Periodic Reporting for period 4 - Space and Motion (Genetics and function of neuronal circuits controlling goal oriented movements) Reporting period: 2020-07-01 to 2021-06-30 Summary of the context and overall objectives of the project Reaching for a cup of coffee or a pen on the desk as well as any object in our surrounding is a simple but fundamental ability that we all share and that, to a large extent, we perform effortlessly. Conditions that impair our motor abilities and impact on such basic motor skills put tremendous strain on the life of individuals. Albeit apparently simple, these actions are composed by multiple simpler steps that range from the cognitive endeavour of choosing a particular target for our actions, to that of coordinating a large number of muscles to produce a final movement that allows us to interact with the target of interest. Our work aims at understanding the neural encoding of such target directed actions with particular focus on the encoding of their metric (i.e. the direction of these movements). To this aim we focus our work on mice and we have chosen to analyse the neural underpinning of target directed head movements as these can be seen as the ethological equivalent of primates’ hand movements. We want to understand what brain networks are involved in the planning and executions of such actions and how the metric of these actions is encoded. Furthermore, we want to understand what neuronal populations are implicated in these processes and define their genetic identity. Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far Understanding how circuits controlling action in space are organised requires the development of tools that allow us to reveal these circuits and manipulate them.Hence, the first part of our work consisted in the development of a novel method to trace and manipulate neural circuits. We designed a self-inactivating Rabies virus (SiR), which retains the ability to jump synapses,but does not cause neuronal death. During the initial viral infection, the neurons in the network are permanently labelled and genetic manipulation is made possible. The virus can modify the neurons so they constantly produce a fluorescent protein, allowing the network to be visualised. Crucially, we modified the virus by adding a tuneable switch – the SiR contains selected proteins which can be targeted for degradation. Without these proteins, the virus is unable to replicate and switches off after the initial infection, thus avoiding death of the neurons and leaving them permanently labelled but virus-free. This will be important at a basic science level to understand how the brain works, and excitingly will also offer insight into how neural networks change upon experience and learning. In addition, this new technique will pave the way to the engineering of the genetic content of selected neurons within a network, which would be extremely useful in neurodegenerative and psychiatric disorders where disease genes could be targeted, with therapeutic potential. Ref. ((Ciabatti et al., 2017; Ciabatti et al., 2020; Lee, Ciabatti et al., in press).We then began to use these tools to dissect circuits controlling goal-oriented actions. We found that the superior colliculus, a region of the midbrain, is where three-dimensional motor space is mapped, and importantly we show how the activity of neurons in this brain region leads to controlled spatially targeted movement. As mice primarily interact with their surroundings by orienting their head, rather than moving their limbs or eyes towards targets, we developed a lightweight head-mounted inertial sensor system to record rotations of the head in three-dimensional space. This system was paired with electrophysiological recordings to determine whether neural activity was related to head movements and whether a map of motor space exists within the superior colliculus. The findings revealed that neurons in the superior colliculus of mice code for head rotations around any of the three rotational axes (the yaw, pitch and roll axes). Neurons whose activity was motor-related showed a preference for a particular angle and direction of rotation, while increased activity of these motion-tuned neurons further modulated the speed of head rotations.These results provide the first evidence of a three-dimensional representation of motor space in the superior colliculus and give new insights into the mechanisms underlying the control of spatially-targeted movement. This finding suggests that neurons in the superior colliculus not only code for the direction of movement, but also the speed depending on the demands of the task.By studying the neural representation of spatially-tuned actions in mice, this work also opens up a door to the study of motor-space representations at a genetic level. This will allow for a better understanding of how networks of neurons represent the relationship between space and motor behaviour. Ref. (Wilson et al., 2018)Following up on this work, we then defined the genetic identity of neurons responsible for the selection of spatially tuned actions and discovered an unexpected anatomical organization of these neurons. Using an arrays of different methodological approaches from in vitro preparations to freely moving mouse models, we dissected the properties, tuning and circuit organisation of neurons controlling the metric of head movements. We identified a neuronal population organised in clusters tiling the entire SC, forming a grid of discrete spatial modules. These modules are responsible for the execution of head movements whose amplitude vary topographically with the activated module. Our work reveals for the first time a motor map for head movements, begins to address the functional relevance of this SC modularity and paves the way for future experiments to investigate principles of sensorimotor integration in SC circuits. Ref. (Masullo, Mariotti et al, 2019).Finally, we moved on to characterise the logic governing the sensory motor alignment in the SC. Sensory-guided behaviours require the transformation of sensory stimuli into goal locations in egocentric space in order to elicit an appropriate motor action. In order to understand the nature and logic of sensory-motor integration, intracellular and extracellular recordings were performed across the SC to assess both the motor and the sensory tuning of individual neurons. Whilst traditional models of sensory motor alignment have centred on the mapping between static spatial features, such as stimulus location and movement endpoints, we show that motor tuned neurons in the deep layers of the superior colliculus respond primarily to kinetic visual features, and reveal the existence of an alignment in vectorial space between the encoded sensory flow and movement vectors rather than between visual receptive fields and movement endpoints as previously hypothesised (Reuda et al., in preparation). 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) Multiple aspects of our project pushed the field way beyond the state of the art. This is particularly evident both in Wilson et al., 2018 as well as in Ciabatti et al., 2017. In the former we developed a completely novel method for tracking motion in 3d using a full sensor based approach. For the latter, the development of Self Inactivating Rabies allows, for the first time, the functional manipulation of neural networks without any time constraints. These works pave the way to the genetic dissection of brain wide circuit for targeted head motion. Indeed, we have achieved for the first time achieve a fairly complete genetic dissection of the collicular populations involved in the control of the metric of head motion. Modular organization of the neurons responsible for the selection of goal-oriented actions in space.