Compartmentation of the reeler cerebellum: segregation and overlap of spinocerebellar and secondary vestibulocerebellar fibers and their target cells.

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 excellent model in which to examine the effect of the abnormal distribution of cerebellar cells on afferent-target relationships. To this effect we studied the organization of the spinocerebellar and secondary vestibulocerebellar afferent projections 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 projection area. Within the CCM, labeled fibers appeared to terminate on distinct groups of Purkinje cells. Thus, the spinocerebellar mossy fibers seem to form both normal and heterologous synapses in the reeler cerebellum. Secondary vestibular injections resulted in both retrograde and anterograde labeling. Retrograde labeling was seen in clusters of Purkinje cells and cerebellar nuclear cells; anterograde labeling 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 posterior region the vestibulocerebellar termination area was ventral to the spinocerebellar area. An area devoid of labeled terminals was also observed ventral to the posterior secondary vestibulocerebellar termination field. Using calretinin immunostaining it was determined that this area contains unipolar brush cells, a cell type found primarily in the vestibulocerebellum of normal mice. Our data indicate that despite of the lack of known landmarks (fissures, lobules) the spinocerebellar and vestibulocerebellar 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 overlapping terminal fields of these afferents in the anterior region, 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.

Inferior olivary-induced expression of Fos-like immunoreactivity in the cerebellar nuclei of wild-type and Lurcher mice.

Earlier behavioural studies have shown that the expression of the immediate-early gene c-fos, as visualized by the immunohistochemical detection of Fos, in the inferior olive (IO) correlated closely with expression in related areas of the cerebellar nuclei. It has been speculated that the expression of c-fos within the cerebellar nuclei may be induced by enhanced spiking activity of the immunopositive neurons in the inferior olive. Two potential mechanisms may play a role in this process: a direct induction by way of the collaterals of the olivary climbing fibres to the cerebellar nuclei, or indirectly, by climbing fibre activity-induced depression of mossy fibre-parallel fibre-induced simple spike frequency of the Purkinje cells resulting in a subsequent disinhibition of the related parts of the cerebellar nuclei. In an attempt to distinguish between these possible mechanisms, we analysed Fos immunoreactivity in the olivocerebellar system of wild-type mice and in the mutant mouse Lurcher which lacks Purkinje cells. The tremorgenic agent harmaline, which is known to induce enhanced and rhythmic firing of olivary neurons was given intraperitoneally to anaesthetized mice of both genotypes. Harmaline application coincides with the induction of Fos-immunoreactive neurons in most areas of the IO in both wild-type and Lurcher mice. Both types of mice also showed enhanced expression in the larger neurons of the cerebellar nuclei. However, in the smaller, GABAergic nucleo-olivary neurons, increased c-fos expression was only observed in the wild-type mice. We conclude that: (i) increased olivary activity indeed may result in increased c-Fos expression in related areas of the cerebellar nuclei; (ii) because the indirect mode of induction is not operative in Lurcher mice, the olivary collateral innervation of the cerebellar nuclei is sufficient for c-fos induction in the larger nucleobulbar neurons in Lurcher and potentially also in wild-type mice; however (iii) for the nucleo-olivary cells an intact cerebellar cortical input is necessary to evoke increased expression of c-fos following harmaline application.

Neonatal Borna disease virus infection in the rat causes a loss of Purkinje cells in the cerebellum.

Viral insults that occur during early postnatal periods, can affect neuronal systems which exhibit significant postnatal development, such as the cerebral cortex and cerebellum. Borna disease virus (BDV) is a single-strand RNA virus which replicates in the nervous system of many species after experimental inoculation and causes acute neurological disease. Neonatal rats infected with BDV do not mount an aggressive response to the virus like their adult counterparts, but instead develop a persistent BDV infection with less overt clinical sequelae. Recently, the cerebellum, a neural structure associated with regulation of motor behavior, and perhaps with higher cognitive functions, has been demonstrated to be a target of neonatal BDV infections in rats (Bautista et al, 1995). In the present study neonatal rats were infected with BDV and their cerebella were analyzed histologically and immunohistochemically at 7 months of age. The cerebella of infected animals were reduced in size but normal foliation and laminar organization was present. However, as visualized with immunohistochemistry for the Purkinje cell-specific antigen calbindin, there were numerous gaps within the Purkinje cell layer and in the molecular layer which contains the Purkinje cell dendritic trees. We estimated the number of Purkinje cells and found there was an approximately 75% loss of PC in adult rats neonatally infected with BDV. These results suggest that neonatal BDV infection may either (1) target the PC and cause the death of these cells directly or (2) acts indirectly by triggering an immune response which is then responsible for the loss of these cells.

