Tool use behaviors have evolved not only in primates but also in some birds (e.g. parrots and crows) and even in some teleost fish (e.g. wrasses and cichlids). As birds and teleosts do not have a six-layered neocortex, these similar cognitive functions must have evolved independently in the lineages of mammals, birds, and teleosts (convergent evolution). Our research project aims to identify the necessary conditions for the emergence of such cognitive abilities during vertebrate evolution. The ability for problem solving and sequential object manipulation is the prerequisite condition for evolving tool use ability. The EVONECTOME project investigates the connectivity (TASK 1) and functions (TASKs 2-4) of associative brain areas involved in a problem solving task and sequential object manipulation in parrots and cichlids. We propose to use Pyrrhura molinae as a parrot model and Amatitlania nigrofasciata as a cichlid model, which are optimal for combining anatomical and behavioral examinations. TASK 1: We will take a connectomic approach using ex-vivo diffusion MRI to visualize neuronal networks with regard to putative associative areas in the pallium. The main goal is to reveal the connectivity of the associative areas, notably with premotor/motor areas. The results will verify whether there is a shared network logic for cognitive-motor integration in primates, parrots, and cichlids. TASK 2: To examine problem solving and object manipulation abilities of parrots and cichlids, we will perform two different behavioral tasks: a puzzle box task and operant conditioning tasks that require object manipulation as responses. After the behavioral tests, the brain areas involved in these behaviors will be examined in TASK 3 and TASK 4. TASK 3: Neuronal activation during the behavioral tasks will be visualized by functional MRI, using Manganese-enhanced MRI (MEMRI). By detecting large-scale brain activity during the behavioral tasks (TASK 2), we expect to identify brain areas involved in problem solving and object manipulation. TASK 4: To assess the role of dopamine (DA) neurotransmission in the behavioral tests, we will destruct DA fibers in the associative areas by a local injection of 6-hydroxydopamine (6-OHDA). DA is known to play a critical role in various higher-order cognitive functions in mammals. If DAergic disruption in non-homologous brain areas leads to the same behavioral effects in mammals, birds, and teleosts, this will further support the importance of DA in the convergent evolution of higher-order cognitive functions. Altogether, our study will give an insight into how morphologically diversified nervous systems achieved similar cognitive functions during evolution. If the same network pattern emerged independently in primates, parrots, and cichlids, this would indicate the existence of a limited degree of biological freedom (high constraints) for the evolution of intelligence in vertebrate brains.

During our daily life, we use a variety of gradually learned motor skills, such as riding a bike, playing guitar, or even speaking. Speech is a fundamental behavior acquired through sensorimotor learning during which a baby discovers the sets of action to execute for saying a new word through motor exploration and perception of her own actions. Previous research indicates that the basal ganglia (BG) are a likely candidate which could evaluate the discrepancy between expected and actual outcome and guide trial-and-error learning. While the BG drive behavioral adaptations that minimize errors early during learning, these adaptations are slowly incorporated in the downstream premotor cortical networks. It was proposed that the BG, through its subcortico-cortical loop, stabilize these adaptations by training premotor cortical areas. Here, we hypothesize that BG-cortical stabilization might occur during sleep through active processes, such as coordination of large neuronal assemblies between BG-cortical areas, which is a putative neurophysiological substrate allowing the long-term imprinting of recently acquired skills. To disentangle the BG-cortical dynamics during motor skill learning and the impact of sleep on these dynamics, we propose to use the songbird model and study the acquisition of their song learning behavior. Song learning is a natural form of sensorimotor learning akin to human speech acquisition during which a juvenile learns his song by imitating an adult bird (a tutor). Sleep in songbirds include mammalian-like features and is crucial for song learning. Moreover, songbirds have a set of interconnected brain nuclei dedicated to song, including a BG-thalamo-cortical loop, and that shares analogies with brain areas involved in human language. Songbirds thus represent an outstanding animal model for understanding the neurobiological bases of motor skill learning and the role of sleep. We hypothesize that sleep plays a critical role in the dynamical reorganization of the BG-cortical network and the consolidation of song. We expect specific events during the song learning stage (songs produced by the juveniles, exposure to the tutor song) to impact offline activity within the BG-cortical network. Offline activity, such as synchronized slow waves or replay bursts over the whole network could consolidate BG-driven adaptation in song in premotor nuclei. We thus expect important changes of coordinated activity within neuronal assemblies of the BG-cortical network during song learning. High-frequency oscillations, similar to mammalian ripples, occur during sleep in the songbird BG and neurons active during these oscillations are also active during singing. We thus hypothesize that offline oscillations contribute to song learning. We will perform longitudinal experiments, monitoring daily changes in the BG-cortical dynamics at the neuronal level and relate it with song acoustic features and song learning performance. We will perform large-scale simultaneous extracellular recordings across the BG and cortical nuclei. We will run experiments to establish cause-effect relationships between epochs of BG activity and song learning performance by chronically disturbing high-frequency oscillations in sleeping birds and assess their impact on birdsong learning We will thus reveal how synchronization between various neuronal populations involved in sensorimotor learning during sleep underlies consolidation in skill learning. The proposed research is at the interface between relevant basic biological issues for understanding sleep-related neurobiological processes at play during learning complex motor skills and pathological issues for assessing the impact both at the neuronal and behavioural level of the alterations of these processes.

