COMPARTMENTATION OF THE REELER CEREBELLUM: SEGREGATION AND OVERLAP OF SPINOCEREBELLAR AND SECONDARY VESTIBULOCEREBELLAR FIBERS AND THEIR TARGET CELLS J. VIG, a D. GOLDOWITZ, b D. A. STEINDLER c AND L. M. EISENMAN d * a Neurobiology Research Group of the Hungarian Academy of Sci- ences and Semmelweis University, Budapest H-1094, Hungary b Department of Anatomy and Neurobiology, University of Tennessee, Medical College, Memphis, TN 38163, USA c Department of Neuroscience, University of Florida, Gainsville, FL 32611, USA d Department of Pathology, Anatomy and Cell Biology, Jefferson Med- ical College, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA Abstract—The cerebellum of the reeler mutant mouse has an abnormal organization; its single lobule is composed of a severely hypogranular cortex and a central cerebellar mass (CCM) consisting of Purkinje cell clusters intermixing with the cerebellar nuclei. As such the reeler represents an excel- lent model in which to examine the effect of the abnormal distribution of cerebellar cells on afferent-target relation- ships. To this effect we studied the organization of the spino- cerebellar and secondary vestibulocerebellar afferent projec- tions in homozygous reeler mice (rl/rl) using anterograde tracing techniques. Spinal cord injections resulted in labeled spinocerebellar mossy fiber rosettes in specific anterior and posterior regions of the cerebellar cortex. Some vestiges of parasagittal organization may be present in the anterior pro- jection area. Within the CCM, labeled fibers appeared to ter- minate on distinct groups of Purkinje cells. Thus, the spino- cerebellar mossy fibers seem to form both normal and het- erologous synapses in the reeler cerebellum. Secondary vestibular injections resulted in both retrograde and antero- grade labeling. Retrograde labeling was seen in clusters of Purkinje cells and cerebellar nuclear cells; anterograde label- ing was distributed in the white matter and in specific regions of the anterior and posterior cortex of the cerebellum. The labeled spinocerebellar and secondary vestibulocerebellar afferents overlapped in the anterior region but in the poste- rior region the vestibulocerebellar termination area was ven- tral to the spinocerebellar area. An area devoid of labeled terminals was also observed ventral to the posterior second- ary vestibulocerebellar termination field. Using calretinin im- munostaining it was determined that this area contains unipolar brush cells, a cell type found primarily in the ves- tibulocerebellum of normal mice. Our data indicate that despite of the lack of known land- marks (fissures, lobules) the spinocerebellar and vestibulo- cerebellar afferent projections in the reeler cerebellum do not distribute randomly but have specific target regions, and the position of these regions, relative to each other, appears to be conserved. Two caveats to this were the finding of over- lapping terminal fields of these afferents in the anterior re- gion, and a posteroventral region that contains unipolar brush cells yet is devoid of secondary vestibulocerebellar afferents. The distribution of Purkinje cells and cerebellar nuclear cells is not random either; those that give rise to cerebellovestibular efferents form distinct groups within the central cerebellar mass. © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: reeler, spinocerebellar afferent, vestibulocerebel- lar afferent, Purkinje cells, unipolar brush cells, vestibular nuclei. The cerebellum has two major afferent systems: the olivo- cerebellar climbing fiber projection and the mossy fiber projections. In the present study we examine two different mossy fiber projections, the spinocerebellar (sc) and the secondary vestibulocerebellar afferents (svc) in the reeler mutant mouse. In the normal mouse sc afferents form a precise topographic map within the cerebellum (Ito, 1984; Gravel and Hawkes, 1990; Sotelo and Wassef, 1991). These fibers project to specific lobules of the anterior and posterior lobe (lobules II–V, and VIII–IX, respectively) where they segregate to parasagittal bands. Terminal fields are ordered with respect to biochemically different Purkinje cell compartments (Gravel and Hawkes, 1990; Ji and Hawkes, 1994; Ji et al., 1997). The arrival of sc mossy fibers occurs early during development [embryonic day 13/14 (E13/14) in the mouse; Grishkat and Eisenman, 1995] and precedes the development of their final cortical postsynaptic targets, the granule cells (Arsenio-Nunes and Sotelo, 1985). Vestibulocerebellar projections consist of primary and secondary vestibulocerebellar fibers (pvc and svc, respectively) and each terminates in discrete areas of the cerebellum. Pvc project to the ipsilateral nodulus and ventral uvula whereas svc from the vestibular nuclei project bilaterally to the nodulus, ventral uvula, flocculus and paraflocculus, and to the ventral part of the anterior lobe (Voogd et al., 1996). The pvc afferents are the earliest to arrive during development [E13 in the rat; Ashwell and Zhang, 1992, 1998]. There is some very recent evidence that the pvc projection may exhibit mediolateral periodicity, at least regarding canal afferents (Maklad and Fritzsch, *Corresponding author. Tel: +1-215-503-1686; fax: +1-215-923-3808. E-mail address: leonard.eisenman@mail.tju.edu. (L. M. Eisenman). Abbreviations: CCM, central cerebellar mass; ECM, extracellular ma- trix; IGL, internal granular layer; IVN, inferior vestibular nucleus; LVN, lateral vestibular nucleus; MVN, medial vestibular nucleus; NHS, nor- mal horse serum; PBS, phosphate buffer saline; pvc, primary vestibu- locerebellar; rl/rl, homozygous reeler; sc, spinocerebellar afferents; svc, secondary vestibulocerebellar afferents; UBC, unipolar brush cell; WGA-HRP, wheat germ agglutinin– horseradish peroxidase. Neuroscience 130 (2005) 735–744 0306-4522/05$30.00+0.00 © 2005 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2004.09.051 735