Regionalization defects in the weaver mouse cerebellum.

The mammalian cerebellum consists of parasagittal bands and transverse zones that are laid down early in development. When the adult cerebellum is immunostained for the Purkinje cell-specific antigen zebrin II (i.e., aldolase C), compartmentation is reflected in alternating zebrin II+ (P+) and zebrin II- bands (P ). The zebrin II phenotype is Purkinje cell autonomous; thus, disruptions in the zebrin pattern may reflect early problems in pattern formation. Zebrin II expression has been examined in the weaver (wv) mouse cerebellum. Both zebrin II- and zebrin II Purkinje cells are present in the homozygous weaver (wv/wv) mouse, but they are not distributed normally. In the posterior vermis, although the zebrin II+ bands are wider and multilaminate, the standard compartmentation is present. However, a large zebrin II+ cell mass is absent from the central vermis, and analysis of the anterior lobe reveals several missing zebrin II- bands. The cytoarchitectonic defects in wv mice are not simply related to the Purkinje cell abnormalities. Instead, serial reconstruction reveals two transverse boundaries-one rostrally in lobule VI and the other caudally in lobule IX-that delineate cytoarchitectonic transverse zones important in cerebellar development. The abnormal zebrin expression pattern in wv/wv mice may be secondary to the deletion of a transverse zone. This is the first demonstration that Purkinje cell compartmentation can be altered by mutation; therefore, the wv mutation should prove valuable in understanding cerebellar regionalization.

Rostral cerebellar malformation (rcm/rcm): a murine mutant to study regionalization of the cerebellum.

A recently described recessive mouse mutant, rostral cerebellar malformation (rcm/rcm), demonstrates a swaying gait at approximately 12 days of age (Lane et al. [1992] J. Hered. 83:315-318). The mutant cerebellar (Cb) phenotype consists of cerebellar tissue that extends rostrally, beyond the usual distinct anterior cerebellar boundary, into the midbrain (Lane et al. [1992] J. Hered. 83:315-318; Ackerman et al. [1997] Nature 386:838-842). Interestingly, the cerebellar ectopia occurs in the absence of any significant alterations in the distribution of nuclear groups within the brainstem. The ectopic Cb tissue is 1) adherent to the posterior and lateral aspects of the inferior colliculus and to the lateral aspect of the rostral brainstem and 2) contains acellular regions within the inner granular layer (igl) and ectopic, calbindin-immunoreactive Purkinje cells (PCs) deep to the igl. Within the Cb proper, PC organization, as revealed by zebrin II immunoreactivity, is generally normal. In the ectopic Cb tissue PCs also exhibit a banded zebrin distribution. Analysis of the spinocerebellar projection in the mutant suggests a lobular distribution similar to that seen in the normal mouse. Within the anterior region, however, the normal parasagittal banding pattern is somewhat obscured. Spinocerebellar innervation of the ectopic Cb tissue exists, but it is almost exclusively to the region adjacent to the caudal inferior colliculus. In conjunction with the recent finding that the mutation appears to affect a UNC-5-like receptor protein for netrin-1, a molecule that may be involved in axonal guidance and cell migration (Ackerman et al. [1997] Nature 386:838-842), our results suggest that this mutant is an important model for the analysis of cerebellar development and regionalization.

Stripes and zones: the origins of regionalization of the adult cerebellum.

