity via both metabolic processes and gliotransmission, but the physiological links between these functions are scantly known. Here we show that endogenous activation of astrocyte type-1 cannabinoid (CB1) receptors determines a shift of glycolysis towards the lactate-dependent production of D-serine, thereby gating synaptic and cognitive functions in male mice. Mutant mice lacking the CB1 receptor gene in astrocytes (GFAP-CB1-KO) are impaired in novel object recognition (NOR) memory. This phenotype is rescued by the gliotransmitter D-serine, by its precursor L-serine, and also by lactate and 3,5-DHBA, an agonist of the lactate receptor HCAR1. Such lactate-dependent effect is abolished when the astrocyte-specific phosphorylated-pathway (PP), which diverts glycolysis towards L-serine synthesis, is blocked. Consistently, lactate and 3,5-DHBA promoted the co-agonist binding site occupancy of CA1 post-synaptic NMDA receptors in hippocampal slices in a PP-dependent manner. Thus, a tight cross-talk between astrocytic energy metabolism and gliotransmission determines synaptic and cognitive processes., (© 2024. The Author(s).)

Tauopathy is a typical feature of Alzheimer's disease of major importance because it strongly correlates with the severity of cognitive deficits experienced by patients. During the pathology, it follows a characteristic spatiotemporal course which takes its origin in the transentorhinal cortex, and then gradually invades the entire forebrain. To study the mechanisms of tauopathy, and test new therapeutic strategies, it is necessary to set-up relevant and versatile in vivo models allowing to recapitulate tauopathy. With this in mind, we have developed a model of tauopathy by overexpression of the human wild-type Tau protein in retinal ganglion cells in mice (RGCs). This overexpression led to the presence of hyperphosphorylated forms of the protein in the transduced cells as well as to their progressive degeneration. The application of this model to mice deficient in TREM2 (Triggering Receptor Expressed on Myeloid cells-2, an important genetic risk factor for AD) as well as to 15-month-old mice showed that microglia actively participate in the degeneration of RGCs. Surprisingly, although we were able to detect the transgenic Tau protein up to the terminal arborization of RGCs at the level of the superior colliculi, spreading of the transgenic Tau protein to post-synaptic neurons was detected only in aged animals. This suggests that there may be neuron-intrinsic- or microenvironment mediators facilitating this spreading that appear with aging., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Huntington's disease is a fatal neurodegenerative disease characterized by striatal neurodegeneration, aggregation of mutant Huntingtin and the presence of reactive astrocytes. Astrocytes are important partners for neurons and engage in a specific reactive response in Huntington's disease that involves morphological, molecular and functional changes. How reactive astrocytes contribute to Huntington's disease is still an open question, especially because their reactive state is poorly reproduced in experimental mouse models. Here, we show that the JAK2-STAT3 pathway, a central cascade controlling astrocyte reactive response, is activated in the putamen of Huntington's disease patients. Selective activation of this cascade in astrocytes through viral gene transfer reduces the number and size of mutant Huntingtin aggregates in neurons and improves neuronal defects in two complementary mouse models of Huntington's disease. It also reduces striatal atrophy and increases glutamate levels, two central clinical outcomes measured by non-invasive magnetic resonance imaging. Moreover, astrocyte-specific transcriptomic analysis shows that activation of the JAK2-STAT3 pathway in astrocytes coordinates a transcriptional program that increases their intrinsic proteolytic capacity, through the lysosomal and ubiquitin-proteasome degradation systems. This pathway also enhances their production and exosomal release of the co-chaperone DNAJB1, which contributes to mutant Huntingtin clearance in neurons. Together, our results show that the JAK2-STAT3 pathway controls a beneficial proteostasis response in reactive astrocytes in Huntington's disease, which involves bi-directional signalling with neurons to reduce mutant Huntingtin aggregation, eventually improving disease outcomes., (© The Author(s) 2022. Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The brain has almost no energy reserve, but its activity coordinates organismal function, a burden that requires precise coupling between neurotransmission and energy metabolism. Deciphering how the brain accomplishes this complex task is crucial to understand central facets of human physiology and disease mechanisms. Each type of neural cell displays a peculiar metabolic signature, forcing the intercellular exchange of metabolites that serve as both energy precursors and paracrine signals. The paradigm of this biological feature is the astrocyte-neuron couple, in which the glycolytic metabolism of astrocytes contrasts with the mitochondrial oxidative activity of neurons. Astrocytes generate abundant mitochondrial reactive oxygen species and shuttle to neurons glycolytically derived metabolites, such as L-lactate and L-serine, which sustain energy needs, conserve redox status, and modulate neurotransmitter-receptor activity. Conversely, early disruption of this metabolic cooperation may contribute to the initiation or progression of several neurological diseases, thus requiring innovative therapies to preserve brain energetics., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

The role played by microglia has taken the center of the stage in the etiology of Alzheimer's disease (AD). Several genome-wide association studies carried out on large cohorts of patients have indeed revealed a large number of genetic susceptibility factors corresponding to genes involved in neuroinflammation and expressed specifically by microglia in the brain. Among these genes TREM2, a cell surface receptor expressed by microglia, arouses strong interest because its R47H variant confers a risk of developing AD comparable to the ε4 allele of the APOE gene. Since this discovery, a growing number of studies have therefore examined the role played by TREM2 in the evolution of amyloid plaques and neurofibrillary tangles, the two brain lesions characteristic of AD. Many studies report conflicting results, reflecting the complex nature of microglial activation in AD. Here, we investigated the impact of TREM2 deficiency in the THY-Tau22 transgenic line, a well-characterized model of tauopathy. Our study reports an increase in the severity of tauopathy lesions in mice deficient in TREM2 occurring at an advanced stage of the pathology. This exacerbation of pathology was associated with a reduction in microglial activation indicated by typical morphological features and altered expression of specific markers. However, it was not accompanied by any further changes in memory performance. Our longitudinal study confirms that a defect in microglial TREM2 signaling leads to an increase in neuronal tauopathy occurring only at late stages of the disease., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Alpha-synuclein (α-syn) and leucine-rich repeat kinase 2 (LRRK2) play crucial roles in Parkinson's disease (PD). They may functionally interact to induce the degeneration of dopaminergic (DA) neurons via mechanisms that are not yet fully understood. We previously showed that the C-terminal portion of LRRK2 (ΔLRRK2) with the G2019S mutation (ΔLRRK2 G2019S ) was sufficient to induce neurodegeneration of DA neurons in vivo, suggesting that mutated LRRK2 induces neurotoxicity through mechanisms that are (i) independent of the N-terminal domains and (ii) "cell-autonomous". Here, we explored whether ΔLRRK2 G2019S could modify α-syn toxicity through these two mechanisms. We used a co-transduction approach in rats with AAV vectors encoding ΔLRRK2 G2019S or its "dead" kinase form, ΔLRRK2 DK , and human α-syn with the A53T mutation (AAV-α-syn A53T ). Behavioral and histological evaluations were performed at 6- and 15-weeks post-injection. Results showed that neither form of ΔLRRK2 alone induced the degeneration of neurons at these post-injection time points. By contrast, injection of AAV-α-syn A53T alone resulted in motor signs and degeneration of DA neurons. Co-injection of AAV-α-syn A53T with AAV-ΔLRRK2 G2019S induced DA neuron degeneration that was significantly higher than that induced by AAV-α-syn A53T alone or with AAV-ΔLRRK2 DK . Thus, mutated α-syn neurotoxicity can be enhanced by the C-terminal domain of LRRK2 G2019 alone , through cell-autonomous mechanisms.

