Neural differentiation promoted by truncated trkC receptors in collaboration with p75(NTR).

trkC receptors, which serve critical functions during the development of the nervous system, are alternatively spliced to yield isoforms containing the catalytic tyrosine kinase domain (TK+) and truncated isoforms which lack this domain (TK-). To test for potential differences in their roles during early stages of neural development, TK+ and TK- isoforms were ectopically expressed in cultures of neural crest, the stem cell population that gives rise to the vast majority of the peripheral nervous system. NT-3 activation of ectopically expressed trkC TK+ receptors promoted both proliferation of neural crest cells and neuronal differentiation. Strikingly, the trkC TK- isoform was significantly more effective at promoting neuronal differentiation, but had no effect on proliferation. Furthermore, the trkC TK- response was dependent on a conserved receptor cytoplasmic domain and required the participation of the p75(NTR) neurotrophin receptor. Antibody-mediated receptor dimerization of TK+ receptors, but not TK- receptors, was sufficient to stimulate differentiation. These data identify a phenotypic response to activation of the trkC TK- receptor and demonstrate a functional interaction with p75(NTR), indicating there may be multiple trkC receptor-mediated systems guiding neuronal differentiation.

4-hydroxynonenal inhibits Na(+)-K(+)-ATPase.

4-Hydroxynonenal binds rapidly to Na(+)-K(+)-ATPase, and this was accompanied by a decrease in measurable sulfhydryl groups and a loss of enzyme activity. The I50 value for Na(+)-K(+)-ATPase inhibition by 4-hydroxynonenal was found to be 120 microM. Although the sulfhydryl groups could be completely restored with beta-mercaptoethanol during the reaction of the Na(+)-K(+)-ATPase-HNE-adduct, the Na(+)-K(+)-ATPase activity was only partially restored by this reducing agent. A combination of hydroxylamine and beta-mercaptoethanol yielded the greatest recovery of enzyme activity, 85% of original. Thus, 4-hydroxynonenal binding to Na(+)-K(+)-ATPase led to an irreversible decrease of enzyme activity under the conditions employed. It is hypothesized that 4-hydroxynonenal reacts with sulfhydryls at sites on the enzyme that are inaccessible by beta-mercaptoethanol. Furthermore, evidence was obtained that 4-hydroxynonenal reacts with other amino acids such as lysine to form adducts that also interfere with protein function.

Expression of a novel nuclear protein is correlated with neuronal differentiation in vivo.

We report the production of a monoclonal antibody (MAb 526) that recognizes a novel, developmentally regulated nuclear protein expressed in neurons throughout the rat nervous system. Analysis of whole brain and cell nuclear extracts by SDS-PAGE and immunoblotting determined that MAb 526 recognizes a single nuclear protein (np) of apparent molecular weight 42 kD, designated np526, as well as a slightly larger (ca. 44 kD) cytoplasmic protein. Light microscopic immunocytochemistry showed np526 to be present in neurons of all types throughout the central and peripheral nervous systems. Nuclei of both fibrous and protoplasmic astrocytes were also immunoreactive, but oligodendrocyte nuclei were negative. Positive, but highly variable immunocytochemical staining of nonneural cell nuclei in a variety of other tissues was also observed. Electron microscopic (EM) immunocytochemistry using pre-embedding peroxidase methods revealed that np526 is associated with euchromatin or with the edges of condensed chromatin bundles in neurons, indicating that it is likely to be a chromosomal protein. Most interestingly, the expression of np526 was found to be developmentally regulated in brain. Immunocytochemical analysis of the developing cerebral cortex from embryonic day (E) 16 to postnatal day (P) 4 and cerebellum from P4 to P18 revealed that np526 first appears in central neurons following the cessation of mitosis and that the intensity of nuclear staining increases during subsequent neuronal maturation. To our knowledge, np526 is the first presumptive chromosomal protein whose expression has been precisely correlated with the early postmitotic differentiation of mammalian neurons.

Monoclonal antibodies identify two novel proteins associated with vasopressin secretory granules of the rat neurohypophysis.

Immunocytochemical and immunoblotting technique have been used to characterize the antigens recognized by two monoclonal antibodies (MAbs C6 and D5) produced against dissociated cells from punches of neonatal supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei of the rat. Peroxidase immunocytochemistry revealed that both MAbs label magnocellular perikarya in the adult and neonatal SON and PVN as well as smaller neurons in the suprachiasmatic nucleus. Axons of the hypothalamo-neurohypophysial tract are also immunolabeled within the hypothalamus and zona interna of the median eminence, and C6 and D5 each bind specifically to both the adult and neonatal neurohypophysis. Dual-label immunofluorescence experiments employing C6 or D5 simultaneously with rabbit antisera specific for either oxytocin, neurophysin or vasopressin neurophysin revealed that C6 binds only to vasopressinergic magnocellular perikarya in the SON, while D5 labels both vasopressinergic and a small subset of oxytocinergic magnocellular neurons. Post-embedding immunogold analysis of MAb binding to the neurohypophysis at the ultrastructural level showed that both C6 and D5 recognize antigens associated with large dense core neurosecretory granules in a subset of neurosecretory axons. Initial biochemical characterization of the antigens recognized by C6 and D5 was performed using SDS-PAGE and Western immunoblotting. MAbs C6 and D5 label single protein bands with apparent molecular weights of 38 and 68 kDa, resp., in blots of reduced extracts from the adult neurointermediate lobe. No cross-reactivity between C6 and D5 and the neurophysins was apparent, nor did anti-neurophysin sera recognize the bands identified by C6 and D5. We have therefore designated these novel antigens as VPGP38 and VPGP68 for VasoPressin Granule Proteins.