The mammalian cerebellum is subdivided into an elaborate, reproducible array of parasagittal stripes and transverse zones. Stripes and zones are most clearly revealed by the patterns of expression of numerous genes and by the consequences of several naturally-occurring mutations. Because the stripe and zone boundaries are orthogonal, they subdivide the cerebellum into a patchwork grid. How is this elaborate topography created during cerebellar development? This article reviews the evidence for cerebellar regionalization and considers various mechanisms by which it might arise during embryogenesis.

Correspondence between L7-lacZ-expressing Purkinje cells and labeled olivocerebellar fibers during late embryogenesis in the mouse.

It has been suggested that Purkinje cells (PC) play a role in organizing topographic relationships of several cerebellar afferent systems, including olivocerebellar fibers. This hypothesis is based on the observation that PC in the rat express biochemical heterogeneities during the presumptive period of olivocerebellar fiber ingrowth to the cerebellum. Previous studies designed to investigate the organization of murine olivocerebellar fibers during embryogenesis have suggested that interactions with PC may play a role in segregating olivocerebellar fibers after they enter the cerebellum. To determine whether PC heterogeneities are related to olivocerebellar fiber organization, transgenic mice carrying a beta-galactosidase (beta-gal) reporter gene linked to the promoter from the PC-specific gene L7/pcp-2 were used in neuroanatomical tracing experiments. Expression of the transgene mirrors endogenous L7/pcp-2 expression, which is upregulated earliest in parasagittal strips of the vermal cortex. Studies were conducted in vitro by using brainstem-cerebellar explants from embryonic day 17/18 (E17/18) and 18/19 mice. Applications of neuroanatomical tracer (horseradish peroxidase or neurobiotin) were made in either the caudal medial accessory olive (cMAO) or the rostral olive. These studies indicate that groups of olivocerebellar fibers and clusters of L7/lacZ+ and L7/lacZ-Purkinje cells respect common distribution boundaries during late embryogenesis. The strong correspondence between the distribution patterns generated by these two markers suggests that expression of L7/pcp-2 and the topographic organization of olivocerebellar (OC) fibers are not interdependent, but may be regulated by a common event or interaction, of a presently unknown nature, which occurs earlier during cerebellar development.

Developmental analysis of GFAP immunoreactivity in the cerebellum of the meander tail mutant mouse.

It is thought that Bergmann glial fibers assist in the inward migration of granule cells. Model systems in which there is a perturbation of either the migrating cells or the glial cell population have been useful in understanding the migratory process. In the meander tail mutant mouse, the anterior cerebellar region is agranular, whereas the posterior cerebellum is relatively unaffected by the mutation. This study presents a qualitative analysis of the development of cerebellar radial glia in mea/mea and +/mea mice aged from postnatal day 0 to adult, using an antibody against the glia specific antigen, glial fibrillary acidic protein. The results indicate a slight delay in the onset of immunoreactivity in the mea/mea cerebellum and abnormal glial formation in the anterior and posterior regions by postnatal day 5. At postnatal day 11, the full complement of labeled fibers appears to be present and although they appear abnormal in formation, they eventually reach the surface and terminate in oddly shaped and irregularly spaced endfeet. In adult mea/mea and +/mea mice, as compared to the early postnatal stages, there is a significant reduction in GFAP immunoreactive fibers. Cresyl violet stained adult mea/mea sections revealed the presence of ectopic granule cells in radial columns and small clumps at the surface of and within the molecular layer of the caudal cerebellum. Quantitative analyses revealed a 4- to 5-fold increase in the number of ectopic granule cells in lobule VIII of the mea/mea when compared with the +/mea cerebellum. These results suggest that the radial glia in the mea/mea cerebellum exhibit some uncharacteristic morphologies, but that these abnormalities are most likely the consequence of environmental alterations produced by the mutant gene.

Morphological correlates of bilateral synchrony in the rat cerebellar cortex.

