Anatomy of the turtle cerebellorubral circuit studied in vitro using neurobiotin and biocytin

We have combined the rapid anterograde and retrograde transport of neurobiotin and biocytin with the extended viability of the isolated turtle brainstem-cerebellum to conduct in vitro studies of the chelonian cerebellorubral circuit. Tracers were pressure injected in 15-25 nl quantities and the optimal transport time was 16 h. Tissue sections were incubated with avidin-biotin-HRP complex and reacted with DAB. Retrogradely labeled soma, dendrites and axons, and anterogradely labeled axons and to a lesser extent terminals were visible with both tracers. Red nucleus injections resulted in dense retrograde label in the contralateral lateral cerebellar nucleus and a heavily labeled contralateral rubrospinal tract. Cerebellar nucleus injections revealed light retrograde and dense terminal label in the contralateral red nucleus, together with retrograde label in a cell cluster in the ipsilateral ventrolateral medullary reticular formation, an area we identify as the lateral reticular nucleus. Injections into this medullary region resulted in heavy mossy fiber input to the ipsilateral cerebellum and moderate retrograde label in the contralateral red nucleus. These results identify prominent recurrent projections between the lateral cerebellar nucleus, red nucleus and lateral reticular nucleus, in addition to revealing other features of the cerebellorubral circuit.