Inherited retinal dystrophies (IRDs) are genetically and clinically heterogeneous. Despite tremendous progress in IRD research over the past 10 years, these diseases still lead to legal blindness due to limited therapeutic options. The recent success of gene supplementation therapy in patients with Leber Congenital Amaurosis (LCA) caused by RPE65 mutations has highlighted the real interest in expanding this approach to other IRDs. Our previous work suggests that diseases due to dominant mutations in CRX, a transcription factor essential to photoreceptor development and maturation, are relevant candidates for gene supplementation. To date, over 50 mutations in the CRX gene are responsible for cone-rod dystrophies (CORD), LCA, and retinitis pigmentosa (RP). Due to the clinical heterogeneity of patients carrying CRX mutations, our proposal aims to restore and/or maintain vision in two mouse models of retinopathies associated with CRX: an LCA model that we have already well characterized, and a novel CORD model. In this context, we will evaluate whether a gene replacement approach is able to compensate for the deleterious effects of CRX mutants for both clinical forms. Towards this aim, a CRX-expressing AAV vector will be administered, and its long-term efficacy evaluated, in both models. The recovery and preservation of retinal function will be tested by electroretinogram and optokinetic reflex recordings, and retinal morphology by optical coherence tomography. This efficacy study will be completed by histological and molecular analyses. Furthermore, in order to better understand disease pathophysiology and to validate a therapeutic approach in humans, we will complement these in vivo studies on iPSC-derived retinal organoids generated from three patients with CRX-associated LCA, CORD, or RP. By analyzing differential gene and protein expression, as well as the morphology of the organoids throughout retinal differentiation and maturation, we will establish a genotype-phenotype correlation and, hence, elucidate the basis for the differential clinical profiles associated with each mutation. Furthermore, we will obtain pertinent read-outs to assay the efficiency of an AAV-mediated gene replacement following the transduction of the patient organoids. In this way, we will determine whether all forms can benefit from our therapeutic approach, without negative effects linked with CRX overexpression. At the end of this preclinical study, our expectation is to have demonstrated the efficacy of our gene therapy in mouse and human models with dominant CRX mutations. The potential clinical value of this project lies in the development of gene therapy to treat a broad spectrum of CRX-associated retinopathies and might serve as proof of concept for other dominant IRDs. This study may serve as a basis for further development of clinical trials.

In mammals, water balance is maintained by physiological processes such as appetite for salt or thirst, as well as by renal secretion or reabsorption of Na+ (natriuresis) or water (diuresis) for which the neuropeptides vasopressin (VP) and oxytocin (OT) play crucial roles. The latter are centrally produced in paraventricular (PVN) and supraoptic nuclei (SON) of the hypothalamus. These nuclei are essential in hydromineral balance regulation as they are able to sense osmotic changes in extracellular fluids on the one hand, and receive inputs from other fluid imbalance sensing areas of the lamina terminalis circumventricular organs devoid of blood brain barrier on the other hand. The hypothalamus is thus an important hub integrating osmotic changes throughout the brain and respond by regulating release of neuropeptides from VP and OT neurons via the neuro-hypophysal system. Neuro-glial interactions play a key role in brain functions, particularly in regions strongly connected to blood flow and regulating vital physiological functions such as the hypothalamus. In this regard, astrocytes sense and integrate changes in blood composition as well as in neuronal activities. Accordingly, recent studies point at astrocytes as primary sensors of osmotic changes in the brain. In spite of this primary physiological importance, our knowledge on the osmoreceptors expressed in astrocytes remain limited, in particular with regard to dehydration/hyperosmotic stress. One line of research regards their ability to respond to activation of mechanosensitive channels by eliciting cytosolic Ca2+ elevations that trigger morphological changes leading to modulation of VP and OT neurons activity. In this regard, lysosomes are underexplored local source of Ca2+ that are involved in astroglial activation. Most interestingly, recent discoveries support that endolysosomes act as osmosensors as there are highly dynamic intracellular compartments that adapt their volume in response to cell swelling or shrinking induced by osmotic challenges. Recently, we have identified that lysosomal Ca2+ permeable two-pore channels (TPC) act as key components in the control of VP and OT secretions. Our preliminary data further show that TPCs are involved in hyperosmolarity-induced modulation of VP/OT neurons activity via a mechanism that remain to be elucidated. Since our current data indicate that TPC partake in hypothalamic astrocytes Ca2+ elevations, we hypothesize they are key contributors to the mechanosensitive machinery allowing astroglial osmosensitivity by driving Ca2+ signals triggering regulatory neuro-glial pathways. To assess this posit we propose to: 1. Characterize in vitro the distribution and mechanosensitive functions of TPC in isolated astrocytes. To do so, we will perform high resolution cell imaging and evaluate the mechanosentivity of endolysosomal channels with an innovative electrophysiological technique developed by C Grimm. 