Neural cell adhesion molecule (NCAM-/-) null mice show impaired sensitization of the startle response.

The neural cell adhesion molecule (NCAM) plays important roles in development of the nervous system and in synaptic plasticity and memory formation in the adult. The present study sought to further investigate the role of NCAM in learning by testing habituation and footshock sensitization learning of the startle response (SR) in NCAM null mutant (NCAM-/-) and wildtype littermate (NCAM+/+) mice. Whereas habituation is a form of non-associative learning, footshock sensitization is induced by rapid contextual fear conditioning. Habituation was tested by repetitive presentation of acoustic and tactile startle stimuli. Although NCAM-/- mice showed differences in sensitivity in both stimulus modalities, habituation learning was intact in NCAM-/- mice, suggesting that NCAM does not play a role in the mechanisms underlying synaptic plasticity in the startle pathway. Footshock sensitization was elicited by presentation of electric footshocks between two series of acoustic stimuli. In contrast to habituation, footshock sensitization learning was attenuated in NCAM-/- mice: the acoustic SR increase after the footshocks was lower in the mutant than in wildtype mice, indicating that NCAM plays an important role in the relevant brain areas, such as amygdala and/or the hippocampus.

Enhanced cortical and hippocampal neuronal excitability in mice deficient in the extracellular matrix glycoprotein tenascin-R.

Mice deficient in the extracellular matrix protein tenascin-R (TN-R-/- mice) show several indices of impaired perisomatic inhibition in hippocampal slices. The present study examined electroencephalograms (EEGs) and auditory-evoked potentials (AEPs) in freely moving TN-R-/- and wild-type control mice, focusing on the hippocampal CA1 field and cerebral cortex. TN-R-/- mice expressed normal high-frequency oscillations (ripples) in CA1 and only a slight reduction of peak theta frequency. In contrast, their hippocampal gamma oscillations were significantly enhanced in amplitude. Also, the amplitude of the cortical EEG of TN-R-/- mice was increased over a wide frequency range. The amplitude of cortical and, to a lesser degree hippocampal, AEPs was clearly enhanced in TN-R-/- mice. In addition, response habituation to repeated sound stimuli was significantly attenuated in TN-R-/- mice. These findings indicate that tenascin-R is involved in the regulation of certain inhibitory mechanisms in the intact brain.

The role of L1 in axon pathfinding and fasciculation.

The neural cell adhesion molecule L1 has been found to play important roles in axon growth and fasciculation. Our main objective was to determine the role of L1 during the development of connections between thalamus and cortex. We find that thalamocortical and corticothalamic axons in mice lacking L1 are hyperfasciculated, a subset of thalamocortical axons make pathfinding errors and thalamocortical axon growth cones are abnormally long in the subplate. These defects occur despite formation of six cortical layers and formation of topographically appropriate thalamocortical connections. The loss of L1 is accompanied by loss of expression of ankyrin-B, an intracellular L1 binding partner, suggesting that L1 is involved in the regulation of Ank2 stability. We postulate that the pathfinding errors, growth cone abnormalities and hyperfasciculation of axons following loss of L1 reflect both a shift in binding partners among axons and different substrates and a loss of appropriate interactions with the cytoskeleton.

The adhesion molecule TAG-1 mediates the migration of cortical interneurons from the ganglionic eminence along the corticofugal fiber system.

Cortical nonpyramidal cells, the GABA-containing interneurons, originate mostly in the medial ganglionic eminence of the ventral telencephalon and follow tangential migratory routes to reach the dorsal telencephalon. Although several genes that play a role in this migration have been identified, the underlying cellular and molecular cues are not fully understood. We provide evidence that the neural cell adhesion molecule TAG-1 mediates the migration of cortical interneurons. We show that the migration of these neurons occurs along the TAG-1-expressing axons of the developing corticofugal system. The spatial and temporal pattern of expression of TAG-1 on corticofugal fibers coincides with the order of appearance of GABAergic cells in the developing cortex. Blocking the function of TAG-1, but not of L1, another adhesion molecule and binding partner of TAG-1, results in a marked reduction of GABAergic neurons in the cortex. These observations reveal a mechanism by which the adhesion molecule TAG-1, known to be involved in axonal pathfinding, also takes part in neuronal migration.

