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.

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Abstract. Muscle development in vivo involves a complex sequence of cell-cell interactions in which secondary myotubes first form in association with primary myotubes and subsequently separate from them. We show here that during this process N-cadherin and the different structural forms of NCAM are regulated in a pattern that involves both temporal changes in expression and localization to particular regions of the muscle cell surface. In particular, levels of N-cadherin on maturing myotubes are decreased, and the form of NCAM synthesized by the muscle changes from a transmembrane non-polysialylated to a lipid-linked polysialylated membrane protein. Moreover, while NCAM was distributed on all myotube surfaces, the polysialylated form of NCAM was restricted to regions of the myotube surface that had recently separated from neighboring cells. We previously found that blockade of nerve-induced activity by d-Tubocurarine perturbed muscle cell interactions, resulting in a failure of myotubes to separate. We now show that this activity blockade also alters adhesion molecule expression. First, N-cadherin was no longer down-regulated in maturing myotubes, and its persistence on the surfaces of mature myotubes may partly explain their failure to separate. Secondly, the developmental switch from transmembrane to lipid-linked NCAM did not occur, and polysialylated NCAM was no longer formed. As the unusual physical properties of PSA have been proposed to impede cell-cell interactions, this alteration would also be expected to compromise cell separation. Together, these results suggest that the regulated expression of both N-cadherin and NCAM isoforms including their polysialylation, is an essential mechanism for the normal separation of secondary myotubes from primary myotubes.

PST and STX are polysialyltransferases that form polysialic acid in the neural cell adhesion molecule (N-CAM), although it is not known why these two polysialyltransferases exist. In the present study, we have first isolated cDNA encoding human STX, which includes 5-untranslated sequence. Northern blot analysis, using this cDNA and PST cDNA previously isolated by us, demonstrated that PST and STX are expressed in different fetal and adult tissues. STX is primarily expressed in embryonic tissues, but only modestly in adult heart, brain, and thymus. PST, on the other hand, is continuously expressed in adult heart, brain, thymus, spleen, small and large intestines, and peripheral blood leukocytes. In various parts of adult brain, the relative amount of PST and STX appears to be substantially different depending on the regions. The analysis by in situ hybridization of mouse adult brain, however, suggests that polysialic acid in the hippocampal formation is synthesized by both STX and PST. HeLa cells doubly transfected with the isolated STX cDNA and N-CAM cDNA supported neurite outgrowth much better than HeLa cells expressing N-CAM alone. However, polysialic acid synthesized by PST appears to be a better substratum than that synthesized by STX. Moreover, the genes for PST and STX were found to reside at chromosome 5, band p21 and chromosome 15, band q26, respectively. These results, taken together, strongly suggest that PST and STX are expressed distinctly in tissue-specific and cell-specific manners and that they apparently have distinct roles in development and organogenesis.Polysialic acid is a developmentally regulated glycan composed of a linear homopolymer of ␣-2,8-linked sialic acid residues. Polysialic acid is mainly attached to the neural cell adhesion molecule (N-CAM) 1 and is more abundant in embryonic brain than adult brain (1-3). Presence of this large negatively charged carbohydrate modulates the adhesive property of N-CAM, and removal of polysialic acid increases binding between N-CAMs (4, 5). During embryonic development, the polysialylated embryonic from of N-CAM is restricted to migrating cells (6, 7), and the removal of polysialic acid from the N-CAM during embryonic development affects motor-axon projection patterns (8). Polysialylated N-CAM was also shown to attenuate cell-cell interactions mediated by other cell adhesion molecules (9 -11).Recently, we and others have cloned cDNAs encoding human, hamster, and mouse polysialyltransferases (PST for human, PST-1 for hamster, and ST8Sia IV for mouse, respectively) (12-14). The amino acid sequences of PST and PST-1 are more than 97% identical. Both PST and PST-1 directed the expression of polysialic acid on the cell surface. The same studies also revealed that PST is highly homologous to STX (sialyltransferase X, or ST8Sia II) (12, 13), and STX and PST have 59% identity at the amino acid level. STX was originally cloned as a developmentally regulated sialyltransferase from the rat fetal brain (15). Although the substrate specificity of STX was not kno...

The inferior olive (IO) appears to be organized functionally in discrete subnuclei that receive transmitter-specific inputs. In particular, the IO receives a GABAergic input that is most densely concentrated in the beta-nucleus. In this experiment, we examined the functional specificity of neurons in the beta-nucleus of the IO of rabbits by recording their activity during natural vestibular and optokinetic stimulation. Rabbits were anesthetized and positioned in a triaxial servo- controlled rate table with the head fixed at the center of rotation. Contour-rich visual stimuli were rear-projected onto a 70 deg tangent screen and moved at constant velocities. Recording sites in the beta-nucleus were verified by subsequent histological analysis of marking microlesions. Neurons in the beta-nucleus responded to roll vestibular stimulation about the longitudinal axis. These neurons were excited when the rabbit was rolled onto the side which was contralateral to the recording site, and inhibited when the rabbit was rolled ipsilaterally. Thirty-eight of the 75 beta-nucleus neurons that were responsive to roll vestibular stimulation also responded to static tilt, indicating an otolithic as well as a vertical semicircular canal origin of the vestibular input. The modulated activity of none of the neurons could be attributed to stimulation of the horizontal semicircular canals. All the recorded neurons were found in a region of the beta-nucleus that was retrogradely labeled following HRP injections into the cerebellar nodulus. Using a "null point" technique, we found that there was a differential projection of information from the anterior and posterior semicircular canals onto to the beta-nucleus. Stimulation of the ipsilateral anterior-contralateral posterior semicircular canals modulates activity of the neurons in the caudal 500 microns of the beta-nucleus. Stimulation of the ipsilateral posterior-contralateral anterior semicircular canals modulates activity of neurons located more rostrally. beta-nucleus neurons and the olivocerebellar circuits in which they participate may constitute an important pathway for the control and adaptive modification of postural reflexes.

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