G protein-coupled receptors (GPCRs) are excellent drug targets. Recent data show that GPCRs are not only expressed on the cell surface but also in intracellular compartments where they have important functions. This raises the question of the specific receptor subpopulation to be targeted to achieve a desired therapeutic effect. The melatonin type 1 (MT1) and cannabinoid type 1 (CB1) receptors are two important GPCRs for which there is convincing data of the functional expression in neuronal mitochondria. Melatonin and endocanabinoids are known to have neuroprotective properties. The goal of our project is to study the contribution of mitochondrial MT1 and CB1 receptors to this neuroprotective effect. The project will increase our knowledge on the function of intracellular GPCRs. Development of drugs targeting either surface receptors or intracellular receptors are likely to show reduced side effects of drugs and improved therapeutic efficacy.

Understanding how the brain achieves higher-order cognitive processing, determining the basic neural architecture underlying such processing, and achieving simultaneous recordings of behavioral decisions and neural activity during complex learning tasks are, so far, ambitious and difficult goals for scholars in the field of neurosciences, psychology, and ethology. Recent studies in an insect model, the honeybee Apis mellifera, have uncovered unsuspected higher-order cognitive processing beyond simple associative learning. Despite their miniature brain, freely-flying bees are capable of non-elemental learning, in the form of object categorization and concept learning of varying complexity. The neural substrates mediating these capacities can be thoroughly investigated by means of a variety of invasive techniques if the insects are immobilized. Yet, the missing link between these two levels (behavior and neurobiology) resides in the fact that neural analyses are difficult in freely moving animals. The present project attempts to fill this gap and to provide a comprehensive view of the neural bases of cognitive processing in the miniature brain of honeybees. Our specific goal is to understand how a relatively ‘simple’ brain forms concepts and learn abstract rules. In the last years, several investigations by the applicant have shown that freely-flying bees can learn abstract concepts (e.g. sameness, above of, larger than, etc) in the visual domain. We will use a combination of behavioral and neurobiological methods in order to quantify and manipulate brain activity in tethered bees that, despite movement restrictions, will be able to solve conceptual discriminations. To this end, we will use a locomotion compensator designed for the analysis of visual orientation of bees, i.e. an experimental setup in which tethered bees will be placed on a hollow Styrofoam sphere whose movements, induced by the walking bee, are recorded by optic sensors thereby allowing reconstruction of the walking trajectory of the bee. Visual stimuli surrounding the setup will be reinforced or non-reinforced so that the bee will have to solve visual tasks that will be arranged in the form of elemental or conceptual discriminations (i.e. rule learning). At the same time, we will access the brain of the tethered bees to study the neural correlates of these two learning forms. Neuroanatomical analyses of neuropile volume and synaptic branching will be performed at the level of the optic lobes, central body and mushroom bodies, both in naïve and in conditioned animals subjected either to elemental or conceptual visual discriminations. In this way, we will determine when and where plastic changes occur in the honeybee brain as a consequence of elemental and higher-order visual discriminations. Reversible and selective inactivation of visual neuropiles (optic lobes and central complex) and mushroom bodies will be achieved by local injection of the anesthetic procaine. In this way, the necessity and sufficiency of these structures for elemental and conceptual visual learning will be determined. Multielectrodes will be implanted in the optic lobes and the mushroom bodies to record neural activity upon elemental and conceptual visual learning. Changes in the neural signatures of the stimuli/rules to be discriminated will be quantified in these regions of the bee brain. The combination of behavioral and neurobiological methods will allow determining 1) the neural structures that are necessary and sufficient for solving elemental and conceptual problems in the visual domain, 2) the dynamics of neural activity underlying conceptual learning in bees and 3) the minimal neural circuit required for achieving such higher-order learning. The knowledge gained in this project will be implemented in a neural-based model of conceptual learning that will constitute a fundamental contribution for scholars in the fields of psychology, neurosciences and informatics.

Under selective pressure, many organisms have developed physiological mechanisms allowing them to avoid, and to cope with stress factors. Studying such mechanisms (stress responses) provides a way to understand adaptive mechanisms, as well as an opportunity to use these mechanisms in order to improve resilience. Still, in a rapidly evolving environment under the influence of human activity, animals encounter increasingly diverse sources of stress, with an increasing frequency. In such conditions, the selected stress responses may not be fully adapted, as suggested by the recent decline of honey bee populations. Given their high ecological and economical importance, many studies have helped to identify multiple factors with proved or potential impact on the fitness of colonies and individuals, and have pointed at synergistic effects of natural parasites, xenobiotics, and the impact of intensive agricultural practices on the diversity and quality of food sources. Given such diversity of negative factors, we claim that focus should be put on physiological processes involved in general stress responses rather than specific defence mechanisms, in order to understand - and possibly improve – stress resilience. Recently, we have unravelled a new role for allatostatins in stress responses of the adult honey bee. These peptides, which can act as neurohormones, target many insects tissues involved in adapted responses to many stressors (muscles, brain, digestive tract, fat body). Our unpublished data show that, in response to stress, they also downregulate juvenile hormones, a key regulator of many physiological and behavioural specificities of the foraging behaviour. Since foragers appear particularly vulnerable to environmental stressors, and as juvenile hormone is also an internal stress signal, we propose to launch a research programme aimed at understanding the role of allatostatins and their receptors in the control of stress resilience, particularly in foragers. To that end, we will examine their capacity to regulate many adaptive processes triggered by a variety of stressors, and test the hypothesis that increased expression levels or activation of allatostatin receptors would promote stress resilience. For this purpose, we plan an integrated approach studying molecular, cellular, physiological and behavioural aspects of stress responses, and linking individual and collective adaptations in this social insect. First, we will assess the susceptibility of foragers to stress, then evaluate the natural variation in expression levels of allatostatin receptors between colonies and correlate it with parameters of the stress response. The causal link between activation of the allatostatin pathway and stress resilience will be addressed through two parallel strategies: genetic manipulation of receptor expression by RNAi treatment, and pharmacological treatments with agonists for which a screening will be performed previously. In all cases, we will measure responses to three stressors believed to be primary causes of bee losses: starvation, infection by Nosema ceranae and ingestion of common pesticides (deltamethrin and fipronil), as a way to account for the diversity of biotic and abiotic stress met by this species. By doing so, we expect to provide a significant breakthrough in the assessment and improvement of general stress response, and thus resilience, of honey bees, which may serve as a starting point for future agronomic applications.

