Abstract
Cell class-specific markers are powerful tools for the study of individual neuronal populations. The peculiar unipolar brush cells of the mammalian cerebellar cortex have only recently been definitively identified by means of the Golgi method, and we have explored markers of cerebellar neurons with the purpose of facilitating the analysis of this new cell population and, especially, its distribution and ultrastructural features. By light microscopic immunocytochemistry, we demonstrate that, in the rat, the unipolar brush cells are the cortical neurons that are most densely immunostained with antiserum to calretinin, a recently discovered calcium-binding protein. The unipolar brush cells are highly concentrated in the flocculo-nodular lobe, the ventral uvula and the ventral paraflocculus, occur at relatively high density in the lingula, at moderate-to-low density in other folia of the vermis and in the narrow intermediate cortex, and at low to very low density, with the exception of a few hot spots, in the lateral regions of the cerebellar hemispheres and in the dorsal paraflocculus. Unipolar brush cells are also found in the cochlear nucleus. In addition to the unipolar brush cells, calretinin antibody distinctly stains certain mossy fibers, and weakly to moderately stains other cerebellar elements, such as granule neurons and climbing fibers. In the lobules containing high densities of unipolar brush cells, the granule cell bodies and the parallel fibers are much less immunoreactive, and there are many more densely immunostained mossy fibers than in the lobules, where these cells are rare, which suggests some relationships between these elements. In the cerebellar nuclei, small neurons are densely immunostained, while large neurons are immunonegative.
The unipolar brush cells reside nearly exclusively in the granular layer. They are small neurons, intermediate in size between granule cells and Golgi cells, and their features are remarkably similar across all lobules. They usually have a single, relatively thick dendrite of varying length that terminates in a brush-like tip consisting of several short branchlets. Utilizing a pre-embedding protocol, we have identified unipolar brush cells with the electron microscope. The cytoplasm of these cells is partially obscured by the electron dense product of calretinin immunoreaction in all regions of the soma and processes. The cells are often covered with non-synaptic appendages and contain a peculiar cytoplasmic inclusion consisting of ringlet subunits. Other characteristic components are numerous neurofilaments, mitochondria and large, dense-core vesicles. Individual brushes enter one or two glomeruli, where the dendritic branchlets establish an unusually extensive synapse with mossy fiber rosettes. In addition to their contact with the mossy rosettes, the branchlets are postsynaptic to boutons presumably belonging to the axonal plexus of Golgi cells and are also presynaptic to small dendrites, displaying small, clear synaptic vesicles at the site of contact. The distinct calretinin-like immunoreactivity of the unipolar brush cells may be related to strong calcium influx at their extensive synapses with the mossy fiber rosettes.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Altman J, Bayer SA (1977) Time of origin and distribution of a new cell type in the rat cerebellar cortex. Exp Brain Res 29:265–274
Andressen C, Blumcke I, Celio MR (1993) Calcium-binding proteins: selective markers of nerve cells. Cell Tissue Res 271:181–208
Arai R, Winsky L, Arai M, Jacobowitz D M (1991) Immunohistochemical localization of calretinin in the rat hindbrain. J Comp Neurol 310:21–44
Barmack NH, Baughman RW, Eckenstein FP (1992 a) Cholinergic innervation of the cerebellum of rat, rabbit, cat and monkey as revealed by choline acetyltransferase activity and immunohistochemistry. J Comp Neurol 317:233–249
Barmack NH, Baughman RW, Eckenstein FP, Shojaku H (1992 b) Secondary vestibular cholinergic projection to the cerebellum of rabbit and rat as revealed by choline acetyltransferase immunohistochemistry, retrograde and orthograde tracers. J Comp Neurol 317:250–270
Barmack NH, Baughman RW, Errico P, Shojaku H (1993) Vestibular primary afferent projections to the cerebellum of the rabbit. J Comp Neurol 327:521–534
Berrebi AS, Mugnaini E (1991) Distribution and targets of the cartwheel cell axon in the dorsal cochlear nucleus of the guinea pig. Anat Embryol 183:427–454
Berrebi AS, Mugnaini E (1993) Alterations in the dorsal cochlear nucleus of cerebellar mutant mice. In: Merchán MA, Juiz JM, Godfrey DA, Mugnaini E (eds) The mammalian cochlear nuclei: organization and function. Plenum Press, New York, pp 107–119
