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Key points • One of the two main inputs to the cerebellum consists of climbing fibres from neurons in the inferior olivary nucleus (IO). • The IO spontaneously forms clusters of co-active neurons in transverse brainstem slices from 1- to 2-week-old animals, coinciding with a critical time period in cerebellar development. • Here, we studied the cluster-forming mechanisms, and find that randomly occurring spontaneous clusters overlap extensively, and contain 10 to hundreds of IO neurons with an average somatodendritic field size that is slightly smaller than the average IO cluster size. • Cluster formation is dependent on sodium action potentials and electrical coupling between IO neurons, and may spread with decreasing velocity and may involve active dendritic properties. • The results help us better understand the basic mechanism underlying cluster formation in the IO, which is an important feature in the generation of patterned input to the cerebellum. Abstract The inferior olivary nucleus (IO) in in vitro slices from postnatal mice (P5.5–P15.5) spontaneously generates clusters of neurons with synchronous calcium transients, and intracellular recordings from IO neurons suggest that electrical coupling between neighbouring IO neurons may serve as a synchronizing mechanism. Here, we studied the cluster-forming mechanism and find that clusters overlap extensively with an overlap distribution that resembles the distribution for a random overlap model. The average somatodendritic field size of single curly IO neurons was ∼6400 μm2, which is slightly smaller than the average IO cluster size. Eighty-seven neurons with overlapping dendrites were estimated to be contained in the principal olive mean cluster size, and about six non-overlapping curly IO neurons could be contained within the largest clusters. Clusters could also be induced by iontophoresis with glutamate. Induced clusters were inhibited by tetrodotoxin, carbenoxelone and 18β-glycyrrhetinic acid, suggesting that sodium action potentials and electrical coupling are involved in glutamate-induced cluster formation, which could also be induced by activation of N-methyl-d-aspartate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors. Spikelets and a small transient depolarizing response were observed during glutamate-induced cluster formation. Calcium transients spread with decreasing velocity during cluster formation, and somatic action potentials and cluster formation are accompanied by large dendritic calcium transients. In conclusion, cluster formation depends on gap junctions, sodium action potentials and spontaneous clusters occur randomly throughout the IO. The relative slow signal spread during cluster formation, combined with a strong dendritic influx of calcium, may signify that active dendritic properties contribute to cluster formation. |
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