Deposits of different abnormal forms of tau in neurons and astrocytes represent key anatomo-pathological features of tauopathies. Although tau protein is highly enriched in neurons and poorly expressed by astrocytes, the origin of astrocytic tau is still elusive. Here, we used innovative gene transfer tools to model tauopathies in adult mouse brains and to investigate the origin of astrocytic tau. We showed in our adeno-associated virus (AAV)-based models and in Thy-Tau22 transgenic mice that astrocytic tau pathology can emerge secondarily to neuronal pathology. By designing an in vivo reporter system, we further demonstrated bidirectional exchanges of tau species between neurons and astrocytes. We then determined the consequences of tau accumulation in astrocytes on their survival in models displaying various status of tau aggregation. Using stereological counting of astrocytes, we report that, as for neurons, soluble tau species are highly toxic to some subpopulations of astrocytes in the hippocampus, whereas the accumulation of tau aggregates does not affect their survival. Thus, astrocytes are not mere bystanders of neuronal pathology. Our results strongly suggest that tau pathology in astrocytes may significantly contribute to clinical symptoms., (© The Author(s) (2021). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Objective: Our aim was to estimate the prevalence of gingival overgrowth (hyperplasia) and to determine whether active molecules affect the severity of overgrowth in a group of epileptic patients., Background: The effects of phenytoin on oral health have been explored in different studies, yet little information is available on other antiepileptic drugs., Methods: Data were collected from 213 subjects of both sexes, from 5 to 80 years. Patients taking the same antiepileptic therapy for at least 1 year and meeting the inclusion criteria of the study (n = 162) were subjected to measurement of gingival overgrowth according to the modified Harris and Ewalt classification and O'Leary's plaque control record (OLR). Descriptive statistics were calculated. Data were analyzed using Pearson's r correlation coefficient and chi-square test. Significance level was set at 5%., Results: The active drugs lamotrigine, oxcarbazepine, and phenobarbital were significantly associated with gingival overgrowth in 61%, 71%, and 53% of cases, respectively, and phenytoin, valproic acid, and carbamazepine in 50%, 44%, and 32% of cases, respectively., Conclusion: Different antiepileptic molecules may be related to gingival overgrowth. In addition to phenytoin, also lamotrigine, oxcarbazepine, and phenobarbital were associated with increased prevalence of gingival overgrowth. In the management of epileptic patients, dentists should take into account different drugs as possible causes for gingival overgrowth and warn for possible alternatives., (© 2020 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Reactive astrocytes are astrocytes undergoing morphological, molecular, and functional remodeling in response to injury, disease, or infection of the CNS. Although this remodeling was first described over a century ago, uncertainties and controversies remain regarding the contribution of reactive astrocytes to CNS diseases, repair, and aging. It is also unclear whether fixed categories of reactive astrocytes exist and, if so, how to identify them. We point out the shortcomings of binary divisions of reactive astrocytes into good-vs-bad, neurotoxic-vs-neuroprotective or A1-vs-A2. We advocate, instead, that research on reactive astrocytes include assessment of multiple molecular and functional parameters-preferably in vivo-plus multivariate statistics and determination of impact on pathological hallmarks in relevant models. These guidelines may spur the discovery of astrocyte-based biomarkers as well as astrocyte-targeting therapies that abrogate detrimental actions of reactive astrocytes, potentiate their neuro- and glioprotective actions, and restore or augment their homeostatic, modulatory, and defensive functions.

