Pharmaco-genetic and opto-genetic analysis of brain microcircuits controlling sleep and arousal. – NEUROGEN

Neurological disorders have an increasing economical and societal impact in developed countries. For example, recent research studies evaluated that more than 200 million individuals are affected by sleep disturbances. A better understanding of brain microcircuits function and the development of innovative techniques to control cerebral activity are both necessary to design new therapeutic strategies. This project aims at developing novel opto-genetic and pharmaco-genetic tools to analyse and manipulate brain microcircuits controling sleep and arousal. The medial thalamus is located in a strategic position, at the core of a neuronal communication highways involving the brainstem, the basal ganglia and the frontal cortex. Deafferentation of the central thalamus is the more frequent cause of long-lasting loss of consciousness following severe brain injuries. In addition, the medial thalamus is closely associated with several pathological mechanisms producing impairments of arousal regulation. The medial thalamus is constituted by several higher order, associative nuclei, the intralaminar and the mediodorsal nuclei. Preliminary experiments from the participant laboratories have shown the existence of a mixed GABAergic/glycinergique innervation to the intralaminar nuclei. Medial thalamus is the only subdivision receiving a dense glycinergic innervation. This specificity is noticeable both from a functional point of view and as a substrate for pharmacological and genetic manipulations. Glycinergic innervation arises from a nucleus of the lower mesencephalic reticular formation, the nucleus pons oralis, or PnO, whihch has recently emerged as a major regulatory locus for the regulation of active sleep and of arousal states. Our project aims at elucidating the function of the microcircuit comprising the PnO/medial thalamus/prefrontal cortex. To this end the french and hungarian members of the consortium will develop innovative opto- and pharmaco-genetic tools, making possible the activation of specific neuronal populations in a fast and reversible manner, and the examination of their role in the circuit. By using transgenic mouse lines, and viral vectors for the insertion, the deletion and the modification of desired genes, optic tools of stimulation, like the light-activated ion channel channelrhodopsin 2, will be expressed specifically in the glycinergic neurons of the PnO. Similarly, the GABAergique inhibition from the PnO will be either specifically potentiated, or completely shut off, in order to understand its role in the regulation of arousal states performed by the medial thalamic nuclei. The analysis of the opto- and pharmaco-genetic tools will be first performed at the morphological level, with electromicroscopical, tracing and immmunohistochemical techniques. At cellular level in acute slices, the cellular and synaptic physiology of the PnO and of the medial thalamus will then be studied via electrophysiological and 2 photon-based calcium imaging techniques. Finally, neuronal activity will be examined in-vivo in the context of wholly intact circuits, and the consequences of the optical and pharmacological stimulations on physiological states will be identified. Throughout the project, this experimental activity will be supported by pharmacological and structure/function studies of molecules involved in the circuit under study. Particular attention will be shed onto atypical, glycine-activated NMDA receptors, which were shown to be functionally active in the PnO/medial thalamic circuit in preliminary experiments. The primary goals of this proposal are, thus, to dissect the function of a complex brain circuit, central to the regulation of sleep and arousal states, and to determine selective pharmacological targets in this circuit. The development of the desired genetic tools, and their optimization for in-vivo applications, will be another milestone of the project. This proposal is, indeed, one of the very first ones based on the in-vitro and in-vivo application of opto- and pharmaco-genetic tools, tools that hold great promise for a major leap in the domain of integrative neuroscience studies.