Simultaneous recordings of the left and right crus IIA of the cerebellar cortex in the rat have demonstrated that Purkinje cells of both sides can be activated synchronously by their climbing fibers. Because climbing fibers arise exclusively from the contralateral inferior olive (IO), this physiological finding seems to contradict the anatomy. To define the structural basis responsible for the bilateral synchrony, we examined the possibilities that bilateral common afferent inputs to the IO and interolivary connections form the underlying mechanisms. The bilaterality of the major afferents of the olivary regions that project to crus IIA was studied using Phaseolus vulgaris leucoagglutinin as an anterograde tracer. We found that the excitatory and inhibitory projections from the spinal trigeminal nucleus and dorsolateral hump of the interposed cerebellar nucleus to the transition area between the principal olive and dorsal accessory olive were bilateral. A second possible mechanism for bilateral synchrony, which is the possibility that axons of olivary neurons provide collaterals to the contralateral side, was investigated using biotinylated dextran amine as an anterograde tracer. Labeled axons were traced and reconstructed from the principal olive and dorsal and medial accessory olive up to the entrance of the contralateral restiform body. None of these axons gave rise to collaterals. The possibility that neurons in the left and right IO are electronically coupled via dendrodendritic connections was investigated by examining the midline region of the IO. The neuropil of the left and right IO is continuous in the dorsomedial cell column. Examination of Golgi impregnations of this subdivision demonstrated that (1) many dendrites cross from one side to the other, (2) neurons close to the midline give rise to dendrites that extend into both olives, and (3) dendrites of neurons in the dorsomedial cell column frequently traverse into adjacent olivary subdivisions such as the medial accessory olive and the transition area between the principal olive and dorsal accessory olive. Sections immunostained for dendritic lamellar bodies or GABAergic terminals showed the same pattern: the neuropils of the dorsomedial cell columns on both sides form a continuum with each other as well as with the neuropil of other adjacent olivary subdivisions. Ultrastructural examination of the dorsomedial cell column demonstrated that the midline area includes many complex glomeruli that contain dendritic spines linked by gap junctions. To verify whether the complex spike synchrony observed between left and right crus IIA could indeed be mediated in part through coupled neurons in the dorsomedial cell column, we recorded simultaneously from crus IIA areas and from left and right vermal lobule IX, which receives climbing fibers from the dorsomedial cell column. In these experiments we demonstrated that the climbing fibers of all four areas, i.e., the left and right crus IIA as well as the left and right lobule IX, can fire synchronously. The present results indicate that synchronous climbing fiber activation of the left and right crus IIA in the rat can be explained by (1) bilateral inputs to the transition areas between the principal olive and dorsal accessory olive and (2) dendrodendritic electrotonic coupling between neurons of the left and right dorsomedial cell column and between neurons of the dorsomedial cell column and adjacent olivary subdivisions.

Further evidence for a unique developmental compartment in the cerebellum of the meander tail mutant mouse as revealed by the quantitative analysis of Purkinje cells.

The cerebellum of the meander tail mutant mouse (mea/mea) is characterized by a relatively normal cytoarchitecture posteriorly with an abrupt transition to an anterior region in which there is abnormal foliation, agranularity, and Purkinje cell (PC) ectopia. This study presents the results of a qualitative and quantitative analysis of the PC in the mea/mea cerebellum. Developmental and morphological analyses reveal that the PC in the anterior region of the mea/mea cerebellum do not form a monolayer during the first week of postnatal development as they do in the wild type mouse. In the adult mea/mea, the dendrites of these ectopic cells are atrophic and disoriented. Quantitative studies in adult animals reveal that while the total number of PC is normal, the number of PC in the affected anterior region of the mea/mea cerebellum is greater than the number of PC in the anterior lobe, as classically defined by the primary fissure, of the normal animal. These data suggest that 1) the developmental morphology of the PC in the anterior region is abnormal, probably due to the lack of granule cells at early postnatal times; 2) the total number of PC in the cerebellum is normal, and 3) the defect is not restricted to the anterior lobe but involves a portion of the posterior lobe. The latter supports the notion that the mutant gene affects a unique developmental compartment in the cerebellum which does not coincide with the classic adult boundary, the primary fissure, between the anterior and posterior lobes.

Development of the spinocerebellar projection in the prenatal mouse.