Brain energy metabolism is often considered as a succession of biochemical steps that metabolize the fuel (glucose and oxygen) for the unique purpose of providing sufficient ATP to maintain the huge information processing power of the brain. However, a significant fraction (10-15 %) of glucose is shunted away from the ATP-producing pathway (oxidative phosphorylation) and may be used to support other functions. Recent studies have pointed to the marked compartmentation of energy metabolic pathways between neurons and glial cells. Here, we focused our attention on the biosynthesis of l-serine, a non-essential amino acid that is formed exclusively in glial cells (mostly astrocytes) by re-routing the metabolic fate of the glycolytic intermediate, 3-phosphoglycerate (3PG). This metabolic pathway is called the phosphorylated pathway and transforms 3PG into l-serine via three enzymatic reactions. We first compiled the available data on the mechanisms that regulate the flux through this metabolic pathway. We then reviewed the current evidence that is beginning to unravel the roles of l-serine both in the healthy and diseased brain, leading to the notion that this specific metabolic pathway connects glial metabolism with synaptic activity and plasticity. We finally suggest that restoring astrocyte-mediated l-serine homeostasis may provide new therapeutic strategies for brain disorders., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Astrocytes are thought to play a pivotal role in coupling neural activity and cerebral blood flow. However, it has been shown that astrocytes undergo morphologic changes in response to brain metastasis, switching to a reactive phenotype, which has the potential to significantly compromise cerebrovascular function and contribute to the neurological sequelae associated with brain metastasis. Given that STAT3 is a key regulator of astrocyte reactivity, we aimed here to determine the impact of STAT3-mediated astrocyte reactivity on neurovascular function in brain metastasis. Rat models of brain metastasis and ciliary neurotrophic factor were used to induce astrocyte reactivity. Multimodal imaging, electrophysiology, and IHC were performed to determine the relationship between reactive astrocytes and changes in the cerebrovascular response to electrical and physiological stimuli. Subsequently, the STAT3 pathway in astrocytes was inhibited with WP1066 to determine the role of STAT3-mediated astrocyte reactivity, specifically, in brain metastasis. Astrocyte reactivity associated with brain metastases impaired cerebrovascular responses to stimuli at both the cellular and functional level and disrupted astrocyte-endothelial interactions in both animal models and human brain metastasis samples. Inhibition of STAT3-mediated astrocyte reactivity in rats with brain metastases restored cerebrovascular function, as shown by in vivo imaging, and limited cerebrovascular changes associated with tumor growth. Together these findings suggest that inhibiting STAT3-mediated astrocyte reactivity may confer significant improvements in neurological outcome for patients with brain metastases and could potentially be tested in other brain tumors. SIGNIFICANCE: These findings demonstrate that selectively targeting STAT3-mediated astrocyte reactivity ameliorates the cerebrovascular dysfunction associated with brain metastasis, providing a potential therapeutic avenue for improved patient outcome., (©2020 American Association for Cancer Research.)

Astrocytes take up glucose from the bloodstream to provide energy to the brain, thereby allowing neuronal activity and behavioural responses 1-5 . By contrast, astrocytes are under neuronal control through specific neurotransmitter receptors 5-7 . However, whether the activation of astroglial receptors can directly regulate cellular glucose metabolism to eventually modulate behavioural responses is unclear. Here we show that activation of mouse astroglial type-1 cannabinoid receptors associated with mitochondrial membranes (mtCB 1 ) hampers the metabolism of glucose and the production of lactate in the brain, resulting in altered neuronal functions and, in turn, impaired behavioural responses in social interaction assays. Specifically, activation of astroglial mtCB 1 receptors reduces the phosphorylation of the mitochondrial complex I subunit NDUFS4, which decreases the stability and activity of complex I. This leads to a reduction in the generation of reactive oxygen species by astrocytes and affects the glycolytic production of lactate through the hypoxia-inducible factor 1 pathway, eventually resulting in neuronal redox stress and impairment of behavioural responses in social interaction assays. Genetic and pharmacological correction of each of these effects abolishes the effect of cannabinoid treatment on the observed behaviour. These findings suggest that mtCB 1 receptor signalling can directly regulate astroglial glucose metabolism to fine-tune neuronal activity and behaviour in mice.