Berrebi AS, Oberdick J, Sangameswaran L, Christakos S, Morgan JI, Mugnaini E (1991) Cerebellar Purkinje cell markers are expressed in retinal bipolar neurons. J Comp Neurol 308:630–649
Braak E, Braak H (1993) The new monodendritic neuronal type within the adult human cerebellar granule cell layer shows calretinin-immunoreactivity. Neurosci Letts 154:199–202
Brodal A, Drablös PA (1963) Two types of mossy fiber terminals in the cerebellum and their regional distribution. J Comp Neurol 121:173–187
Cajal S, Ramón y (1911) Histologie du système nerveux de l'homme et des vertébrés, vols I and II. Maloine, Paris, Reprinted 1955. Consejo Superior de Investigaciones Cientificas, Madrid
Chan-Palay V, Palay SL (1971) The synapse en marron between Golgi type II neurons and mossy fibers in the rat's cerebellar cortex. Z Anat Entwicklungsgesch 133:274–287
Cozzi MG, Rosa P, Greco A, Hille A, Huttner WB, Zanini A, DeCamilli P (1989) Immunohistochemical localization of secretogranin II in the rat cerebellum. Neuroscience 28:423–441
De Camilli P, Jahn R (1990) Pathways to regulated exocytosis in neurons. Ann Rev Physiol 52:625–645
Dechesne CJ, Winsky L, Kim HN, Goping G, Vu TD, Wenthold RJ, Jacobowitz DM (1991) Identification and ultrastructural localization of a calretinin-like calcium-binding protein (protein 10) in the guinea pig and rat inner ear. Brain Res 560:139–148
Eccles JC, Ito M, Szentágothai J (1967) The cerebellum as a neuronal machine. Springer, New York Heidelberg Berlin
Floris A, Dunn ME, Berrebi AS, Jacobowitz DM, Mugnaini E (1992) Pale cells of the flocculo-nodular lobe are calretinin-positive. Soc Neurosci Abstr 18:853
Friedrich VL Jr, Mugnaini E (1981) Preparation of neural tissues for electron microscopy. In: Heimer L, Robards MJ (eds) Neuroanatomical tract-tracing methods. Plenum Press, New York, pp 345–375
Hámori J, Szentágothai J (1966) Participation of Golgi neuron processes in the cerebellar glomeruli: an electron microscopic study. Exp Brain Res 2:65–81
Harris J, Moreno S, Shaw G, Mugnaini E (1993) Unusual neurofilament composition in cerebellar unipolar brush neurons. J Neurocytol 22:663–681
Hockfield S (1987) A Mab to a unique cerebellar neuron generated by immunosuppression and rapid immunization. Science 237:67–70
Ito M (1984) The cerebellum and neural control. Raven Press, New York, pp 1–508
King JS, Cummings SL, Bishop GA (1992) Peptides in cerebellar circuits. Progr Neurobiol 39:423–442
Korte GE, Mugnaini E (1979) The cerebellar projection of the vestibular nerve in the cat. J Comp Neurol 184:265–278
Lange W (1974) Regional differences in the distribution of Golgi cells in the cerebellar cortex of man and other mammals. Cell Tissue Res 153:219–226
Mugnaini E (1972) The histology and cytology of the cerebellar cortex. In: Larsell O, Jansen J (eds) The comparative anatomy and histology of the cerebellum. The human cerebellum, cerebellar connections, and cerebellar cortex. The University of Minnesota Press. Minneapolis, pp 201–264
Mugnaini E, Dahl AL (1983) Zinc-aldehyde fixation for light-microscopic immunocytochemistry of nervous tissues. J Histochem Cytochem 31:1435–1438
Mugnaini E, Floris A (1994) Unipolar brush cell: a neglected neuron of the mammalian cerebellar cortex. J Comp Neurol 339:174–180
Mugnaini E, Morgan JI (1987) The neuropeptide cerebellin is a marker for two similar neuronal circuits in rat brain. Proc Natl Acad Sci USA 84:8692–8696
Mugnaini E, Nelson BJ (1989) Corticotropin-releasing Factor (CRF) in the olivo-cerebellar system and the feline olivary hypertrophy. In: Strata P (ed) The olivocerebellar system in motor control. Exp Brain Res 17:187–197
Mugnaini E, Oertel WH (1985) An atlas of the distribution of GABAergic neurons and terminals in the rat CNS as revealed by GAD immunohistochemistry. In: Bioeklund A, Hoekfelt T (eds) Handbook of chemical neuroanatomy. GABA and neuropeptides in the CNS, vol 4, Part 1. Elsevier, Amsterdam, pp 463–622
Mugnaini E, Osen KK, Dahl AL, Friedrich VL Jr, Korte G (1980) Fine structure of granule cells and related interneurons (termed Golgi cells) in the cochlear nuclear complex of cat, rat, and mouse. J Neurocytol 9:537–570
Mugnaini E, Floris A, Wright-Goss M (1994) The extraordinary synapse of the unipolar brush cells of the rat cerebellum: a study by standard electron microscopy. Synapse 16:284–311
Munoz D G (1990) Monodendritic neurons: a cell type in the human cerebellar cortex identified by chromogranin A-like immunoreactivity. Brain Res 528:335–338
Nelson B, Mugnaini E (1991) The GABAergic cerebello-olivary projection in the rat. Anat Embryol 184:225–243
