Transsynaptic tracing of eyeblink-related motor pathways in the mouse cerebellum [electronic resource]
Keywords: Biological Sciences

The eyeblink is one of the most basic motor behaviors and has served as a longstanding model for the study of adaptive and associative forms of motor conditioning. However, cerebellar pathways that support blinking behaviors remain poorly studied in the mouse, an increasingly popular animal model for motor learning. The precise location of eyeblink-related neurons is poorly defined, particularly in the red nucleus and the cerebellar nuclei and cortex. To pinpoint these neurons, I injected the GFP-expressing retrograde transsynaptic viral tracer Pseudorabies (PRV-152) into the orbicularis oculi muscle in the eyelids of live C57Bl6 mice. PRV travels retrograde across multiple synapses in the motor pathway and infects cells ultimately projecting to the injection site. By registering anatomical sites of the virally labeled cells, I created detailed maps of neurons presumably involved in controlling blink-related behavior. In the facial nucleus, viral labeling was strictly ipsilateral to the injection site and restricted to the dorsolateral rostral segment. The red nucleus is bilaterally labeled at the lateral rim with heavier labeling in the contralateral nucleus. Labeling was bilaterally distributed across all three deep cerebellar nuclei. In the cerebellar cortex, a characteristic spatiotemporal pattern was observed. Posterior vermal and floccular Purkinje cells were labeled first, followed by mid- and anterior vermal cells, and finally cells in the hemispheric Crus 1/2 and Simple lobules. Based on these results, I hypothesized that PRV injections into the eyelid uncover multiple distinct, cerebellar-modulated motor output pathways. These pathways have not previously been explored in the mouse; additionally, several of the putative pathways were poorly defined in other model systems as well. This hypothesis was then tested with single-step retrograde tracings from each of the deep cerebellar nuclei. The following circuits were confirmed: 1) from the ansiform lobule to the dentate nucleus 2) from the simple lobule to the anterior interpositus nucleus 3) from the anterior vermal and paravermal cerebellar cortex to the posterior interpositus nucleus, and 4) from the posterior vermis and paravermis to the fastigial nucleus. In prior literature, pathways 1 and 2 have been implicated in classical conditioning, and pathway 3 in cerebellar modulation of reflex blinks. Based on circuit analysis, pathway 4 is putatively associated with eye-eyelid coordination, but there is little existing research on this possibility. To complete my investigation of the mouse cerebellar eyeblink circuit, I traced anterograde from the vestibular nucleus to the red nucleus and retrograde from the red nucleus to the vestibular nucleus, establishing the existence of a previously unexplored vestibulorubral pathway in the mouse which may serve as the output circuit of pathway 4. As a whole, this body of work explores the location and distribution of eyeblink-related neurons in the mouse, identifies multiple parallel motor circuits in the mouse cerebellum, and offers evidence toward functional organization of the cerebellum.