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The nucleus is located in the midbrain at the level of the inferior colliculus ventral and medial periaqueductal grey and dorsal to the medial longitudinal fasciculus. It lies just caudal to the oculomotor nucleus and Edinger-Westphal nucleus. The nucleus forms a longitudinal paramedian column of somatic motor nuclei along with the abducens, oculomotor and hypoglossal nuclei.
Trochlear efferent fibers leave the nucleus dorsally and laterally passing around the periaqueductal grey matter and descend medial to the mesencephalic nucleus where they decussate at the level of the superior medullary velum. The fibers emerge just caudal and lateral to the inferior colliculus.