In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. The consequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD, we developed viral vectors targeting astrocytes that either activate or inhibit the Janus kinase-signal transducer and activator of transcription 3 (JAK2-STAT3) pathway, a central cascade controlling astrocyte reaction. We aimed to evaluate whether reactive astrocytes contribute to tau as well as amyloid pathologies in the hippocampus of 3xTg-AD mice, an AD model that develops tau hyper-phosphorylation and amyloid deposition. JAK2-STAT3 pathway-mediated modulation of reactive astrocytes in 25% of the hippocampus of 3xTg-AD mice did not significantly influence tau phosphorylation or amyloid processing and deposition at early, advanced, and terminal disease stage. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did not improve spatial memory in the Y maze but it did reduce anxiety in the elevated plus maze. Our unique approach to specifically manipulate reactive astrocytes in situ show they may impact behavioral outcomes without influencing tau or amyloid pathology., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Protoplasmic astrocytes (PrA) located in the mouse cerebral cortex are tightly juxtaposed, forming an apparently continuous three-dimensional matrix at adult stages. Thus far, no immunostaining strategy can single them out and segment their morphology in mature animals and over the course of corticogenesis. Cortical PrA originate from progenitors located in the dorsal pallium and can easily be targeted using in utero electroporation of integrative vectors. A protocol is presented here to label these cells with the multiaddressable genome-integrating color (MAGIC) Markers strategy, which relies on piggyBac/Tol2 transposition and Cre/lox recombination to stochastically express distinct fluorescent proteins (blue, cyan, yellow, and red) addressed to specific subcellular compartments. This multicolor fate mapping strategy enables to mark in situ nearby cortical progenitors with combinations of color markers prior to the start of gliogenesis and to track their descendants, including astrocytes, from embryonic to adult stages at the individual cell level. Semi-sparse labeling achieved by adjusting the concentration of electroporated vectors and color contrasts provided by the Multiaddressable Genome-Integrating Color Markers (MAGIC Markers or MM) enable to individualize astrocytes and single out their territory and complex morphology despite their dense anatomical arrangement. Presented here is a comprehensive experimental workflow including the details of the electroporation procedure, multichannel image stacks acquisition by confocal microscopy, and computer-assisted three-dimensional segmentation that will enable the experimenter to assess individual PrA volume and morphology. In summary, electroporation of MAGIC Markers provides a convenient method to individually label numerous astrocytes and gain access to their anatomical features at different developmental stages. This technique will be useful to analyze cortical astrocyte morphological properties in various mouse models without resorting to complex crosses with transgenic reporter lines.

The 18 kDa translocator protein (TSPO) is the main molecular target to image neuroinflammation by positron emission tomography (PET). However, TSPO-PET quantification is complex and none of the kinetic modelling approaches has been validated using a voxel-by-voxel comparison of TSPO-PET data with the actual TSPO levels of expression. Here, we present a single case study of binary classification of in vivo PET data to evaluate the statistical performance of different TSPO-PET quantification methods. To that end, we induced a localized and adjustable increase of TSPO levels in a non-human primate brain through a viral-vector strategy. We then performed a voxel-wise comparison of the different TSPO-PET quantification approaches providing parametric [ 18 F]-DPA-714 PET images, with co-registered in vitro three-dimensional TSPO immunohistochemistry (3D-IHC) data. A data matrix was extracted from each brain hemisphere, containing the TSPO-IHC and TSPO-PET data for each voxel position. Each voxel was then classified as false or true, positive or negative after comparison of the TSPO-PET measure to the reference 3D-IHC method. Finally, receiver operating characteristic curves (ROC) were calculated for each TSPO-PET quantification method. Our results show that standard uptake value ratios using cerebellum as a reference region (SUV CBL ) has the most optimal ROC score amongst all non-invasive approaches.