Our recent and remote memories define both our identity and the range of our knowledge and capabilities. Before becoming lasting, they undergo gradual consolidation, a process supported by changes at both synaptic and systems-levels. We recently discovered that fiber-sparing lesions of the reuniens and rhomboid nuclei (ReRh), which belong to the ventral midline thalamus, alter spatial memory persistence in the Morris water maze, but neither its initial acquisition nor its recent retrieval (Loureiro et al., J Neurosci 32, 2012). Neuroanatomical and electrophysiological evidence (Cassel et al., Prog Neurobiol 111, 2013, for review) place the ReRh in a hub position between the medial prefrontal cortex (mPFC) and the hippocampus (HIP), with which it has reciprocal connections. Therefore, the ReRh are likely to support functional interactions between the mPFC and the HIP, both believed to be essential to systems-level consolidation and memory persistence. While research on memory consolidation is highly competitive, there is currently no other published work regarding the implication of the ReRh nuclei in spatial memory persistence. Complementary approaches by 4 Partner-laboratories (Strasbourg-UMR7364, Marseille-UMR7291, Toulouse-UMR5169, Bordeaux-UMR5287) will decipher the role played by ReRh in memory persistence. Our objectives are the following: a) the establishment of a spatial memory requires accurate early information processing (encoding, hippocampal-triggered consolidation…); when inaccurate, it could lead to non-lasting memories. In addition, optimal memory performance requires an accurate use of the memory during goal-oriented navigation. Therefore, electrophysiological recordings of place cells in the HIP and of goal-encoding cells in the mPFC will be performed in rats with permanent, fiber-sparing ReRh lesions; b) we will examine how the ReRh is functionally connected with both the HIP and the mPFC to promote spatial memory persistance and/or retrieval. To do so, we will combine tract-tracing, including state-of-the-art viral-mediated monosynaptic tracing, and cellular imaging techniques; c) retrieval of remote memory is accompanied by an engagement of the mPFC. This engagement could be disrupted after ReRh lesions, which effects will be investigated using immediate early-gene expression (c-fos, zif268, arc); d) consolidation is supported by neuronal network remodeling which implies formation of new synapses in the mPFC when memories become remote. Thus, recent and remote memory-related synapse formation will be assessed in the mPFC and HIP after ReRh lesions; e) systems-level consolidation seems to involve adult-generated hippocampal neurons. As ReRh projections to the HIP (on CA1) could participate in the regulation of hippocampal neurogenesis or/and in the recruitment of newly formed neurons, the impact of ReRh lesions on neurogenesis and new neurons maturation will be explored (preliminary results suggest an impact of ReRh lesions on the maturation of new neurons); f) consolidation and memory persistence is supported by epigenetic regulations such as histone acetylation on chromatin. As ReRh lesions could disrupt such regulations, their epigenetic effects will be investigated in the mPFC and the HIP. When relevant, the aforementioned ReRh lesion-induced changes will be assessed under baseline and spatial learning conditions. This innovative project aims to unravel the mechanisms by which the ReRh nuclei of the ventral midline thalamus do indeed contribute to spatial memory persistence. Our approach could point to these nuclei as an ideal target for deep brain stimulations in future preclinical investigations aiming to improve memory functions after brain disease.

How pollinators, such as bees, exploit resources in their environment is a fundamental question in biology with deep biological, ecological and societal implications. When foraging, bees transfer pollen between flowers which mediates the reproduction of plants, a pollination service on which most plants and animals (including humans) rely on. Studying the movements and interactions of bees at the scale of landscapes is a vital scientific challenge to understand the functioning and maintenance of terrestrial ecosystems. My ERC project (BEE-MOVE) aims at unravelling the mechanisms by which the movements of pollinators shape the reproduction patterns of plants over large spatial scales, through a detailed study of bee behaviour and pollen flow in the field. To achieve this goal, I will develop a new automated tracking methodology for combining robotic plants and radars to track bees in the field. This Tremplin – ERC project will demonstrate the validity of the methodology. I will develop a prototype of the tracking system and accumulate preliminary data on a model species: the buff-tailed bumblebee. The validation of the methodology will demonstrate the feasibility of the experiments planned in my ERC project. Beyond the exploration of new grounds in pollination ecology (by integrating research on pollinator behaviour and plant reproduction), my research may help improve conservation plans, practices in agro-ecology, and green development, in the alarming context of widespread pollinator declines.