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Retrograde transneuronal tracing with rabies virus from the right orbicularis oculi muscle was used to identify neural networks underlying spontaneous, reflex, and learned blinks. The kinetics of viral transfer was studied at sequential 12 hr intervals between 3 and 5 d after inoculation. Rabies virus immunolabeling was combined with the immunohistochemical detection of choline acetyltransferase expression in brainstem motoneurons or Fluoro-Ruby injections in the rubrospinal tract. Virus uptake involved exclusively orbicularis oculi motoneurons in the dorsolateral division of the facial nucleus. At 3–3.5 d, transneuronal transfer involved premotor interneurons of trigeminal, auditory, and vestibular reflex pathways (in medullary and pontine reticular formation, trigeminal nuclei, periolivary and ventral cochlear nuclei, and medial vestibular nuclei), motor pathways (dorsolateral quadrant of contralateral red nucleus and pararubral area), deep cerebellar nuclei (lateral portion of interpositus nucleus and dorsolateral hump ipsilaterally), limbic relays (parabrachial and Kölliker–Fuse nuclei), and oculomotor structures involved in eye–eyelid coordination (oculomotor nucleus, supraoculomotor area, and interstitial nucleus of Cajal). At 4 d, higher order neurons were revealed in trigeminal, auditory, vestibular, and deep cerebellar nuclei (medial, interpositus, and lateral), oculomotor and visual-related structures (Darkschewitsch, nucleus of the posterior commissure, deep layers of superior colliculus, and pretectal area), lateral hypothalamus, and cerebral cortex (particularly in parietal areas). At 4.5 and 5 d the labeling of higher order neurons occurred in hypothalamus, cerebral cortex, and blink-related areas of cerebellar cortex. These results provide a comprehensive picture of the premotor networks mediating reflex, voluntary, and limbic-related eyelid responses and highlight potential sites of motor learning in eyelid classical conditioning.

the journal of neuroscience/the journal of neuroscience

Neuronal Premotor Networks Involved in Eyelid Responses: Retrograde Transneuronal Tracing with Rabies Virus from the Orbicularis Oculi Muscle in the Rat