Oberdick J, Schilling K, Smeyne RJ, Corbin JG, Bocchiaro C, Morgan JI (1992) Control of segment-like patterns of gene expression in the mouse cerebellum. Neuron 10:1007–1018
Oertel WH, Schmechel DE, Mugnaini E, Tappaz ML, Kopin IJ (1981) Immunocytochemical localization of glutamate decarboxylase in rat cerebellum with a new antiserum. Neuroscience 6:2715–2735
Ojima H, Kawajiri S, Yamasaki T (1989) Cholinergic innervation of the rat cerebellum: qualitative and quantitative analyses of elements immunoreactive to a monoclonal antibody against choline acetyltransferase. J Comp Neurol 290:41–52
Ottersen O, Storm-Mathisen J, Somogyi P (1988) Colocalization of glycine-like and GABA-like immunoreactivities in Golgi cell terminals in the rat cerebellum: a postembedding light and electron microscopic study. Brain Res 450:342–353
Palay SL, Chan-Palay V (1974) Cerebellar cortex. Cytology and organization. Springer, New York Heidelberg Berlin
Palkovits M, Magyar P, Szentágothai J (1971) Quantitative histological analysis of the cerebellar cortex in the cat. II. Cell numbers and densities in the granular layer. Brain Res 32:15–30
Peters A, Palay SL, Webster FH de (1991) The fine structure of the nervous system. Oxford University Press, New York
Résibois A, Rogers JH (1992) Calretinin in rat brain: an immunohistochemical study. Neuroscience 46:101–134
Rogers JH (1989a) Two CaBPs mark many chick sensory neurons. Neuroscience 31:697–709
Rogers JH (1989b) Immunoreactivity for calretinin and other calcium binding proteins in cerebellum. Neuroscience 31:711–721
Rogers JH, Khan M, Ellis J (1989) Calretinin and other CaBPs in the nervous system. In: Pochet R, Lawson DEM, Heizman CW (eds) Calcium binding proteins in normal and transforned cells. Plenum Press, New York, pp 195–203
Rossi DJ, Mugnaini E, Slater NT (1994) Glutamate receptor-mediated transmission at a novel giant synapse in rat cerbellum: the mossy fibre-unipolar brush cell synapse. Brain Res Assoc Abstr (in press)
Sahin M, Hockfield S (1990) Molecular identification of the Lugaro cell in the cat cerebellar cortex. J Comp Neurol 301:575–584
Smeyne RJ, Oberdick J, Schilling K, Berrebi AS, Mugnaini E, Morgan JI (1991) Dynamic organization of developing Purkinje cells revealed by transgene expression. Science 254:719–721
Sotelo C (1977) Formation of presynaptic dendrites in the rat cerebellum following neonatal X-irradiation. Neuroscience 2:275–283
Sotelo C, Palay SL (1968) The fine structure of the lateral vestibular nucleus in the rat. I. Neurons and neuroglial cells J Cell Biol 36:151–179
Sotelo C, Wassef M (1991) Cerebellar development: afferent organization and Purkinje cell heterogeneity. Philos Trans R Soc Lond Biol 331:315–322
Storm-Mathiesen J, Leknes AK, Bore A, Vaaland JL, Edminson P, Haug FMS, Ottersen OP (1983) First visualization of glutamate and GABA in neurones by immunocytochemistry. Nature 301:517–520
Sturrock RR (1990) A quantitative histological study of Golgi II neurons and pale cells in different cerebellar regions of the adult and ageing mouse brain. Z Mikrosk Anat Forsch 104:705–714
Voogd J, Bigaré F (1980) Topographical distribution of olivary and cortico-nuclear fibers in the cerebellum: a review. In: Courville J, Montigny C de, Lamarre Y (eds) The olivary nucleus: anatomy and physiology. Raven Press, New York, pp 207–234
Welker W (1987) Spatial organization of somatosensory projections to granule cell cerebellar cortex: functional and connectional implications of fractured somatotopy (summary of Wisconsin studies). In: King JS (ed) New concepts in cerebellar neurobiology. Liss, New York, pp 239–280
Winkler H, Fischer-Colbrie R (1992) The chromogranins A and B: the first 25 years and future perspectives. Neuroscience 49:497–528
Winsky L, Jacobowitz DM (1991) Purification, identification and regional distribution of a brain-specific calretinin-like calcium-binding protein (protein 10). In: Heizmann C (ed) Novel calcium-binding proteins: Fundamentals and clinical implications. Springer, Heidelberg Berlin New York, pp 277–300
Winsky L, Nakata H, Martin BM, Jacobowitz DM (1989) Isolation, partial amino acid sequence, and immunocytochemical localization of a brain-specific calcium binding protein. Proc Natl Acad Sci USA 86:10139–10143
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Floris, A., Diño, M., Jacobowitz, D.M. et al. The unipolar brush cells of the rat cerebellar cortex and cochlear nucleus are calretinin-positive: a study by light and electron microscopic immunocytochemistry. Anat Embryol 189, 495–520 (1994). https://doi.org/10.1007/BF00186824
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00186824