Netrin-1 promotes thalamic axon growth and is required for proper development of the thalamocortical projection.

The thalamocortical axon (TCA) projection originates in dorsal thalamus, conveys sensory input to the neocortex, and has a critical role in cortical development. We show that the secreted axon guidance molecule netrin-1 acts in vitro as an attractant and growth promoter for dorsal thalamic axons and is required for the proper development of the TCA projection in vivo. As TCAs approach the hypothalamus, they turn laterally into the ventral telencephalon and extend toward the cortex through a population of netrin-1-expressing cells. DCC and neogenin, receptors implicated in mediating the attractant effects of netrin-1, are expressed in dorsal thalamus, whereas unc5h2 and unc5h3, netrin-1 receptors implicated in repulsion, are not. In vitro, dorsal thalamic axons show biased growth toward a source of netrin-1, which can be abolished by netrin-1-blocking antibodies. Netrin-1 also enhances overall axon outgrowth from explants of dorsal thalamus. The biased growth of dorsal thalamic axons toward the internal capsule zone of ventral telencephalic explants is attenuated, but not significantly, by netrin-1-blocking antibodies, suggesting that it releases another attractant activity for TCAs in addition to netrin-1. Analyses of netrin-1 -/- mice reveal that the TCA projection through the ventral telencephalon is disorganized, their pathway is abnormally restricted, and fewer dorsal thalamic axons reach cortex. These findings demonstrate that netrin-1 promotes the growth of TCAs through the ventral telencephalon and cooperates with other guidance cues to control their pathfinding from dorsal thalamus to cortex.

Activity-dependent cortical target selection by thalamic axons.

Connections in the developing nervous system are thought to be formed initially by an activity-independent process of axon pathfinding and target selection and subsequently refined by neural activity. Blockade of sodium action potentials by intracranial infusion of tetrodotoxin in cats during the early period when axons from the lateral geniculate nucleus (LGN) were in the process of selecting visual cortex as their target altered the pattern and precision of this thalamocortical projection. The majority of LGN neurons, rather than projecting to visual cortex, elaborated a significant projection within the subplate of cortical areas normally bypassed. Those axons that did project to their correct target were topographically disorganized. Thus, neural activity is required for initial targeting decisions made by thalamic axons as they traverse the subplate.

Many major CNS axon projections develop normally in the absence of semaphorin III.

The semaphorins constitute a large gene family of transmembrane and secreted molecules, many of which are expressed in the nervous system. Genetic studies in Drosophila have revealed a role for semaphorins in axon guidance and synapse formation, and several in vitro studies in mice have demonstrated a dramatic chemorepellent effect of semaphorin III (Sema III) on the axons of several populations of neurons. To investigate the function of Sema III during in vivo axon guidance in the mammalian CNS, we studied the development of axonal projections in mutant mice lacking Sema III. Projections were studied for which either the in vitro evidence suggests a role for Sema III in axon guidance (e.g., cerebellar mossy fibers, thalamocortical axons, or cranial motor neurons) or the in vivo expression suggests a role for Sema III in axon guidance (e.g., cerebellar Purkinje cells, neocortex). We find that many major axonal projections, including climbing fiber, mossy fiber, thalamocortical, and basal forebrain projections and cranial nerves, develop normally in the absence of Sema III. Despite its in vitro function and in vivo expression, it appears as if Sema III is not absolutely required for the formation of many major CNS tracts. Such data are consistent with recent models suggesting that axon guidance is controlled by a balance of forces resulting from multiple guidance cues. Our data lead us to suggest that if Sema III functions in part to guide the formation of major axonal projections, then it does so in combination with both other semaphorins and other families of guidance molecules.

Activity-dependent regulation of NMDAR1 immunoreactivity in the developing visual cortex.

NMDA receptors have been implicated in activity-dependent synaptic plasticity in the developing visual cortex. We examined the distribution of immunocytochemically detectable NMDAR1 in visual cortex of cats and ferrets from late embryonic ages to adulthood. Cortical neurons are initially highly immunostained. This level declines gradually over development, with the notable exception of cortical layers 2/3, where levels of NMDAR1 immunostaining remain high into adulthood. Within layer 4, the decline in NMDAR1 immunostaining to adult levels coincides with the completion of ocular dominance column formation and the end of the critical period for layer 4. To determine whether NMDAR1 immunoreactivity is regulated by retinal activity, animals were dark-reared or retinal activity was completely blocked in one eye with tetrodotoxin (TTX). Dark-rearing does not cause detectable changes in NMDAR1 immunoreactivity. However, 2 weeks of monocular TTX administration decreases NMDAR1 immunoreactivity in layer 4 of the columns of the blocked eye. Thus, high levels of NMDAR1 immunostaining within the visual cortex are temporally correlated with ocular dominance column formation and developmental plasticity; the persistence of staining in layers 2/3 also correlates with the physiological plasticity present in these layers in the adult. In addition, visual experience is not required for the developmental changes in the laminar pattern of NMDAR1 levels, but the presence of high levels of NMDAR1 in layer 4 during the critical period does require retinal activity. These observations are consistent with a central role for NMDA receptors in promoting and ultimately limiting synaptic rearrangements in the developing neocortex.

Individual axon morphology and thalamocortical topography in developing rat somatosensory cortex.

