Nogo-A is a potent neurite growth inhibitor in vitro and plays a role both in the restriction of axonal regeneration after injury and in structural plasticity in the CNS of higher vertebrates. The regions that mediate inhibition and the topology of the molecule in the plasma membrane have to be defined. Here we demonstrate the presence of three different active sites: (1) an N-terminal region involved in the inhibition of fibroblast spreading, (2) a stretch encoded by the Nogo-A-specific exon that restricts neurite outgrowth and cell spreading and induces growth cone collapse, and (3) a C-terminal region (Nogo-66) with growth cone collapsing function. We show that Nogo-A-specific active fragments bind to the cell surface of responsive cells and to rat brain cortical membranes, suggesting the existence of specific binding partners or receptors. Several antibodies against different epitopes on the Nogo-A-specific part of the protein as well as antisera against the 66 aa loop in the C-terminus stain the cell surface of living cultured oligodendrocytes. Nogo-A is also labeled by nonmembrane-permeable biotin derivatives applied to living oligodendrocyte cultures. Immunofluorescent staining of intracellular, endoplasmic reticulum-associated Nogo-A in cells after selective permeabilization of the plasma membrane reveals that the epitopes of Nogo-A, shown to be accessible at the cell surface, are exposed to the cytoplasm. This suggests that Nogo-A could have a second membrane topology. The two proposed topological variants may have different intracellular as well as extracellular functions.

Nogo-A is a neurite growth inhibitor involved in regenerative failure and restriction of structural plasticity in the adult CNS. Three major protein products (Nogo-A, -B, and -C) are derived from the nogo gene. Here we describe the embryonic and postnatal expression of the three Nogo isoforms in the rat by in situ hybridization and immunohistochemistry. Northern and Western blot analysis indicated that Nogo-A is predominantly expressed in the nervous system with lower levels also present in testis and heart. In CNS myelin, confocal and immunoelectron microscopy revealed that Nogo-A is expressed in oligodendrocyte cell bodies and processes and localized in the innermost adaxonal and outermost myelin membranes. Additionally, we find Nogo-A to be expressed by projection neurons, in particular during development, and by postmitotic cells in the developing cortex, spinal cord, and cerebellum. The expression levels of Nogo-A/B were not changed significantly after traumatic lesions to the cortex or spinal cord. Nogo-B showed widespread expression in the central and peripheral nervous systems and other peripheral tissues. Nogo-C was mainly found in skeletal muscle, but brain and heart were also found to express this isoform. The localization of Nogo-A in oligodendrocytes fits well with its role as a myelin-associated inhibitor of regenerative fiber growth and structural plasticity. However, expression of Nogo-A in other tissues and, in particular, in neurons and the widespread expression of the two shorter isoforms, Nogo-B and -C, suggest that the Nogo family of proteins might have function(s) additional to the neurite growth-inhibitory activity.

The zebrafish epithalamus, consisting of the pineal complex and flanking dorsal habenular nuclei, provides a valuable model for exploring how left-right differences could arise in the vertebrate brain. The parapineal lies to the left of the pineal and the left habenula is larger, has expanded dense neuropil, and distinct patterns of gene expression from the right habenula. Under the influence of Nodal signaling, positioning of the parapineal sets the direction of habenular asymmetry and thereby determines the left-right origin of habenular projections onto the midbrain target, the interpeduncular nucleus (IPN). In zebrafish with parapineal reversal, neurons from the left habenula project to a more limited ventral IPN region where right habenular axons would normally project. Conversely, efferents from the right habenula adopt a more extensive dorsoventral IPN projection pattern typical of left habenular neurons. Three members of the leftover-related KCTD (potassium channel tetramerization domain containing) gene family are expressed differently by the left and right habenula, in patterns that define asymmetric subnuclei. Molecular asymmetry extends to protein levels in habenular efferents, providing additional evidence that left and right axons terminate within different dorsoventral regions of the midbrain target. Laser-mediated ablation of the parapineal disrupts habenular asymmetry and consequently alters the dorsoventral distribution of innervating axons. The results demonstrate that laterality of the dorsal forebrain influences the formation of midbrain connections and their molecular properties.