An abundance of information is available concerning the spinocerebellar projection in adult mammals. However, only a few studies have attempted a developmental analysis of this important projection system in early postnatal and/or prenatal animals. The present study provides an analysis of the development of the projection from the spinal cord to the cerebellum in fetal mice using anterograde tracing techniques in an in vitro preparation. After applications of biocytin to the caudal cervical spinal cord, anterogradely labelled fibers were present in the brainstem of embryonic day 12 (E12/13) mice, however, there was no indication of label in the cerebellum. At E13/14, labelled fibers were evident in the rostrolateral portions of the cerebellum/isthmus region. By E15/16, labelled spinocerebellar fibers had progressed farther into the cerebellum and were seen crossing the midline in a very superficial position. At older ages, the number of crossing fibers increased, and they became more ventrally positioned within the cerebellum. At E17/18 and E18/19, labelled spinocerebellar fibers were observed to branch and invade deeper portions of the cerebellum including the cerebellar nuclei. However, at E18/19, there was no indication of the parasagittal organization characteristic of this projection in the adult animal. The results of this study indicate that spinocerebellar fibers are present within the cerebellum significantly earlier than the development and differentiation of their primary targets, the granule cells. Furthermore, these data suggest that spinocerebellar fibers may form associations with cerebellar nuclear cells during fetal development.

Postnatal neurophysiologic effects of prenatal X-irradiation.

Histological and neurophysiological effects of in utero irradiation were examined following exposure of pregnant Wistar rat to 2.0 Gy X-irradiation or sham-irradiated on the 17th day of gestation. The 234 newborns were monitored for the age of appearance of four selected physiologic markers and the age of acquisition of five selected reflexes. Offspring were evaluated as young adults using selected behavioural tests. Postnatal growth was monitored weekly. Selected offspring were autopsied to determine the presence of morphologic central nervous system alterations. The results indicated that 2.0 Gy X-irradiation during the foetal period in rat gestation caused permanent alterations in the mature adult organism, which include non-recuperable growth retardation, morphologic changes in the brain such as microcephaly, abnormal cerebellar cortical cellular patterns, and alterations in the cell architecture of the hippocampus; diminished attainment of selected reflexes; alterations in the appearance of selected physiologic markers; and changes in adult test performance indicating significant hyperactivity among the irradiated offspring. Such exposure to X-irradiation during this period results in behavioural and morphologic alterations, which persist throughout life.

External cuneocerebellar projection and Purkinje cell zebrin II bands: a direct comparison of parasagittal banding in the mouse cerebellum.

The parasagittal parcellation of the mammalian cerebellar cortex has been revealed using anatomical, electrophysiological, histological and immunological techniques. Correlation studies have been carried out to determine whether a common organizational plan encompassing the various afferent, efferent and intrinsic maps may exist in the mammalian cerebellum. Many of these studies utilized the parasagittal Purkinje cell antigenic banding pattern as revealed by the monoclonal antibody against zebrin antigens as a standard reference. In this study, the pattern of labelled mossy fiber terminals originating from the external cuneate nucleus was determined and compared with the Purkinje cell antigenic zebrin bands in the same sections. External cuneocerebellar fibers in the mouse were observed to project in well-delineated parasagittal terminal distribution zones, primarily to the ipsilateral vermal cerebellar cortex. However, there was a very minor, but consistent contralateral component. The external cuneocerebellar fiber termination pattern in the mouse differed from that seen in other rodents such as the rat. Comparison of the external cuneucerebellar terminal zones in sections immunohistochemically stained for the zebrin II antigen revealed that the boundaries of the terminal fields of the external cuneocerebellar projection do not always align with those of the zebrin II antigenic bands. These results strongly suggest that mossy fibers have a complicated relationship to zebrin defined compartments. Therefore, the designation 'functional unit' of the cerebellum or 'cerebellar module' remains uncertain at this time.

Antigenic compartmentation in the mouse cerebellar cortex: zebrin and HNK-1 reveal a complex, overlapping molecular topography.

