Internuclear neurons in the ocular motor system of frogs

Medial and lateral rectus motoneurons of frogs were localized after retrograde labeling with horseradish peroxidase (HRP) injected in the medial rectus muscle or applied on the cut end of the abducens nerve. Coordinates of these cell columns were used as target areas for the injection of small amounts of HRP (20-60 nl) and [H-3]leucine (25-40 nl) and as search areas for retrogradely and anterogradely labeled internuclear neurons (INT) in in vivo and in vitro experiments. HRP injection in the medial rectus subdivision of the oculomotor nucleus (n = 6) resulted in retrograde labeling of cell bodies in the contralateral principal abducens nucleus. On the average about 16 cells per animal were found. Somatic diameters were about 13.5 +/- 2.8-mu-m (n = 32). The number and the size of these abducens internuclear neurons (AbINT) are smaller than those of lateral rectus motoneurons (n = 75;diameter: 19 +/- 3.2-mu-m). A crossed projection of AbINT to medial rectus motoneurons in the contralateral oculomotor nucleus is further supported by autoradiographic results. Following injection of [H-3]leucine into the abducens nucleus, a high density of silver grains was visible within the contralateral oculomotor nucleus, mainly in the caudal part of the oculomotor nucleus, where medial rectus motoneurons are located. Injection of [H-3]leucine in vivo (n = 4) and in vitro (n = 3) resulted in a similar high density of silver grains within the contralateral oculomotor nucleus, but the background level of silver grains was significantly higher after in vitro (264 +/- 38/2,500-mu-m2) than after in vivo injections (195 +/- 17/2,500-mu-2). HRP injection in the principal abducens nucleus (n = 9) resulted in retrograde labeling of cell bodies in the medial rectus subdivisions of the bilateral oculomotor nuclei. Ipsilateral projections predominated, with about 10 (+/- 8) labeled cells over contralateral projections (about 3 +/- 2). Average diameters of these oculomotor internuclear neurons (OcINT) were again smaller (10.8 +/- 2-mu-m;n = 18) than those of medial rectus motoneurons (14.4 +/- 3-mu-m;n = 52). In addition, retrogradely labeled cells were consistently encountered in the bilateral vestibular nuclei, the cerebellar nuclei, the dorsal brainstem caudal to the abducens nuclei, and ipsilaterally in the pretectum. Most of the vestibular neurons were located in the rostral part of the vestibular nuclear complex. These neurons might constitute part of the three-neuronal arc of the vestibulo-ocular reflex in the frog. Labeled cells in the pretectum were restricted to the ipsilateral posterior thalamic nucleus (P). After abducens injections the number of retrogradely labeled neurons in the P differed on the average by a factor of ten, depending on the micropipette's direction to the injection site. A ventral approach through the medial longitudinal fasciculus (MLF) resulted (as in other studies) in a false-positive overestimation of the number of P neurons projecting to the abducens nucleus, probably due to labeling of fibers of passage in the MLF. Many axons of these P neurons descend more caudally and project to an area where retrogradely labeled neurons are found after oculomotor or abducens HRP injections. Location and input-output relations of these neurons are compatible with a prepositus hypoglossi nucleus in frogs.