The morphology of individual thalamocortical axons in developing rat primary somatosensory cortex was studied using lipophilic tracers. Anterograde labeling with lipophilic dyes demonstrated a topographical organization of thalamocortical projections exiting the thalamus as early as embryonic day (E) 16; retrograde labeling studies demonstrated topography of these projections as they reached the cortex as early as E18. At E17, axons course tangentially within the intermediate zone and turn or branch near the deepest layer of cortex (layer VIb), suggesting the presence of guidance cues in this region. Axons appear to grow and branch progressively within layers VIb and VIa during the following days; axons in the intermediate zone may give rise to radially directed branches. Individual axons appear to grow steadily and progressively into the cortex, with the leading front of axons at the transition zone between the cortical plate (CP) and the differentiating cortical layers. At birth (P0), thalamocortical axons extend radially through layers VIa and V and emit branches within these layers; some axons reach the CP. By P1, layer IV has begun to differentiate and axons begin to form a few simple branches in the vicinity of the layer IV cells. Over the ensuing week, axons generate more branches within layer IV, but the tangential extent of individual axon arbors does not exceed the width of a barrel. By P7, individual axons overlap within barrel clusters, and individual axons span the width of a cluster. These observations indicate that thalamic afferents develop by progressive growth of arbors that remain spatially restricted, rather than by overbranching and retracting arbors.

Rapid alteration of thalamocortical axon morphology follows peripheral damage in the neonatal rat.

The effect of day of birth (postnatal day 0; P0) infraorbital nerve section on the morphology of individual thalamocortical axons in rat somatosensory cortex was examined on P3. Thalamic fibers were labeled in fixed brains with the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, and individual photo-converted thalamocortical fibers were reconstructed. In normal animals on P3, axon arbor terminal formation within layer IV has commenced and terminal arbor width is comparable to that of a cortical "barrel." After infraorbital nerve section, the average width of thalamocortical terminal arbors is significantly greater than is the average arbor width of normal rats of the same age; however, neither the number of branches per terminal arbor nor total arbor length differs between groups. These observations suggest that the role of the periphery in guiding terminal arbor formation is exerted both very rapidly and at the level of the single thalamic axon. Further, these results indicate a close association between individual axon terminal arbor morphology and pattern formation in the rat somatosensory cortex.

Early ingrowth of thalamocortical afferents to the neocortex of the prenatal rat.

The initial ingrowth of thalamocortical afferents into the presumptive somatosensory cortex was examined in the fetal rat. Thalamic fibers were labeled in fixed brains with the carbocyanine dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI). On embryonic day 16, thalamocortical afferents arrive in the neocortex and course tangentially within the intermediate zone immediately underneath the cortical plate. By embryonic day 17, thalamocortical fibers have begun their radial growth into cortex and their arbors span the cell-sparse zone between layer VIb and the bottom of the cortical plate. By the day of birth (embryonic day 21), thalamocortical fibers from a dense plexus within layers VI and V below the dense cortical plate. Our observations indicate that in the rat thalamic afferents arrive in the cortex at a very early age and arborize within the forming cortical layers without an apparent "waiting" period.

Early climbing fiber interactions with Purkinje cells in the postnatal mouse cerebellum.

The time and place of initial contacts between afferent axons and their target cells are not known for most regions of the mammalian CNS. To address this issue, we have selectively visualized afferent climbing fiber axons together with their synaptic targets, Purkinje cells, in postnatal mouse cerebellum. Climbing fibers were orthogradely labeled by injection of rhodamine isothiocyanate into their brainstem source, the inferior olivary nucleus. Purkinje cells were localized with an antibody to a calcium-binding protein, calbindin D-28k (CaBP), in the same section or in adjacent sections. A novel view of the olivocerebellar projection and the morphology of climbing fiber arbors prior to the well-known "nest" stage has emerged from this analysis. At birth, climbing fibers project into the zone of Purkinje cells, before these cells have aligned into a monolayer. During this phase, climbing fibers have simple morphologies consisting of relatively unbranched terminal arbors and small tapered growing tips. Purkinje cells are arranged 3-6 cells deep and have tufted dendrites and relatively smooth somata. By postnatal days 3-4, climbing fibers branch over several adjacent Purkinje cell perikarya, which are still organized in a band several cells thick. From postnatal days 5-7, when climbing fibers subsequently make focused nests on individual cells, Purkinje somata are smoother and form a more distinct monolayer. Up to this time, however, climbing fibers continue to associate with Purkinje perikarya, even though Purkinje cell dendrites have emerged and branched extensively. By postnatal days 8-10, climbing fiber terminals climb onto the trunk of the relatively mature Purkinje dendritic tree. At birth, mossy fibers originating from the pontine nuclei resemble immature climbing fibers in that they also have a simple unbranched morphology and growing tips, but project only so far as the internal granule cell layer. Occasional individual fibers reach into the Purkinje zone both at postnatal day 0 and postnatal day 4, confirming that the fibers formerly described as "combination fibers" (Mason and Gregory, S4. J. Neurosci, 4:1715-1735) can be mossy in origin. These data demonstrate that climbing fibers project among Purkinje cells earlier than suspected, before these afferents begin to arborize and form pericellular nests. Our observations are not in accord with the view derived from autoradiographic tracing studies that as in other cortical areas, climbing afferents wait in the vicinity of Purkinje cells in the early neonatal period, then advance onto these cells in synchrony with Purkinje cell alignment into a monolayer and dendritic maturation.(ABSTRACT TRUNCATED AT 400 WORDS)