High speed voltage imaging of Purkinje cells for the interrogation of cerebellar circuit dynamics - PROJECT SUMMARY Understanding how neuronal circuits give rise to behavior requires recording their activity at a high temporal and spatial resolution with cell type specificity. This is particularly critical in the case of the cerebellum, which in addition to being essential for effective control of movement, has ever emerging roles in cognitive functions and neurological disorders. However, such recordings have historically been challenging, due to the complex and conserved cytoarchitecture of the cerebellar cortex, and the firing properties of its primary output neurons, Purkinje cells, which exhibit two distinct activity modalities: simple spikes and complex spikes. Optical interrogation of Purkinje cell Ca2+ can report complex spike events due to their low frequency (0.5-2Hz), but the slow kinetics of Ca2+ indicators make them unviable for resolving simple spikes. Conversely, while microelectrode arrays offer high temporal resolution, they provide limited spatial information. Here, we propose to leverage ultrasensitive genetically encoded voltage indicators (GEVIs) in order to perform the first optical voltage recordings of Purkinje cell simple and complex spike activity during behavior. Our approach will allow us to examine the encoding properties of Purkinje cell simple and complex spiking modalities during behavior and interrogate their interactions relative to the geometry of their inputs and overarching functional organization. In Aim 1 of this proposal, we will establish the instrumentation and procedures for ultra-high-speed voltage imaging of cerebellar Purkinje cells in awake behaving mice. Using this approach, we will examine spike level dynamics of both simple and complex spike firing, computing spatiotemporal correlation and activation patterns of neighboring Purkinje cells. We will also interrogate the kinematic encoding properties of these two activity modalities. In Aim 2, we will combine our approach for voltage imaging with simultaneous Calcium imaging of dendritic activity, to compare Purkinje cell encoding properties and responses to sensorimotor mismatches, whose processing is considered a hallmark of cerebellar function, with their overarching functional dendritic organization. Together, our work will shed new light on the population dynamics of Purkinje cell spiking activity and contributions to behavior, as well as provide new potential links between the functional and structural organization of the cerebellar cortex.