Alteration of brain aerobic glycolysis is often observed early in the course of Alzheimer's disease (AD). Whether and how such metabolic dysregulation contributes to both synaptic plasticity and behavioral deficits in AD is not known. Here, we show that the astrocytic l-serine biosynthesis pathway, which branches from glycolysis, is impaired in young AD mice and in AD patients. l-serine is the precursor of d-serine, a co-agonist of synaptic NMDA receptors (NMDARs) required for synaptic plasticity. Accordingly, AD mice display a lower occupancy of the NMDAR co-agonist site as well as synaptic and behavioral deficits. Similar deficits are observed following inactivation of the l-serine synthetic pathway in hippocampal astrocytes, supporting the key role of astrocytic l-serine. Supplementation with l-serine in the diet prevents both synaptic and behavioral deficits in AD mice. Our findings reveal that astrocytic glycolysis controls cognitive functions and suggest oral l-serine as a ready-to-use therapy for AD., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The G2019S substitution in the kinase domain of LRRK2 (LRRK2 G2019S ) is the most prevalent mutation associated with Parkinson's disease (PD). Neurotoxic effects of LRRK2 G2019S are thought to result from an increase in its kinase activity as compared to wild type LRRK2. However, it is unclear whether the kinase domain of LRRK2 G2019S is sufficient to trigger degeneration or if the full length protein is required. To address this question, we generated constructs corresponding to the C-terminal domain of LRRK2 (ΔLRRK2). A kinase activity that was increased by G2019➔S substitution could be detected in ΔLRRK2. However biochemical experiments suggested it did not bind or phosphorylate the substrate RAB10, in contrast to full length LRRK2. The overexpression of ΔLRRK2 G2019S in the rat striatum using lentiviral vectors (LVs) offered a straightforward and simple way to investigate its effects in neurons in vivo. Results from a RT-qPCR array analysis indicated that ΔLRRK2 G2019S led to significant mRNA expression changes consistent with a kinase-dependent mechanism. We next asked whether ΔLRRK2 could be sufficient to trigger neurodegeneration in the substantia nigra pars compacta (SNc) in adult rats. Six months after infection of the substantia nigra pars compacta (SNc) with LV-ΔLRRK2 WT or LV-ΔLRRK2 G2019S , the number of DA neurons was unchanged. To examine whether higher levels of ΔLRRK2 G2019S could trigger degeneration we cloned ΔLRRK2 in AAV2/9 construct. As expected, AAV2/9 injected in the SNc led to neuronal expression of ΔLRRK2 WT and ΔLRRK2 G2019S at much higher levels than those obtained with LVs. Six months after injection, unbiased stereology showed that AAV-ΔLRRK2 G2019S produced a significant ~30% loss of neurons positive for tyrosine hydroxylase- and for the vesicular dopamine transporter whereas AAV-ΔLRRK2 WT did not. These findings show that overexpression of the C-terminal part of LRRK2 containing the mutant kinase domain is sufficient to trigger degeneration of DA neurons, through cell-autonomous mechanisms, possibly independent of RAB10., (Copyright © 2019. Published by Elsevier Inc.)

Astrocytes play essential roles in the neural tissue where they form a continuous network, while displaying important local heterogeneity. Here, we performed multiclonal lineage tracing using combinatorial genetic markers together with a new large volume color imaging approach to study astrocyte development in the mouse cortex. We show that cortical astrocyte clones intermix with their neighbors and display extensive variability in terms of spatial organization, number and subtypes of cells generated. Clones develop through 3D spatial dispersion, while at the individual level astrocytes acquire progressively their complex morphology. Furthermore, we find that the astroglial network is supplied both before and after birth by ventricular progenitors that scatter in the neocortex and can give rise to protoplasmic as well as pial astrocyte subtypes. Altogether, these data suggest a model in which astrocyte precursors colonize the neocortex perinatally in a non-ordered manner, with local environment likely determining astrocyte clonal expansion and final morphotype.

The Morris Water Maze (MWM) is a behavioral test widely used in the field of neuroscience to evaluate spatial learning memory of rodents. However, the interpretation of results is often impaired by the common use of statistical tests based on independence and normal distributions that do not reflect basic properties of the test data, such as the constant-sum constraint. In this work, we propose to analyze MWM data with the Dirichlet distribution, which describes constant-sum data with minimal hypotheses, and we introduce a statistical test based on uniformity (equal amount of time spent in each quadrant of the maze) that evaluates memory impairments. We demonstrate that this test better represents MWM data and show its efficiency on simulated as well as in vivo data. Based on Dirichlet distribution, we also propose a new way to plot MWM data, showing mean values and inter-individual variability at the same time, on an easily interpretable chart. Finally, we conclude with a perspective on using Bayesian analysis for MWM data., Competing Interests: No competing interests were disclosed., (Copyright: © 2019 Maugard M et al.)