Binding of mistletoe lectins to cutaneous malignant melanoma: implications for prognosis and therapy.

BACKGROUND: Glycoconjugates, as detected by lectin histochemistry, have been implicated in metastasis formation in many neoplasias. However, no data concerning the three mistletoe lectins (MLs) and the spread of malignant melanoma have been published. MATERIALS AND METHODS: The binding status of ML-I, -II and -III was histochemically assessed in 100 malignant melanomas and correlated with metastasis in a 10 year follow-up period. Furthermore, the staining intensity of the three MLs, scored from negative (-) to very intense (+ + +), was evaluated. RESULTS: Kaplan-Meier analsis revealed that very intense binding (+ + +) of ML-I was positively-correlated with metastasis (p=0.044). CONCLUSION: Since ML-I is specific for galactose, high density galactose expression in malignant melanoma is a predictor of poor prognosis.

Molecular cloning and expression of an N-acetylgalactosamine-4-O-sulfotransferase that transfers sulfate to terminal and non-terminal beta 1,4-linked N-acetylgalactosamine.

We have identified and characterized an N-acetylgalactosamine-4-O-sulfotransferase designated GalNAc-4-ST2 (GenBank(TM) accession number ) based on its homology to HNK-1 sulfotransferase (HNK-1 ST). The cDNA predicts an open reading frame encoding a type II membrane protein of 443 amino acids with a 12-amino acid cytoplasmic domain, a 23-amino acid transmembrane domain, and a 408-amino acid luminal domain containing four potential N-linked glycosylation sites. GalNAc-4-ST2 displays a high degree of amino acid sequence identity with GalNAc-4-ST1 (46%), HNK-1 ST (23%), chondroitin 4-O-sulfotransferase-1 (C4ST-1) (27%), and chondroitin 4-O-sulfotransferase-2 (C4ST-2) (24%). GalNAc-4-ST2 transfers sulfate to the C-4 hydroxyl of terminal beta1,4-linked GalNAc in the sequence GalNAc-beta1,4GlcNAcbeta-R found on N-linked oligosaccharides and nonterminal beta1,4-linked GalNAc in chondroitin and dermatan. The translated region of GalNAc-4-ST2 is encoded by five exons located on human chromosome 18q11.2. Northern blot analysis reveals a 2.1-kilobase transcript. GalNAc-4-ST2 message is most highly expressed in trachea and to a lesser extent in heart, liver, pancreas, salivary gland, and testis. The I.M.A.G.E. cDNA clone 49547 contains a putative GalNAc-4-ST2 splice form with an open reading frame encoding a protein of 358 amino acids that lacks the transmembrane domain and the stem region. This form of GalNAc-4-ST2 is not retained by transfected cells and is active against chondroitin but not terminal beta1,4-linked GalNAc. Thus, as with GalNAc-4-ST1, sequences N-terminal to the catalytic domain contribute to the specificity of GalNAc-4-ST2 toward terminal beta1,4-linked GalNAc.

The cell recognition molecule CHL1 is strongly upregulated by injured and regenerating thalamic neurons.

Close homologue of L1 (CHL1) is a cell recognition molecule known to promote axonal growth in vitro. We have investigated the expression of CHL1 mRNA by regenerating central nervous system (CNS) neurons, by using in situ hybridisation 3 days to 10 weeks following the implantation of living and freeze-killed peripheral nerve autografts into the thalamus of adult rats. At all survival times after implantation of living grafts, neurons of the thalamic reticular nucleus (TRN), close to the graft tip and up to 1 mm away from it, displayed strong signal for CHL1 mRNA, even though TRN neurons show very low levels of CHL1 mRNA expression in unoperated animals. When the cell bodies of regenerating neurons were identified by retrograde labelling from the distal portion of the grafts, 4-6 weeks after operation, most of the labelled cells were found in the TRN and could be shown to haveupregulated CHL1 mRNA. In addition, some neurons in dorsal thalamic nuclei near the graft tip transiently upregulated CHL1 mRNA during the first 3 weeks after graft implantation, and glial cells showing CHL1 mRNA expression were present at the brain/graft interface 3 days to 2 weeks after operation. Freeze-killed grafts, into which axons do not regenerate, caused a transient upregulation of CHL1 in very few TRN neurons near the graft tip and in glial cells at the brain/graft interface but did not produce prolonged CHL1 mRNA expression. CHL1 can therefore be added to the list of molecules (including GAP-43, L1, and c-jun) strongly expressed by CNS neurons that regenerate their axons into nerve grafts, but not by those neurons that fail to regenerate their axons.