Two monoclonal antibodies--anti-zebrin I and anti-HNK-1--have been used to study the compartmentation of the mouse cerebellar cortex. As in other species, the pattern of localization of the Purkinje cell specific antigen zebrin I is confined to a subset of Purkinje cells that are organized into parasagittal bands. The basic pattern consists of two abutting paramedian bands (P1+) and up to three additional vermal bands on either side (P2(+)-P4+). This pattern is altered in the vermal regions of lobules X and VI-VII where all Purkinje cells are immunoreactive. In the hemisphere there are three additional bands present (P5(+)-P7+) plus two shorter bands in the paravermal area (P4b+ and P5a+) that extend from the paramedian lobule through the lobulus simplex. This pattern is very similar, but perhaps not identical, to that previously described for the rat. These results suggest a common mammalian plan for the expression and localization of zebrin I. By using a monoclonal antibody to an epitope associated with HNK-1, we have now identified a novel pattern of compartmentation in mouse cerebellum. The HNK-1 epitope is expressed most notably on Purkinje cells and Golgi cells. The molecular layer immunoreactivity associated with the Purkinje cell dendrites varies in intensity in a systematic and reproducible fashion. This reveals a novel cerebellar compartmentation that is sometimes complementary, sometimes overlapping, to that revealed by anti-zebrin. As a result, it is now possible to subdivide the cerebellar cortex into a still finer mosaic of antigenic patches and bands than was possible by using zebrins alone.

Developmental analysis of the external granular layer in the meander tail mutant mouse: do cerebellar microneurons have independent progenitors?

The cerebellum of the meander tail mutant mouse (mea/mea) is characterized by an apparently normal cytoarchitecture posteriorly with an abrupt transition to an abnormal anterior region. Anteriorly, there is abnormal foliation, a drastic reduction in the granule cells (GC) population, disorganization of the Purkinje cells (PC), and a virtual absence of Bergmann glial processes. In this paper we analyze the prenatal and postnatal development of the cerebellum in the mea/mea and attempt to determine the phenotypic onset of the mutation in the anterior region. Hematoxylin and eosin stained sections reveal a morphological difference in the cerebellum of the mea/mea as early as embryonic day 16 characterized by a reduction in the external granule cell layer (EGL). The reduction in the EGL becomes increasingly apparent as development proceeds. This deficit in the EGL most probably results in the absence of GC, but it is unclear at this point whether reduced migration, proliferation, and/or increased cell death is the major factor. Interestingly, immunohistochemical staining with a monoclonal antibody against parvalbumin reveals that the basket and stellate cells, which are also thought to arise from the EGL, are present in the anterior region of the mea/mea cerebellum. These results suggest that the lack of GC in the meander tail is due to an early expressed abnormality of the EGL. However, the presence of the basket and/or stellate cells raises some interesting questions concerning the lineage of the cerebellar microneurons.

Evidence of early topographic organization in the embryonic olivocerebellar projection: a model system for the study of pattern formation processes in the central nervous system.

Many projection systems within the peripheral and central nervous system are topographically organized, and it has become increasingly clear that interactions which occur during development determine the projection patterns these systems exhibit in the adult. The olivocerebellar system was chosen as a model system for this study of afferent pattern formation because it has several characteristics which lend themselves to a study of this type. Applications of horseradish peroxidase were made to both the cerebellar primordium and to the inferior olive of embryonic and neonatal mice using an in vitro perfusion system to support the tissue during the transport period. Fibers labeled after restricted olivary applications are limited to particular mediolateral regions of the cerebellum. Similarly, olivary cells retrogradely labeled after discrete cerebellar applications are restricted to particular olivary subdivisions. The results indicate that the olivocerebellar projection displays elements of topographic organization as early as E15 and that the pattern displayed is roughly comparable to that of the adult mammal. The observed trajectories of olivocerebellar fibers and their concomitant association with both Purkinje and cerebellar nuclear cells during embryonic development suggests a role for either or both cell types in the pattern formation process.

Analysis of protein variations in adult and postnatal day 11 staggerer and lurcher mutant mice.

