Olivier Raineteau

Cortical circuits are built at perinatal times and gradually refined in an activity-dependent manner during a so-called postnatal period of critical plasticity. Although lesions of the central nervous system (CNS) happening during this period typically recover better than those occurring later in life, they are often associated with long-term behavioral deficits. This suggests that neuronal circuits rewiring, in particular within the cortex, may either be incomplete or inappropriate. To address this possibility, we used chronic perinatal hypoxia, a mouse model of very premature birth. We confirmed that chronic hypoxia induced a decrease in cortical thickness frequently observed in very preterm babies, which rapidly recovered 8 days later. To explore the transcriptional correlates of this recovery we next performed singlenuclei transcriptomic analysis of the cortex at short (P11) and long (P45) timepoints following hypoxia. This revealed persistent transcriptional changes within neurons, including of genes involved in mitochondrial metabolism, axonogenesis and synaptogenesis. Further, histological analysis using anterograde and retrograde tracing as well as mitochondrial labelling support persistent alterations in upper cortical neurons resulting in increased corticocortical connectivity within the cortex of adult hypoxic mice. Finally, behavioral testing revealed altered social behavior in mice exposed to chronic hypoxia, which amplified with age. Altogether, our results unravel how brain lesions happening early in life, alter normal cortical development and have long term consequences on cortical wiring, contributing to the observed behaviors defects appearing later in life.

Oligodendrocytes are critical for CNS myelin formation and are involved in preterm-birth brain injury (PBI) and multiple sclerosis (MS), both of which lack effective treatments. We present a pharmacogenomic approach that identifies compounds with potent pro-oligodendrogenic activity, selected through a scoring strategy (OligoScore) based on their modulation of oligodendrogenic and (re)myelination-related transcriptional programs. Through in vitro neural and oligodendrocyte progenitor cell (OPC) cultures, ex vivo cerebellar explants, and in vivo mouse models of PBI and MS, we identify FDA-approved leucovorin and dyclonine as promising candidates. In a neonatal chronic hypoxia mouse model mimicking PBI, both compounds promote neural progenitor cell proliferation and oligodendroglial fate acquisition, with leucovorin further enhancing differentiation. In an adult MS model of focal de/remyelination, they improve lesion repair by promoting OPC differentiation while preserving the OPC pool. Additionally, they shift microglia from a pro-inflammatory to a pro-regenerative profile and enhance myelin debris clearance. These findings support the repurposing of leucovorin and dyclonine for clinical trials targeting myelin disorders, offering potential therapeutic avenues for PBI and MS.

Transcardiac perfusion of fixative agent is generally recommended for quality preparations for cerebral histology, ensuring rapid and deep penetration in the tissue to preserve the most fragile brain structures. Despite being performed under anesthesia and with proper analgesia, this procedure is cumbersome for the experimenter and raises ethical questions. Recently, alternative protocols have been proposed, based on prior sacrifice of the animal followed by an injection of a fixative agent into the circulation. These so-called post-mortem perfusion protocols should in theory ensure an equivalent quality of tissue fixation, without exposing live animals to a procedure. Before adopting this new method, it is necessary to validate that sample quality is equivalent, ensuring the validity of scientific results. We performed a parallel comparison of several protocols of tissue fixation, by transcardiac or post-mortem perfusion, and measured the impact on the maintenance of axonal structures, dendritic spines, and mitochondrial morphology. Our results showed that histological parameters show variable sensitivity to perfusion condition and fixative used. For instance, axon fragmentation and altered mitochondrial morphology were observed in post-mortem perfusion groups. We furthermore determined that fixation condition had a variable effect on immunostaining, impacting detected expression level or pattern. Our results serve as a guide to orient the experimenter in selecting the best condition for optimal tissue fixation, which minimizes animal suffering while guaranteeing the integrity of the biological results obtained.

Behaviors are encoded by widespread neural circuits within the brain that change with age and experience. Immunodetection of the immediate early gene c-Fos has been successfully used for decades to reveal neural circuits active during specific tasks or conditions. Our objectives here were to develop and benchmark a workflow that circumvents classical temporal and spatial limitations associated with c-Fos quantification. We combined c-Fos immunohistochemistry with c-Fos driven Cre-dependent tdTomato expression in the TRAP2 mice, to visualize and perform a direct comparison of neural circuits activated at different times or during different tasks. By using open-source software (QuPath and ABBA), we established a workflow that optimize and automate cell detection, cell classification (e.g. c-Fos vs. c-Fos/tdTomato) and whole brain registration. We demonstrate that this workflow, based on fully automatic scripts, allows accurate cell number quantification with minimal interindividual variability. Further, interrogation of brain atlases at different scales (from simplified to detailed) allows gradually zooming on brain regions to explore spatial distribution of activated cells. We illustrate the potential of this approach by comparing patterns of neuronal activation in various contexts (two vigilance states, complex behavioral tasks…), in separate groups of mice or at two time points in the same animals. Finally, we explore software (BrainRender) for intuitive representation of the results. Altogether, this automated workflow accessible to all labs with some expertise in histology, allows an unbiased, fast and accurate analysis of the whole brain activity pattern at the cellular level, in various contexts.

Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS), with oligodendroglial pathologies leading to strong disabilities, from early preterm-birth brain injury (PBI) to adult multiple sclerosis (MS). No medication presenting convincing repair capacity in humans has been approved for these pathologies so far. Here, we present a pharmacogenomic approach leading to the identification of small bioactive molecules with a large pro-oligodendrogenic activity, selected through an expert curation scoring strategy ( OligoScore ) of their large impact on transcriptional programs controlling oligodendrogenesis and (re)myelination. We demonstrate the pro-oligodendrogenic activity of these compounds in vitro, using neural and oligodendrocyte progenitor cell (OPC) cultures, as well as ex vivo, using organotypic cerebellar explant cultures. Focusing on the two most promising molecules, i.e. leucovorin and dyclonine, we tested their therapeutic efficacy using a mouse model of neonatal chronic hypoxia, which faithfully mimics aspects of PBI. In this model, both compounds promoted proliferation and oligodendroglial fate acquisition from neural stem/progenitor cells, with leucovorin also promoting their differentiation. We extended these findings to an adult focal de/remyelination mouse MS model, in which both compounds improved lesion repair by promoting OPC differentiation while maintaining the pool of OPCs, and in parallel, by accelerating the transition from pro-inflammatory to pro-regenerative microglial profiles and myelin debris clearance. This study paves the way for clinical trials aimed at repurposing these FDA-approved compounds to treat myelin pathologies such as PBI and MS. Graphical Pharmacogenomic screening identifies and repurposes dyclonine and leucovorin as pro-oligodendrogenic and pro-myelinating compounds. Schematics of the pharmacogenomic approach leading to the identification of small bioactive molecules (compounds) with potential pro-oligodendrogenic activity, followed by the in vitro validation of the top compounds using neural and oligodendrocyte progenitor cell (OPC) cultures as well as organotypic cerebellar explants. The therapeutic efficacy of the top two compounds, leucovorin and dyclonine, both approved by the Food and Drug Administration (FDA), was assessed in vivo using two clinically relevant mouse models of myelin pathologies. In the neonatal hypoxia mouse model, mimicking some aspects of preterm brain injury, both leucovorin and dyclonine promoted neural stem cell (NSC) differentiation into OPCs and OPC proliferation, with leucovorin additionally restoring the density of myelinating OLs found in normoxic conditions. In an adult focal de/remyelination mouse model of multiple sclerosis, both compounds significantly improved lesion repair in adult mice by promoting OPC differentiation while preserving the pool of OPCs, and by accelerating myelin debris clearance and shifting microglia from pro-inflammatory to pro-regenerative profiles.

The ventricular-subventricular zone (V-SVZ) is the largest neurogenic region of the postnatal forebrain, containing neural stem cells (NSCs) that emerge from both the embryonic pallium and subpallium. Despite of this dual origin, glutamatergic neurogenesis declines rapidly after birth, while GABAergic neurogenesis persists throughout life. We performed single-cell RNA sequencing of the postnatal dorsal V-SVZ for unraveling the mechanisms leading to pallial lineage germinal activity silencing. We show that pallial NSCs enter a state of deep quiescence, characterized by high bone morphogenetic protein (BMP) signaling, reduced transcriptional activity and Hopx expression, while in contrast, subpallial NSCs remain primed for activation. Induction of deep quiescence is paralleled by a rapid blockade of glutamatergic neuron production and differentiation. Last, manipulation of Bmpr1a demonstrates its key role in mediating these effects. Together, our results highlight a central role of BMP signaling in synchronizing quiescence induction and blockade of neuronal differentiation to rapidly silence pallial germinal activity after birth.

The ventricular-subventricular zone (V-SVZ) is the largest neurogenic region of the postnatal forebrain, containing neural stem cells (NSCs) that emerge from both the embryonic pallium and subpallium. Despite of this dual origin, glutamatergic neurogenesis declines rapidly after birth, while gabaergic neurogenesis persists throughout life. Here, we performed single-cell RNA-sequencing (scRNA-Seq) of the postnatal dorsal V-SVZ for unravelling the mechanisms leading to pallial lineage germinal activity silencing. We identify cell lineage-specific NSCs primed for the generation of neurons or glial cells, as well as a large population of so far uncharacterized quiescent NSCs (qNSC). Pallial qNSCs enter a state of deep quiescence, characterized by persistent TGFbeta/BMP signalling, reduced transcriptional activity and Hopx expression, whilst in contrast, subpallial qNSCs remain transcriptionally primed for activation. Induction of deep pallial quiescence is paralleled by a rapid blockade of glutamatergic neuron production and differentiation. Finally, manipulation of the TGFbeta/BMP receptor Bmpr1a demonstrate its key role in mediating these effects at early postnatal times. Together, our results highlight a central role of TGFbeta/BMP-signalling in synchronizing quiescence induction and blockade of neuronal differentiation to rapidly silence pallial germinal activity after birth.