Myelination, the process by which glial cells ensheath and electrically insulate axons, has been investigated intensely. Nevertheless, knowledge of how myelination is regulated or how myelinating cells communicate with neurons is still incomplete. As a prelude to genetic analyses of these processes, we have identified zebrafish orthologues of genes encoding major myelin proteins and have characterized myelination in the larval zebrafish. Expression of genes corresponding to proteolipid protein (PLP/DM20), myelin protein zero (P0), and myelin basic protein (MBP) is detected at 2 days postfertilization (dpf), first in the ventral hindbrain, close to the midline. During the next 8 days, expression spreads rostrally to the midbrain and optic nerve, and caudally to the spinal cord. DM20 is expressed in the CNS only, while MBP transcripts are detected both in the CNS and in Schwann cells of the lateral line, cranial nerves, and spinal motor nerves. Unlike its closest homologue, trout IP1, zebrafish P0 transcripts were restricted to the CNS. Ultrastructurally, the expression of myelin genes correlated well with myelination, although myelination showed a temporal lag. Myelinated axons were first detected at 4 dpf in the ventral hindbrain, where they were loosely wrapped by processes of glia cells. By 7 dpf, bundles of heavily myelinated axons were observed in the same region. Axons in the lateral line and optic nerves were also surrounded by compact myelin. Conservation in gene expression patterns and the early appearance of myelinated axons, support using the zebrafish to dissect the process of myelination by a genetic approach.

deficits [1]. Small injuries can result in transient impairments, but the mechanisms of recovery are poorly understood [2]. At the cortical level, rearrangements of the sensory and motor representation maps often parallel recovery [3, 4]. Results and discussion In the sensory system, studies have shown thatAdult female Lewis rats were anesthetized, and their left cortical and subcortical mechanisms contribute to motor cortex was exposed and stimulated by a tungsten map rearrangements [5, 6], but for the motor system microelectrode as described in the supplemental materials the situation is less clear. Here we show that large-section, with the aim to identify an area that yields consisscale structural changes in the spared rostral part tent and exclusive hind-limb (HL) responses. Consistent of the spinal cord occur simultaneously with shifts with previous studies [8, 9], this was found to be the case of a hind-limb motor cortex representation after for stimulations at 2 mm caudal to bregma and 1.5-2.5 mm traumatic spinal-cord injury. By intracortical lateral to the midline. Stimulation at these coordinates microstimulation, we defined a cortical area that usually activated hind-limb flexor muscles and, more consistently and exclusively yielded hind-limb rarely, extensor muscles. In two animals, muscles of the muscle responses in normal adult rats. Four weeks tail base were activated. We determined the stimulus after a bilateral transsection of the corticospinal threshold by increasing stimulus intensities and recording tract (CST) in the lower thoracic spinal cord, we again the lowest intensity at which a consistent movement was stimulated this cortical field and found forelimb, observed [7]. The average threshold was 49.6 A (Ϯ 18.1 whisker, and trunk responses, thus demonstrating standard deviation [SD], n ϭ 32), and animals with threshreorganization of the cortical motor olds above 100 A were not used for physiological evaluarepresentation. Anterograde tracing of corticospinal tion. After the stimulation, the animals received an addifibers originating from this former hind-limb area tional dose of anaesthetic (xylazine), and the scalp wasrevealed that sprouting greatly increased the sutured. Subsequently, the spinal cord was exposed at a normally small number of collaterals that lead into mid-thoracic level (T8), and the dorsal columns, including the cervical spinal cord rostral to the lesion. We the main component of the corticospinal tract (CST), were conclude that the corticospinal motor system has transsected bilaterally as described [10]. Care was taken greater potential to adapt structurally to lesions than not to compromise the dorsolaterally running rubrospinal was previously believed and hypothesize that this tract (Figure 1). In a control group, the spinal cord was spontaneous growth response is the basis for the exposed but not transsected (sham operation). The tissue observed motor representation rearrangements overlying the spinal cord was sutured, and the animals and contributes to functional recov...

