Axon guidance and target recognition depend on neuronal cell surface receptors that recognize and elicit selective growth cone responses to guidance cues in the environment. Contactin, a cell adhesion/recognition molecule of the immunoglobulin gene superfamily, regulates axon growth and fasciculation in vitro, but its role in vivo is unknown. To assess its function in the developing nervous system, we have ablated contactin gene expression in mice. Contactin-/- mutants displayed a severe ataxic phenotype consistent with defects in the cerebellum and survived only until postnatal day 18. Analysis of the contactin-/- mutant cerebellum revealed defects in granule cell axon guidance and in dendritic projections from granule and Golgi cells. These results demonstrate that contactin controls axonal and dendritic interactions of cerebellar interneurons and contributes to cerebellar microorganization.

Brevican is a nervous system-specific chondroitin sulfate proteoglycan that belongs to the aggrecan family and is one of the most abundant chondroitin sulfate proteoglycans in adult brain. To gain insights into the role of brevican in brain development, we investigated its spatiotemporal expression, cell surface binding, and effects on neurite outgrowth, using rat cerebellar cortex as a model system. Immunoreactivity of brevican occurs predominantly in the protoplasmic islet in the internal granular layer after the third postnatal week. Immunoelectron microscopy revealed that brevican is localized in close association with the surface of astrocytes that form neuroglial sheaths of cerebellar glomeruli where incoming mossy fibers interact with dendrites and axons from resident neurons. In situ hybridization showed that brevican is synthesized by these astrocytes themselves. In primary cultures of cerebellar astrocytes, brevican is detected on the surface of these cells. Binding assays with exogenously added brevican revealed that primary astrocytes and several immortalized neural cell lines have cell surface binding sites for brevican core protein. These cell surface brevican binding sites recognize the C-terminal portion of the core protein and are independent of cell surface hyaluronan. These results indicate that brevican is synthesized by astrocytes and retained on their surface by an interaction involving its core protein. Purified brevican inhibits neurite outgrowth from cerebellar granule neurons in vitro, an activity that requires chondroitin sulfate chains. We suggest that brevican presented on the surface of neuroglial sheaths may be controlling the infiltration of axons and dendrites into maturing glomeruli.

Immunocytochemical detection of glutamate decarboxylase (GAD), the predominant biosynthetic enzyme of gamma-aminobutyric acid (GABA), reveals the presence of a dense GABAergic innervation in all parts of the inferior olive. One brain center that provides a substantial projection to the inferior olive is the cerebellar nuclei, which contain many small GABAergic neurons. These neurons were tested as a source of GABAergic olivary afferents by combining retrograde tract tracing with GAD immunocytochemistry. As expected from previous studies, injections of wheat germ agglutinin-conjugated horseradish peroxidase (WGA-HRP) into the inferior olive retrogradely label many small neurons in the interposed and lateral cerebellar nuclei and the dorsal part of the lateral vestibular nucleus, and fewer neurons in the ventro-lateral region of the medial cerebellar nucleus. These projections are predominantly crossed and are topographically arranged. The vast majority, if not all, of these projection neurons are also GAD-positive. The relative contribution of this projection to the GABAergic innervation of the inferior olive was tested by lesion of the cerebellar nuclei, or the superior cerebellar peduncle. Within 10 days the lesion eliminates most GAD-immunoreactive boutons in the principal olive, the rostral lamella of the medial accessory olive, the ventrolateral outgrowth, and the lateral part of the dorsal accessory olive ventral fold. Thus, the effectiveness of this depletion demonstrates that the cerebellar nuclei provide most of the GABAergic innervation to regions of the inferior olive known to receive a cerebellar projection. Moreover, when the lateral vestibular nucleus is damaged, the dorsal fold of the dorsal accessory olive is depleted of GABAergic boutons. The synaptic relations that boutons of the GABAergic cerebello-olivary projection share with olivary neurons were investigated at the electron microscopic level by GAD-immunocytochemistry, anterograde degeneration of the cerebellar axons or anterograde transport of WGA-HRP. All of these methods confirm that GABAergic, cerebello-olivary axon terminals contain pleomorphic vesicles, and synapse on various portions of olivary neurons, and especially on dendritic spines within glomeruli, often in very close proximity to the gap junctions that characteristically couple the dendritic profiles. These results demonstrate four major points: that virtually all of the GABAergic, and presumably inhibitory, neurons of the cerebellar and dorsal lateral vestibular nuclei are projection neurons; that a large portion of the inferior olive receives GABAergic afferents from the cerebellar nuclei; that a portion of the dorsal accessory olive receives GABAergic afferents from the dorsal lateral vestibular nucleus; and that cerebello-olivary fibers often synapse near gap junctions, and therefore could influence electrical coupling of olivary neurons.

No abstract