ALL-OPTICAL FUNCTIONAL MAPPING OF THE FORELIMB MOTOR CORTEX REVEALS TWO DISTINCT GRASPING CORTICAL REPRESENTATIONS

The anatomical and functional organization of the motor cortex and its role in forelimb movement control were deeply investigated over the past years. Extensive research showed two spatially segregated functional areas related to forelimb control: the caudal forelimb area (CFA) and the rostral forelimb area (RFA). Many studies suggest that these two areas are a part of a highly integrated computational unit with distinct motor functions. Recently, optogenetic motor mapping revealed that distinct complex movements are related to segregated cortical functional modules. Although studying optogenetically-evoked behaviors already provides a powerful way to investigate the neuronal pathways related to motor output, decoding the functional engagement and interdependence of cortical motor circuits are still two largely unexplored field. The “all-optical” interrogation of neuronal circuits constitute a successful strategy to causally dissect the functional organization of the motor cortex, since it combines optogenetics and optical indicators to simultaneously record and manipulate the activity of selected neuronal populations using light. During my Ph.D., I contributed to develop a one-photon all-optical strategy to causally investigate the neuronal activity patterns in RFA and CFA driving optogenetically-evoked complex movements in awake head-fixed mice. To this aim, we first examined four different red-shifted Genetically Encoded Calcium Indicator (GECI) , identifying jRCaMP1a as the best indicator for detecting in vivo neuronal activity on multiple cortical areas simultaneously. Then, we combined the widely used blue-sensitive opsin, ChannelRhodopsin2 (ChR2) with jRCaMP1a for detecting in vivo large-scale stimulated cortical dynamics. Once we demonstrated that our one-photon all-optical approach was cross-activation free, we exploited it to causally investigate RFA and CFA cortical activity patterns associated with two optogenetically-evoked complex movements, the grasp-like and the locomotion-like movement. We showed stereotyped and reproducible spatiotemporal propagation patterns of calcium dynamics per movement category highlighting a direct contribution of defined patterns to complex movement execution. Furthermore, we demonstrated that movement-specific cortical activity maps were bounded on discrete function modules centred on the related light-based motor maps, providing clear evidence of their independent functional organization. Importantly, the visualization of the cortical activity elicited by optogenetic stimulation allowed us to identify a third cortical functional module evoking grasp, which is characterized by segregated large-scale cortical dynamics. We named it Lateral Forelimb Area (LFA). In conclusion, our one-photon large-scale all-optical system led to a robust classification of the connectivity, independence, and hierarchy of three functional cortical regions involved in performing evoked complex movements. The results obtained during my Ph.D. provide important insights on the physiological interplay of brain activity and motor control which could be further applied to the investigation of the altered cortical activity patterns in pathological conditions.