The close homologue of the neural adhesion molecule L1 (CHL1): patterns of expression and promotion of neurite outgrowth by heterophilic interactions.

The close homologue of L1 (CHL1), a member of the L1 family of neural adhesion molecules, is first expressed at times of neurite outgrowth during brain development, and is detectable in subpopulations of neurons, astrocytes, oligodendrocyte precursors and Schwann cells of the mouse and rat. Aggregation assays with CHL1-transfected cells show that CHL1 does not promote homophilic adhesion or does it mediate heterophilic adhesion with L1. CHL1 promotes neurite outgrowth by hippocampal and small cerebellar neurons in substrate-bound and soluble form. The observation that CHL1 and L1 show overlapping, but also distinct patterns of synthesis in neurons and glia, suggests differential effects of L1-like molecules on neurite outgrowth.

Expression of a Na,K-ATPase beta 3 subunit during development of the zebrafish central nervous system.

Zebrafish beta 3, a full length cDNA clone encoding a zebrafish Na,K-ATPase beta subunit, was isolated. The protein shares highest homology with the beta 3 subunits of amphibians and mammals, slightly less homology with the beta 2 subunits, and is distinct from the beta 1 subunits. The fish beta subunit co-assembled with alpha subunits to form Na,K-ATPase enzymes when expressed in Xenopus oocytes. Embryonic expression was first detected by whole-mount in situ hybridization between 8-12 hr post-fertilization (hpf) in the head mesoderm. Subsequently, and up to 24 hpf, the mRNA was confined to four dorsal domains in the anterior neural tube. After a transient downregulation during the second day, expression was again conspicuous in the nervous system of 3-day-old larvae. Based on its distribution pattern, the fish beta subunit could be involved in setting up regional identities in the developing fish CNS and in the differentiation of distinct cell types.

Effects of NMDA receptor blockade in the developing rat somatosensory cortex on the expression of the glia-derived extracellular matrix glycoprotein tenascin-C.

The patterning of synaptic connections during development is thought to be influenced by the correlation of neuronal impulse activity. N-methyl-D-aspartate (NMDA) receptors have been implicated in the reorganization of thalamocortical afferents in the visual system. The topographic mapping of the periphery of sensory systems onto the somatosensory cortex in the whisker-barrel field of rodents has served as another important paradigm in the study of extrinsic influences on synaptic rearrangements. In a search for the molecular cues that may contribute to synaptic plasticity, we have investigated the distribution of the glia-derived extracellular matrix glycoprotein tenascin-C, which is highly expressed during the formation of the barrel field map around birth and delineates the boundaries between barrel fields after segregation of afferent inputs. Here we show that systemic and local application of NMDA receptor antagonists at postnatal day 2 inhibited the down-regulation of tenascin mRNA and protein by postnatal day 6 and prevented the appearance of tenascin-positive barrel field boundaries. Furthermore, barrels were not distinguishable by Nissl staining, and segregation of thalamocortical afferents as monitored by anterograde Dil tracing and acetylcholinesterase histochemistry was not complete. These observations indicate that expression of tenascin-C and segregation of afferent inputs are modified by NMDA receptor-dependent neuronal activity.

Transient expression of NADPH diaphorase activity in the mouse whisker to barrel field pathway.

