Unusual neurofilament composition in cerebellar unipolar brush neurons

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Abstract
During antibody screening on sections of rat cerebellum, we noticed a group of small neurons which exhibited unusual staining properties. They were robustly immunopositive for the high molecular weight neurofilament protein, moderately immunostained with antibodies to the low molecular weight neurofilament protein and α-internexin, but only faintly immunoreactive (in PAP sections) or essentially immunonegative (in immunofluorescent sections) with all members of a panel of antibodies directed against the middle molecular weight neurofilament protein. Since neurons generally react equally well with phosphate-independent, (antibodies to) low, middle and high molecular weight neurofilament protein, we conclude that middle molecular weight neurofilament protein is present in these cells in an unusually low relative amount. These cells are found in the granular layer and appear concentrated in the flocculus, ventral paraflocculus, and vermis, particularly in the ventral uvula and nodulus (lobules IXd and X). Previous studies performed by Hockfield defined a population of neurons of similar appearance and distribution using the monoclonal antibody Rat-302, which recognized an uncharacterized 160 kDa protein. We show here that the cells described by Hockfield are identical to those we have found and furthermore that the Rat-302 antibody specifically recognizes the dephosphorylated form of the lysine-serine-proline repeated sequences of high molecular weight neurofilament protein. These cells were studied by pre-embedding immunoelectron microscopy. The nucleus is deeply indented and shows little condensed chromatin. The cytoplasm contains scattered microtubules and a larger number of neurofilaments than expected in a small cell. There are numerous large dense core vesicles, an unusual organelle consisting of ringlet subunits, and relatively little granular endoplasmic reticulum. A thin axon and a single stout dendritic trunk emanate from the perikaryon. Although the cell body and the dendritic shaft may form either complex contacts with mossy fibres (resembling those previously termed en marron synapses) or simple symmetric synapses with small boutons containing pleomorphic vesicles, most of the synaptic relations are established on the shafts of brush-like branchlets that form at the tip of the dendrite and enter one or two glomeruli. Each branchlet forms an extraordinarily extensive asymmetric synapse with the mossy fibre rosette and the subsynaptic region shows a microfibrillar web connected to the postsynaptic density. In addition to other organelles, the branchlets contain numerous mitochondria and large dense core vesicles. Short, non-synaptic appendages with few cytoplasmic organelles emanated from the cell body, dendritic shaft and branchlets. The immunoreaction products of all neurofilament antibodies were similarly distributed within the small cells, and were absent from the granular reticulum, the Golgi apparatus, the appendages and the subsynaptic region. These high molecular weight neurofilament protein rich small cells correspond to the pale cells, the calretinin and secretogranin positive small cells and the unipolar brush neurons newly described with the Golgi method. Unlike the multipolar Golgi neurons, unipolar brush cells are not immunopositive for the inhibitory neurotransmitters GABA or glycine. The unusual concentration of secretogratin and two different types of calcium-binding protein (calretinin and high molecular weight neurofilament protein) along with the high content of mitochondria suggest that these cells subserve a function that requires an unusual degree of metabolic activity, perhaps as a result of their unusually rich synaptic connections.