Periodic Reporting for period 1 - MoThal (Functional exploration of the contributions of brainstem-motor thalamic pathways to motor execution and learning) Reporting period: 2018-01-01 to 2019-12-31 Summary of the context and overall objectives of the project The ability to learn and to adapt a motor program is of fundamental importance to all species, since it enables performance of complex movements, but also to adapt these learned movements to perturbation in the environment. However, the circuits involved in these processes are poorly known. An important brain structure in this context is the cerebellum, and in particular the deep cerebellar nuclei (DCN), which are the sole output of the cerebellum. Interestingly, the DCNs project strongly to the brainstem and to the motor thalamus, two major structures in motor function. Indeed, premotor brainstem areas with projections to the spinal cord are a major locus needed for the control of movement. A recent study has shown that different brainstem nuclei connect to motor neurons innervating forelimb and/or hindlimb muscles in very specific patterns. For example, the manipulation of glutamatergic neurons in the medullary reticular formation ventral part (MdV) in mice led to specific deficits in the grasp behavior in a motor learning task, the single-food-pellet retrieval task. This same manipulation also led to deficits in an accelerating rotarod task, another motor learning task. Therefore, these results begin to suggest that specific brainstem nuclei have functional contributions to a specific aspect of a movement sequence. By their direct impact on an ongoing movement, the brainstem nuclei seems to represent unavoidable actors in the learning and adaptation of movements.On the other side of the fence is the Mthal, a ventral structure of the thalamus composed of several nuclei. These nuclei received strong projections from DCNs, and project differentially to motor cortical areas. The research on Mthal nuclei is very limited, mostly due to technical limitations needed to surmount the complexity of the intricate multi-nuclei organization of Mthal. Nevertheless, some lesion studies have shown a major contribution of Mthal in motor learning, in particular the target-reaching task and rotarod task, mirroring effects of transient manipulation of brainstem nuclei in mice in similar tasks. As such, these data suggest a complementary role of the Mthal to the brainstem in the learning and adaptation of the movement. It is therefore the purpose of this present project to elucidate the specific role of the DCNs to brainstem and DCNs to Mthal pathways in the aforementioned motor function. 3 major objectives have been set: 1, to study the anatomical connectivity between the DCNs, the brainstem and the Mthal. Not only this will bring new fundamental knowledge about poorly studied pathways, but it will also enable us to postulate and test a hypothesis on the role of a DCN-brainstem-Mthal pathway identified in motor execution adaptation. To probe these hypotheses, the 2 objective will consisted to correlate the activity of these pathways when mice is performing motor tasks, by means of in vivo calcium imaging and electrophysiological recordings. 3, the functional role of the different brainstem-Mthal pathways will be assessed through circuit manipulation and loss of function. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Regarding the 1 objective several experiments have been done to describe the network between the DCNs, the Mthal and the brainstem. Injection of retrograde and anterograde tracers in the different brain regions have enabled to draw a map of projections between the 3 structures. The major finding is that neuronal populations inside the DCNs send specific projections to subregions of the brainstem and the Mthal. The anatomical analysis for the retrograde experiment consisted in schematic reconstructions of neuronal populations at the level of the DCNs, with count of the total number of neurons in each DCNs regions. Regarding the anterograde experiment, schematic reconstructions of the synaptic population (originating from DCNs projections) at the level of the brainstem and the Mthal have been made. The following experiment showed that single neurons in DCNs send collaterals to both the Mthal and the brainstem. Two experiments have been realized to come to this conclusion. First, two distinct retrogrades tracers have been injected in the Mthal and the brainstem, and the number of neurons expressing both tracers in the DCNs has been recorded. The second experiment consisted in a first injection of a retrograde virus in the brainstem. At the level of the DCNs, a second virus has been injected, for which his expression depends on the presence of the first injected virus in the same neurons. In that case, the second virus will express a synaptic tag, enable to see if the infected DCN neuron, projecting to the brainstem, send collaterals to the Mthal as well.These previous experiment identified the intDL, a nucleus of the DCNs, as an interesting candidate to focus on. Indeed, it sends projections to both the Mthal and the PCRt, a region of the brainstem. Because of his projection on the PCRt (see Esposito et al., 2014), the intDL-Mthal-PCRt pathway could be involved in forelimb movements. Moreover, the pathway from the intDL to the Mthal could transmit a copy of the motor command send to the PCRt to the motor cortex. To bring first confirmations of this hypothesis, two different viral strategies have been used in the mice, to show that the IntDL send projections to the thalamocortical neurons. Both experiments confirmed that hypothesis.These anatomical experiments showed that the DCNs send projections from specific neuronal subpopulations to distinct regions inside the Mthal and the brainstem. Moreover, these DCNs populations displayed collaterals projecting to both the Mthal and the brainstem. Inside the DCNs, the IntDL represents an interesting candidate to focus on because of his projections on the Mthal and the PCRt. Finally, the IntDL could send motor information to the motor cortex through the Mthal.Concerning the 2 and 3 objectives, only preliminary results have been obtained. Electrophysiological recordings of the intDL and the Mthal have shown a neuronal activity while mice were performing a forearm tasks, the single pellet reaching tasks. However these first results need to be confirmed on a larger population, and carefully analyzed. Optogenetic experiments have also been realized, consisting in activating acutely the different brain structures. Mthal activation didn’t trigger any movement, but the activation of the intDL led to a retraction of the arm and the closing of the fist. Moreover, the activation of IntDL terminals in the PCRt resulted in distinct arm movements, depending on the position of the fibers on the anteroposterior axis of the PCRt.These works have enabled to draw a map of the connexion between the DCNs, the brainstem and the Mthal. Preliminary functional results indicated a possible implication of the intDL-Mthal-brainstem pathway in the forelimb movements. Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far) These results bring a better comprehension of the connectivity between the DCNs, the Mthal and the brainstem. They able to postulate new hypothesis regarding the way that a motor program can be learned and adapted.This tripartite organization could suggests that the DCNs send a motor command to the brainstem to perform or adjust an ongoing motor program.Through the collateral to the Mthal, a copy of this motor command can be send to the Mthal and the motor cortex, for further processing. From a larger perspective, this project could lead to a better comprehension of how a motor program is learned and adapted.