Transsynaptic tracing of conditioned eyeblink circuits in the mouse cerebellum

The eyeblink has long served as a model for motor learning and modulation. However, cerebellar pathways underlying conditioned blinks remain poorly studied in the mouse, and the location of blink-related neurons has never been transsynaptically mapped in the cerebellar cortex. This study aims to rectify this gap in our knowledge. By injecting GFP-expressing Pseudorabies virus (PRV-152) into the mouse orbicularis oculi muscle, neurons in the mouse eyeblink motor control circuit are transsynaptically labeled. In the facial nucleus, labeling was strictly ipsilateral to the injection site and restricted to the dorsolateral rim, consistent with previous studies. The red nucleus is bilaterally labeled at the lateral rim with clear contralateral preference. Previously unreported labeling was found in the ventrolateral red nucleus. Single-step tracing confirmed this area receives projections from eyeblink-related portions of the anterior interpositus and sends projections to eyelid-controlling portions of the facial nucleus. In the deep cerebellar nuclei, blink-related neurons were labeled both in areas associated with blink conditioning and in areas associated with other blink modulation. Finally, novel maps of the cerebellar cortex revealed a characteristic spatiotemporal pattern of labeling. Posterior vermal Purkinje cells were labeled first, followed by anterior vermal cells, then by hemispheric cells.