Development of the topographic map of the somatosensory cortex of rodents appears to depend on fine-tuned patterns of neuronal activity. Nitric oxide (NO) has been described as a potent messenger in the modulation of neural activity associated with synaptic plasticity. To evaluate the role of NO in the murine somatosensory pathway, we investigated NO synthase activity by NADPH diaphorase histochemistry at crucial developmental stages. At birth, NADPH diaphorase activity was detected in the cortical plate of the developing somatosensory cortex. At day 3, diffuse NADPH diaphorase activity increased within the emerging layer 4 in the future barrel field hollows. This staining was most intense at day 6 in the barrel field hollows and became undetectable by the end of the second postnatal week. The appearance of the diffuse NADPH diaphorase staining pattern was also observed in a similar time course and topography in the ascending relays of the somatosensory cortex, specifically in the barreloids within the ventrobasal nucleus of the thalamus and the barrelettes of the trigeminal nucleus of the brainstem. Lesioning the C row of whiskers at day 1 (i.e. during the critical period of barrel formation) led to fused C barrels of diffuse NADPH diaphorase activity in the barrel fields. In addition, highly NADPH diaphorase activity-positive individual cells present in the deeper layers of the somatosensory cortex at days 0 and 3 became visible in the upper layers at day 6 and remained until day 15. In layer 4, these cells were predominantly localized in the septa at day 6 and 9. No positive individual cells were detected in barrelettes or barreloids at any age. We conclude that NADPH diaphorase activity is present during experience-dependent consolidation of synaptic contacts in the somatosensory pathway.

Zebrafish neurons express two L1-related molecules during early axonogenesis.

Partial clones coding for two L1-related genes, zebrafish L1.1 and L1.2, were isolated from a zebrafish embryonic cDNA library. The homology analysis, based on the deduced amino acid sequences of L1.1 and L1.2, revealed that the two molecules are most closely related to each other and to mouse L1. Analysis by in situ hybridization revealed that during embryonic development of the nervous system the L1.1 and L1.2 messages are restricted to postmitotic neurons and that the onset of expression correlates with the initiation of axonogenesis. L1.1 is expressed by all known classes of neurons, consistent with an important general function during axonal outgrowth. Most of the neurons also express L1.2. However, L1.2 either is undetectable or is expressed at very low levels in the neurons of the olfactory placodes, anterior lateral line/acoustic ganglia complex, posterior lateral line ganglion, and in late developing hindbrain neurons. In the spinal cord, L1.2 message is detected only in a subpopulation of Rohon-Beard cells. This suggests the possibility that different levels of L1.2 expression may serve to distinguish different populations of neurons and their axons.

Molecular basis of interactions between regenerating adult rat thalamic axons and Schwann cells in peripheral nerve grafts. II. Tenascin-C.

Tenascin-C is a developmentally regulated extracellular matrix component. There is evidence that it may be involved in axon growth and regeneration in peripheral nerves. We have used in situ hybridization and immunocytochemistry to investigate the association of tenascin-C with central nervous system axons regenerating through a peripheral nerve autograft inserted into the thalamus of adult rats. Between 3 days and 4 weeks after implantation, tenascin-C immunoreactivity was increased in the grafts, first at the graft/brain interface, then in the endoneurium of the graft, and finally within the Schwann cell columns of the graft. By electron microscopy, reaction product was present around collagen fibrils and basal laminae in the endoneurium, but the heaviest deposits were found at the surface of regenerating thalamic axons within Schwann cell columns. Schwann cell surfaces were not associated with tenascin-C reaction product except where they faced the tenascin-rich basal lamina or were immediately opposite axons surrounded by tenascin-C. By 8 weeks after graft implantation tenascin-C in the endoneurium and around axons of the graft was decreased. In the brain parenchyma around the proximal part of the graft, axonal sprouts associated with tenascin-C could not be identified earlier than 2 weeks after grafting and were sparse at this stage. Larger numbers of such axons were present at 8-13 weeks after grafting and were located predominantly where the glia limitans between brain and graft appeared to be incomplete, suggesting that the tenascin-C may have penetrated the brain parenchyma from the graft. By in situ hybridization, cells expressing tenascin-C mRNA (probably Schwann cells) appeared first at the brain/graft interface 3 days after grafting and thereafter were mainly located within the grafts. Lightly labelled cells containing tenascin-C mRNA (probably glial cells) were scattered in the thalamic parenchyma both ipsilateral and contralateral to the graft and a few heavily labelled cells were located very close to the tip of the graft. These results show that regenerating adult thalamic axons, unlike regenerating peripheral axons, become intimately associated with peripheral nerve graft-derived tenascin-C, suggesting that they express a tenascin-C receptor, as many neurons do during development, and that tenascin-C derived from Schwann cells may play a role in the regenerative growth of such axons through the grafts.

