In vivo imaging of inferior olive neurons reveals roles of co-activation and cerebellar feedback in olivocerebellar signaling

Complex spikes (CSs), generated by inferior olive (IO) neurons, are foundational to most theories of cerebellar function and motor learning. Despite their importance, recordings from IO neurons in living animals have been limited to single-electrode methods, providing no insights into multineuron dynamics within intact circuits. Here, we used a novel ventral surgical approach that allows calcium imaging-based monitoring of multicellular activity in the IO of anesthetized mice. This method provides direct optical access to the ventral medulla, enabling simultaneous recording of spontaneous and sensory-evoked activity within localized clusters of IO neurons, specifically in the principal (PO) and dorsal accessory olives (DAO). Our findings reveal that spontaneous activity rates and event magnitudes differ between the PO and DAO, consistent with observations from cerebellar cortex recordings in zebrin-positive (Z+) and zebrin-negative (Z-) zones, respectively. We further demonstrate that spontaneous event amplitudes are influenced by co-activation among neighboring neurons, so that events occurring in clusters are larger than single ones. Event co-activation is more pronounced in the PO than in the DAO, potentially explaining the differences in complex spike sizes observed in Z+ and Z-zones. Sensory-evoked events induced by periocular airpuff stimulation were larger than spontaneous ones, as expected. However, this difference diminishes when accounting for the higher levels of co-activation during sensory stimulation. By comparing spontaneous and sensory-evoked events categorized as clustered or single, we find no intrinsic differences in amplitudes, emphasizing the role of co-activation in shaping event magnitude. Next, we optogenetically activated cerebellar nucleo-olivary (N-O) axons, a pathway central to theories of CS generation. To our surprise, while this robustly suppressed spontaneous IO activity, sensory-evoked events showed no reduction in either their probability or waveform. Our findings suggest that the traditional view of the N-O pathway as purely inhibitory or desynchronizing might be complemented with selective suppression of background activity while preserving sensory-driven responses. Together with the role of local co-activation in shaping IO event magnitudes, this work offers new insight into the timing and variation in complex spikes and their functional significance for behavior.