Inactivation of cerebellar output axons impairs acquisition of conditioned eyeblinks

Acquisition of classically conditioned eyeblink responses (CRs) in the rabbit critically depends on intermediate cerebellum-related neural circuits. A highly efficient method for determining possible sites of plasticity within eyeblink circuits is the reversible inactivation of circuit components during learning. Inactivation of either the HVI region of the cerebellar cortex or the cerebellar interposed nuclei (IN) during learning is known to prevent CR acquisition. In contrast, inactivating cerebellar efferent axons in the brachium conjunctivum (BC) with small injections of tetrodotoxin (TTX) has been reported to have no effect on CR acquisition. This suggested that the intermediate cerebellum is essential for learning CRs and that activity mediated by the BC is not required for this process. Since we previously found that BC inactivation blocks CR extinction we re-examined its role in CR acquisition. To ensure complete and long-lasting inactivation of the BC, we injected before each training session doses of TTX that were larger than those in the previous acquisition study. Contrary to the previous negative findings, we found that this temporary block of axons in the brachium conjunctivum prevented normal acquisition of CRs. Injecting TTX directly in the adjacent lateral lemniscus, which could possibly influence CR acquisition, had no effect on learning. In addition, a functional test of TTX diffusion around the BC indicated that the inactivation did not affect other known parts of eyeblink circuits, such as the cerebellar interposed nuclei, the middle cerebellar peduncle or the contralateral red nucleus. We conclude that this form of associative learning in the rabbit eyeblink system requires extra-cerebellar learning and/or cerebellar learning that depends on the operation of cerebellar feedback loops.