As an initial attempt at uncovering the molecular basis of cerebellar synaptogenic defects in the lurcher and staggerer mutant mice, the pattern of protein expression was compared between cerebellar and non-cerebellar tissues, between normal and mutant mice and between early postnatal and adult developmental stages. While cerebellar and mutation-specific alterations in the expression of proteins could be easily observed in adult tissues, no such alterations were observed in early postnatal lurcher mice and only one qualitatively unique protein species could reproducibly be identified in early postnatal staggerer mice.

The olivocerebellar projection in normal (+/+), heterozygous weaver (wv/+), and homozygous weaver (wv/wv) mutant mice: comparison of terminal pattern and topographic organization.

Olivocerebellar organization and topography were analyzed in adult normal (+/+), heterozygous weaver (wv/+), and homozygous weaver (wv/wv) mutant mice. The two genotypes (wv/+ and wv/wv) of the weaver mutant present a gradation of abnormal cerebellar morphology. Purkinje cell (PC) ectopia ranges from mild (wv/+) to moderate (wv/wv), and regional PC loss is also graded in the two types. To determine olivocerebellar organization and topography, tritiated amino acids were placed into different regions of the inferior olivary complex (IO) in normal, heterozygous, and homozygous weaver mice. Despite some PC loss and ectopia, olivocerebellar fiber (OCF) terminals in both homozygous and heterozygous weaver mice have an orthogonal distribution and topography similar to that seen in normal mice. Differences in OCF termination, such as an increased density of OCF terminal label in the lower portion of the molecular layer, the PC, and granule cell layers, are seen in homozygous weaver mice. In some heterozygous weaver and normal cases, multiple injections labeling most IO cells on one side of the IO resulted in continuous OCF terminal labeling in many regions of the contralateral cerebellar cortex, suggesting that all PCs receive OCF input. Retrograde analysis involving injections of horseradish peroxidase conjugated to wheat germ agglutinin into different mediolateral cerebellar regions in homozygous weaver mice further demonstrates a generally normal olivocerebellar topography.

Novel developmental boundary in the cerebellum revealed by zebrin expression in the lurcher (Lc/+) mutant mouse.

The cerebellar cortex contains at least two classes of Purkinje cells, which are organized into alternating arrays of parasagittal bands. The clearest demonstration of this compartmentation is the pattern of expression of a family of polypeptide antigens, the zebrins, which are expressed selectively by Purkinje cell subsets. Furthermore, anterograde tracing experiments show that the zebrin compartments are closely correlated with both afferent and efferent projection maps. The further subdivision of long parasagittal bands into smaller modules may occur through several different mechanisms, including the intrinsic cerebellar lobulation and the selective distribution of afferent terminal fields. However, while the longitudinal subdivisions are straightforwardly shown, the mediolateral boundaries are more subtle. In this report we describe a novel mediolateral and anteroposterior compartmentation boundary in mice, running across lobule VIII, that is revealed by the consequences of the lurcher (Lc/+) allele for zebrin expression. In normal mice zebrin compartmentation develops in several discrete stages: until postnatal day 5 (PD5) there is no zebrin expression; from PD5-PD7 zebrin is found only in the posterior lobe vermis, with immunoreactive Purkinje cells in lobules X, IX, and VIII but not elsewhere; from PD7-PD12 most Purkinje cells in the vermis become zebrin+; from PD12-PD15 immunoreactivity also appears in the hemispheres so that almost all Purkinje cells now are zebrin+; and finally, from PD15-PD25 zebrin is gradually suppressed in those Purkinje cells that are zebrin- in the adult until the mature pattern of parasagittal compartments is revealed. In the Lc/+ mutant the normal developmental progression is interrupted at around PD7. As a result, the pattern of zebrin expression becomes frozen at that stage when immunoreactive Purkinje cells are confined exclusively to the posterior lobe vermis. A reproducible boundary between expressing and nonexpressing zones runs mediolaterally across the dorsal surface of lobule VIII. Apart from zebrin expression itself, there are no obvious structural correlates of this transition. This mediolateral boundary identifies a developmental unit in the posterior lobe vermis of the cerebellum, and provides further evidence that the cerebellum is a highly heterogeneous structure.