Molecular basis of interactions between regenerating adult rat thalamic axons and Schwann cells in peripheral nerve grafts I. Neural cell adhesion molecules.

To gain insight into the possible molecular mechanisms underlying axonal regeneration of neurons of the adult central nervous system (CNS), we have investigated, by in situ hybridization and by immunocytochemistry, the localization and sites of synthesis of the neurite outgrowth-promoting cell surface molecules L1, N-CAM and its highly sialylated form, N-CAM-PSA, in and around peripheral nerve grafts implanted into the thalamus of adult rats. Normal unoperated adult rat thalamus contains N-CAM and L1 but no N-CAM-PSA immunoreactive axons. Between 7 days and 13 weeks after graft implantation, L1, N-CAM and N-CAM-PSA were all present at the surface of axonal sprouts in the brain parenchyma close to grafts and in the central parts of Schwann cell columns within grafts. Schwann cell membranes were L1 and N-CAM positive at all postgraft survival times, more strongly at 2-4 weeks than other times, but were associated with N-CAM-PSA reaction product only where they abutted N-CAM-PSA positive axons. Schwann cell membranes apposed to basal laminae (which were avoided by regenerating CNS axons) were L1, N-CAM and N-CAM-PSA negative. Between 3 days and 8 weeks after grafting, N-CAM and L1 mRNA were generally weakly upregulated in neurons of the ipsilateral thalamus, but, most conspicuously, L1 mRNA was strongly upregulated in the neurons of the thalamic reticular nucleus; these neurons are known to regenerate axons very effectively into peripheral nerve grafts and are the probable source of most of the axons which enter thalamic grafts. N-CAM and L1 mRNA were also strongly upregulated in presumptive Schwann cells in the graft. These results show that regenerating CNS axons (re)express N-CAM-PSA and upregulate L1 and N-CAM, suggesting that all of these molecules may play a role in cellular interactions during the regeneration of CNS axons. Furthermore L1 synthesis appears to be particularly well correlated with the ability of CNS neurons to regenerate axons into peripheral nerve grafts.

The adhesion molecule on glia (AMOG/beta 2) and alpha 1 subunits assemble to functional sodium pumps in Xenopus oocytes.

The adhesion molecule on glia, AMOG, an integral cell surface glycoprotein highly expressed by cerebellar astrocytes and involved in neuron to astrocyte adhesion and granule neuron migration (Antonicek, H., Persohn, E., and Schachner, M. (1987) J. Cell Biol. 104, 1587-1595) has been identified as a beta 2 subunit isoform of the mouse sodium pump (Gloor, S., Antonicek, H., Sweadner, K.J., Pagliusi, S., Frank, R., Moos, M., and Schachner, M. (1990) J. Cell Biol. 110, 165-174). Here we demonstrate that AMOG/beta 2 expressed by cRNA injection in Xenopus oocytes is capable of combining with endogenous Xenopus alpha 1 subunits or coexpressed Torpedo alpha 1 subunits to yield a functional alpha 1/AMOG sodium pump isozyme. Determinations of the number of ouabain binding sites and ouabain-sensitive 86Rb+ uptake suggest that the alpha 1/AMOG isozyme has slightly lower maximum transport rate and apparent affinity for external K+ than the alpha 1/beta 1 isozyme. Immunoprecipitation of alpha 1/AMOG complexes from digitonin extracts of [35S]methionine-labeled oocytes with a monoclonal anti-AMOG antibody provides direct evidence for a stable association between AMOG and the alpha 1 subunits of Xenopus and Torpedo.