Afferent Projections from Motoneurons Innervating Extraocular Muscles to the Cerebellum Demonstrated by the Retrograde Double-Labeling Technique

The objective of this study was to investigate the characteristics and distributions of neuronal origin of cerebellar afferents from motor cranial nerve nuclei innervating extraocular muscles by the method of retrograde transport of two fluorescence tracers in rats. Under deep anesthesia and aseptic conditions, 5l of 3 % solution of Fluoro-Gold (FG) in phosphate buffer solution (PBS) was injected into the bellies of the six extraocular muscles to study the labeling of motoneu-rons innervating corresponding extraocular muscles. The cerebellum was exposed by craniotomy, and 0.3 l of 10 % solution of Dextran Tetramethyl Rhodamine Biotin (Micro Ruby: or MR) in PBS was injected into many regions of the anterior vermis (lobule I, II) and the posterior vermis (lobule VI, VII, IX, X),the flocculus, the paraflocculus and the deep cerebellar nuclei. Multiple injections were made to cover the entire cerebellum in order to obtain a near maximum labeling of cerebellar afferent neurons. In other cases, only small single or a few injections were made in specific areas of the cerebellum to study specific distributions and topographic organization. In one group of rats, injections were made both in the extraocular muscles with FG and in the cerebellum with MR to study the double labeling of neurons, which project their axons to both the extraocular muscle and the cerebellum. Another group of rats were injected in both sites with only PBS and served as the control for auto-fluorescence background. After 3 days postoperative survival time, all animals were deeply reanesthetized and perfused with heparinized normal saline solution, followed by 4 % paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, and 30 % sucrose solution in PBS. The brainstem and the cerebellum were removed immediately, and stored in sucrose solution in PBS at 4C