Axons in the CNS of higher vertebrates generally fail to regenerate after injury. This lack of regeneration is crucially influenced by neurite growth inhibitory protein constituents of CNS myelin. We have shown previously that a monoclonal antibody (mAb IN-1) capable of binding and neutralizing Nogo-A, a myelin-associated inhibitor of neurite growth, can induce long-distance axonal regeneration and increased structural plasticity with improved functional recovery in rat models of CNS injury. In this paper we demonstrate that a partially humanized, recombinant Fab fragment (rIN-1 Fab) derived from the original mAb IN-1, was able to promote long-distance regeneration of injured axons in the spinal cord of adult rats. When infused into a spinal cord injury site, regrowth of corticospinal fibers in 11 of 18 animals was observed after a survival time of 2 weeks. Regenerating fibers grew for Ͼ9 mm beyond the lesion site and arborized profusely in the distal cord. Regenerated fibers formed terminal arbors with varicosities in the spinal cord gray matter, strongly resembling synaptic points of contact to neurons in the spinal cord distal to the lesion. In animals that had received a bovine serum albumin solution or a recombinant IN-1 fragment that had been mutated in the antigen binding site (mutIN-1 Fab), no significant growth beyond normal lesion-induced sprouting was observed. Neutralization of endogenous nerve growth inhibitors represents a novel use of recombinant antibody technology with potential therapeutic applications after traumatic CNS lesions.

The cells of origin, the course, and termination patterns of the ventral, uncrossed component of the rat corticospinal tract (CST) was investigated by using retrograde and anterograde tracing methods. Anterograde tracing with biotin dextran-amine (BDA) revealed the position and detailed morphology of CST fibers in the spinal cord. Cross sections on spinal levels C4, T8, and L4 showed labeled fibers in the ipsilateral ventral funiculus on all levels. Although ipsilateral ventral CST fibers run close to the midline in the cervical cord, they were found to disperse more in the ventromedial funiculus at lower spinal levels. To study the termination patterns of the ipsilateral ventral projection, a dorsal spinal cord hemisection was performed at level T8, severing the crossed dorsomedial and dorsolateral components but leaving ipsilateral ventral running fibers intact. These fibers were observed to have sometimes several collaterals with terminal arbors extending into different spinal segments, innervating mostly laminae III-VI. Structures closely resembling synaptic boutons were identified in these arbors. By retrograde tracing in animals with dorsal spinal cord hemisection, we found labeled cells equally distributed throughout the spinally projecting cortical areas corresponding to the level of tracer injection. Labeled cells were found in layer V. The diameter of the labeled cells was not significantly different from other spinally projecting cortical neurons. In summary, a neuroanatomically complete ipsilateral, ventral corticospinal projection down to low spinal levels was found. The large extension of the terminal arborizations in intermediate laminae of the spinal cord suggests a modulatory role of this CST component.

The zebrafish has become an important model organism to study myelination during development and after a lesion of the adult central nervous system (CNS). Here, we identify Claudin k as a myelin-associated protein in zebrafish and determine its localization during development and adult optic nerve regeneration. We find Claudin k in subcellular compartments consistent with location in autotypic tight junctions of oligodendrocytes and myelinating Schwann cells. Expression starts in the hindbrain at 2 days (mRNA) and 3 days (protein) postfertilization and is maintained in adults. A newly generated claudin k:green fluorescent protein (GFP) reporter line allowed us to characterize oligodendrocytes in the adult retina that express Claudin k and olig2, but not P0 and uniquely only form loose wraps of membrane around axons. After a crush of the adult optic nerve, Claudin k protein levels were first reduced and then recovered within 4 weeks postlesion, concomitant with optic nerve myelin de- and regeneration. During optic nerve regeneration, oligodendrocytes, many of which were newly generated, repopulated the lesion site and exhibited increasing morphological complexity over time. Thus, Claudin k is a novel myelin-associated protein expressed by oligodendrocytes and Schwann cells from early stages of wrapping and myelin formation in zebrafish development and adult regeneration, suggesting important functions of the gene for myelin formation and maintenance. Our Claudin k antibodies and claudin k:GFP reporter line represent excellent ways to visualize oligodendrocyte and Schwann cell differentiation in vivo.