An in vitro brain stem preparation from turtles exhibits a neural correlate of eyeblink classical conditioning during pairing of auditory (CS) and trigeminal (US) nerve stimulation while recording from the abducens nerve. The premotor neuronal circuits controlling abducens nerve-mediated eyeblinks in turtles have not been previously described, which is a necessary step for understanding cellular mechanisms of conditioning in this preparation. The purpose of the present study was to neuroanatomically define the premotor pathways that underlie the trigeminal and auditory nerve-evoked abducens eyeblink responses. The results show that the principal sensory trigeminal nucleus forms a disynaptic pathway from both the trigeminal and auditory nerves to the principal and accessory abducens motor nuclei. Additionally, the principal abducens nucleus receives vestibular inputs, whereas the accessory nucleus receives input from the cochlear nucleus. The late R2-like component of abducens nerve responses is mediated by the spinal trigeminal nucleus in the medulla. Both the principal sensory trigeminal nucleus and the abducens motor nuclei receive CS-US convergence and therefore both, or either, might be considered potential sites of synapse modification during in vitro abducens conditioning. Further data are required to determine the role of the principal sensory trigeminal nucleus in in vitro conditioning.

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