Organization of retinopetal axons in the optic nerve of the cichlid fish, Herotilapia multispinosa.

We have studied the position and numbers of retinopetal axons in the rainbow cichlid fish, Herotilapia multispinosa, to determine the response of related parts of the brain of fish to the continual addition of new neurons in the retina. The retinopetal axons were traced by using the retrograde tracers HRP and cobaltous lysine and an immunocytochemical probe, antibodies to FMRF amide, the molluscan cardioexcitatory peptide. One population of cells with retinopetal axons was found in the telencephalon (in the nucleus olfactoretinalis) and the other was scattered in the diencephalon. Some of the cells in the nucleus olfactoetinalis with retinopetal axons were FMRF amide positive; antibodies were used to trace the axons of these cells into the retina. All the retinopetal axons, from the nucleus olfactoretinalis and the diencephalon, were confined to the portion of the optic nerve that contains axons from the central retinal ganglion cells, that is, the oldest ganglion cells. This result suggests that the retinopetal axons grow into the optic nerve and retina early in the life of the fish, and no new ones are added later in life despite the extensive addition of cells in the retina. Counts of the cells in the nucleus olfactoretinalis that project to the retina in 3-month-old and adult fish support this interpretation. We conclude that retinopetal axons grow into the retina early in the life of the fish and respond to the formation of new retina by extending their arbors toward the new retina.

Development of retinopetal projections in the cichlid fish, Herotilapia multispinosa.

This paper reports a study of the development of cells that project to the retina from the telencephalic nucleus olfactoretinalis and the diencephalon. Stell et al. (Proc. Natl. Acad. Sci. USA 81:940-944, '84) have shown that the FMRFamide-immunoreactive (FMRFamide-ir) cells in the nucleus olfactoretinalis project to the retina. Therefore, we used immunocytochemistry to study the development of these cells in the nucleus olfactoretinalis. Twenty hours after fry hatched, FMRFamide-ir cells were unambiguously seen in the nucleus olfactoretinalis. At this time the axons of these cells could be traced into the optic nerve. A few hours later the axons were visible in the retina and soon attained their adult position in the inner plexiform layer near the amacrine cells. In older frey, tracers were used to fill retinopetal cells in both the nucleus olfactoretinalis and the diencephalon. Counts of these cells demonstrated that over one-third of the adult number of retinopetal cells in the nucleus olfactoretinalis are present and have axons in the retina when the fry is 9 days old, and the percentage grows to one-half by the time the fry is 1 month old. Development of the retinopetal cells in the diencephalon lags behind that of the cells in the nucleus olfactoretinalis. However, about one-third of the adult number extend their axons into the optic nerve by 1 month of age. These results support our suggestion that the retinopetal cells have axons in the old part of the optic nerve because these cells were born and extended axons early in the life of the fish.

A flow cytometric method for intracellular labeling and purification of rare neuronal populations: isolation of fixed neurophysin neurons.

A method is described for flow cytometric analysis and fluorescence-activated cell sorting of small populations of neurons following dissociation of fixed brain tissue and immunofluorescent labeling of intracellular antigens. This method has been successfully applied to neurophysin-containing magnocellular neurons of the rat supraoptic (SON) and paraventricular (PVN) hypothalamic nuclei. These neurons constitute a rare population in the context of flow cytometry, comprising less than 2% of all cells present in dissociated tissue punches of SON and PVN. Following labeling with anti-neurophysins sera and fluorescein-conjugated second antibody, a highly enriched population containing 80-85% neurophysin-positive neurons was isolated by fluorescence-activated cell sorting. Recovery of 29% of all neurophysin-containing neurons in the SON/PVN was achieved. Perikarya were recovered largely intact, frequently with attached proximal dendritic processes. Applications of this method include purification of specific neuronal types for use as immunogens in production of monoclonal antibodies to cell-type-specific antigens, and rapid surveys of fluorescent lectin or other ligand binding to cell populations identified by the presence of particular intracellular antigens.

Rhodamine immunohistofluorescence applied to plant tissue.

Tissue slices from the roots and seeds of sanifoin (Onobrychis viciifolia, Scop.) exhibit bright autofluorescence when illuminated with blue (495 nm) light. This autofluorescence is indistinguishable from the fluorescence emission of fluorescein, the commonly used fluorochrome in immunohistochemical staining procedures. Rhodamine isothiocyanate, when coupled to immunoglobulin, and excited with green light at 546 nm, exhibits a reddish-orange fluorescence with an emission maximum at 590 nm. Plant tissue has little or no autofluorescence when illuminated at this wavelength and viewed with a 580 nm barrier filter. Therefore, use of rhodamine for immunohistochemical localization in plant tissue avoids interpretative complications due to inherent autofluorescence.