2. Identify in vivo the implication of astroglial TPC in VP/OT-driven physiological control of water balance, water intake and the associated mechanisms. To this end, water intake will be evaluated together with the in vivo imaging of OT and VP release under conditions of hyperosmotic stress in mice deleted for astroglial TPCs. In all, the current project will provide unprecedented information on the identity of the osmo/mechanosentive machinery at play in the control of the hydromineral balance.

Mutations in genes belonging to the RHO GTPase pathway are responsible for intellectual disability (ID), psychiatric disorders and brain development anomalies. The great heterogeneity of phenotypes associated with these gene mutations renders the development of therapeutic strategies strenuous. Studying PAK3, a central gene of the RHO GTPase pathway, will help us establish a genotype/phenotype correlation, which is essential to 1- define the rules behind mutation pathogenicity, 2- understand the underlying mechanisms and 3- propose adapted therapeutic approaches. 1- Our project is to define the genotype/phenotype correlation using about 20 different PAK3 mutations in order to understand the origin of PAK3-linked ID degree of severity, as well as why ID may sometimes be associated with other neurodevelopmental defects. Thus, we will establish and characterise the broadest cohort of patients bearing PAK3 mutations ever built. In parallel, we will assess the functional defects of mutated PAK3 variants and their effects on cell biology (shape, adhesion, migration) as well as neuron differentiation (neurite growth, dendritic spine formation). Our hypothesis states that mutation pathogenicity is not simply a loss or gain of function but may involve more complex mechanisms of signalling interference. Indeed, the presence of a mutated protein is often more deleterious than the lack of a protein. 2- To go further in analysing the severe forms of PAK3-linked ID, we created a new knock-in model bearing a mutation clinically responsible for a severe ID associated with secondary microcephaly. This mouse model presents strong behavioural and cognitive anomalies, as well as secondary microcephaly, reminiscent of the clinical case. Our project consists in a more thorough analysis of the mouse model behavioural and cognitive defects, in order to compare our results with the patient’s clinical traits. Our ex-vivo and in-vitro preliminary analyses allowed us to propose a new molecular mechanism of mutation pathogenicity, which we will investigate thoroughly. 3- We will test two phenotypical rescue strategies with the aim of further developing therapeutic solutions. The first strategy concerns severe forms of the disease. The degradation of stable pathogenic PAK3 proteins should, at least partially, restore phenotypic anomalies usually associated with severe ID. This strategy of specifically degrading stable pathogenic variants was never explored in the context of neurodevelopmental disorders, even while it is being developed as potential cancer treatment. It would also be applicable to over-activating mutations in genes belonging to the RHO GTPase pathway. The second rescue approach targets Cofilin, a convergence point of the RHO GTPase pathway. Several strategies targeting this actin polymerisation regulator were already explored to rescue behavioural anomalies and synaptic plasticity defects. We aim to demonstrate that this approach would also correct neuronal differentiation anomalies appearing during post-natal development. Thus, the efficiency of a cofilin-blocking peptide to restore neuritic arborisation and dendritic spine formation in mutated mice will be evaluated. This project is based on strong preliminary results and an already operational consortium composed of 2 clinician teams and 3 research teams (1 team being knowledgeable in the two fields). This project will allow us to understand the genotype/phenotype relations regarding PAK3 gene mutations as well as mutations on other genes belonging to the RHO-GTPase pathway. Our results will greatly help advance genetic counselling and patient monitoring. The post genomic and preclinical aspects of this project will also enable us to pave the way for new therapeutic approaches in the optic of personalised medicine.