Multiple origins of cerebellar cholinergic afferents from the lower brainstem in the gerbil

The possible origins of cerebellar cholinergic afferents from the lower brainstem of the gerbil were examined using immunohistochemistry combined with retrograde neuronal labelling techniques. Choline acetyltransferase (ChAT) monoclonal antibody was used in conjunction with a retrogradely transported tracer, horseradish peroxidase (HRP). The use of this technique allowed an unequivocal localisation of cholinergic neurons in different parts of the lower brainstem projecting to the cerebellum. In addition, single labelling of acetylcholinesterase (AChE), ChAT and HRP was carried out to elucidate the efferent projection from the lower brainstem to the cerebellum as well as the cholinergic distribution in these two areas. Our results showed the presence of HRP/ChAT double-labelled neurons in (1) the midline medulla: the periventricular gray beneath the 4th ventricle, C3 adrenergic area, raphe obscurus nucleus and medial longitudinal fasciculus, (2) the reticular formation: the medullary, lateral, intermediate, gigantocellular, lateral paragigantocellular and dorsal paragigantocellular reticular nuclei and gigantocellular reticular nucleus ventralis, and (3) sensory nuclei: the gracile nucleus, cuneate nucleus, external cuneate nucleus, spinal trigeminal nucleus interpolaris, prepositus hypoglossal nucleus and medial vestibular nucleus. In the cerebellum, AChE-positive mossy fibres were chiefly localised in the vermian lobules VIb,c, VII and X, paramedian lobule, crura I and II, paraflocculus and flocculus, and they were distributed in the white matter and granular layer of the cortex. The 3 above-mentioned cerebellar cholinergic afferent systems associated with the unique AChE distribution pattern in the cerebellum may be of important functional significance.