SUMMARY A mechanistic understanding of neural computation requires determining how information is processed as it passes through neurons and across synapses. However, it has been challenging to measure membrane potential changes in axons and dendrites in vivo. We use in vivo, two-photon imaging of novel genetically encoded voltage indicators, as well as calcium imaging, to measure sensory stimulus-evoked signals in the Drosophila visual system with subcellular resolution. Across synapses, we find major transformations in the kinetics, amplitude, and sign of voltage responses to light. We also describe distinct relationships between voltage and calcium signals in different neuronal compartments, a substrate for local computation. Finally, we demonstrate that ON and OFF selectivity, a key feature of visual processing across species, emerges through the transformation of membrane potential into intracellular calcium concentration. By imaging voltage and calcium signals to map information flow with subcellular resolution, we illuminate where and how critical computations arise.

Highlights d For population analyses, spike sorting has a minor impact on estimates of neural state d Multiunit activity creates a random projection of the lowdimensional neural state d Results from three prior studies in macaque PMd/M1 are replicated without spike sorting d Neuropixels probes provided high-quality unit isolation to validate findings

Animals have a remarkable capacity to learn new motor skills, but it remains an open question as to how learning changes neural population dynamics underlying movement 1 . Specifically, we asked whether changes in neural population dynamics relate purely to newly learned movements or if additional patterns are generated that facilitate learning without matching motor output. We trained rhesus monkeys to learn a curl force field 2 task that elicited new arm-movement kinetics for some but not all reach directions 3,4 . We found that along certain neural dimensions, preparatory activity in motor cortex reassociated existing activity patterns with new movements. These systematic changes were observed only for learning-altered reaches. Surprisingly, we also found prominent shifts of preparatory activity along a nearly orthogonal neural dimension. These changes in preparatory activity were observed uniformly for all reaches including those unaltered by learning. This uniform shift during learning implies formation of new neural activity patterns, which was not observed in other short-term learning contexts [5][6][7][8] . During a washout period when the curl field was removed, movement kinetics gradually reverted, but the learning-induced uniform shift of preparatory activity persisted and a second, orthogonal uniform shift occurred. This persistent shift may retain a motor memory of the learned field 9-11 , consistent with faster relearning of the same curl field observed behaviorally and neurally. When multiple different curl fields were learned sequentially, we found distinct uniform shifts, each reflecting the identity of the field applied and potentially separating the associated motor memories 12,13 . The neural geometry of these shifts in preparatory activity could serve to organize skill-specific changes in movement production, facilitating the acquisition and retention of a broad motor repertoire..

Calcium imaging is a powerful tool for recording from large populations of neurons in vivo. Imaging in rhesus macaque motor cortex can enable the discovery of fundamental principles of motor cortical function and can inform the design of next generation brain-computer interfaces (BCIs). Surface two-photon imaging, however, cannot presently access somatic calcium signals of neurons from all layers of macaque motor cortex due to photon scattering. Here, we demonstrate an implant and imaging system capable of chronic, motion-stabilized two-photon imaging of neuronal calcium signals from macaques engaged in a motor task. By imaging apical dendrites, we achieved optical access to large populations of deep and superficial cortical neurons across dorsal premotor (PMd) and gyral primary motor (M1) cortices. Dendritic signals from individual neurons displayed tuning for different directions of arm movement. Combining several technical advances, we developed an optical BCI (oBCI) driven by these dendritic signalswhich successfully decoded movement direction online. By fusing two-photon functional imaging with CLARITY volumetric imaging, we verified that many imaged dendrites which contributed to oBCI decoding originated from layer 5 output neurons, including a putative Betz cell. This approach establishes new opportunities for studying motor control and designing BCIs via two photon imaging.