Intermediate filaments in nervous tissues

Intermediate filaments have been isolated from rabbit intradural spinal nerve roots by the axonal flotation method. This method was modified to avoid exposure of axons to low ionic strength medium. The purified filaments are morphologically 75-80 percent pure. The gel electrophoretogram shows four major bands migrating at 200,000, 145,000, 68,000, and 60,000 daltons, respectively. A similar preparation from rabbit brain shows four major polypeptides with mol wt of 200,000 145,000, 68,000, and 51,000 daltons. These results indicate that the neurofilament is composed of a triplet of polypepetides with mol wt of 200,000, 145,000, and 68,000 daltons. The 51,000-dalton band that appears in brain filament preparations as the major polypeptide seems to be of glial origin. The significance of the 60,000- dalton band in the nerve root filament preparation is unclear at this time. Antibodies raised against two of the triplet proteins isolated from calf brain localize by immunofluorescence to neurons in central and peripheral nerve. On the other hand, an antibody to the 51,000-dalton polypeptide gives only glial staining in the brain, and very weak peripheral nerve staining. Prolonged exposure of axons to low ionic strength medium solubilizes almost all of the triplet polypeptides, leaving behind only the 51,000- dalton component. This would indicate that the neurofilament is soluble at low ionic strength, whereas the glial filament is not. These results indicate that neurofilaments and glial filaments are composed of different polypeptides and have different solubility characteristics.     

Zinc-aldehyde fixation for light-microscopic immunocytochemistry of nervous tissues

Two procedures are described for vascular perfusion of the nervous system with a zinc-aldehyde fixative. The procedures, simple and economical, combine the advantages of perfusion fixation with an aldehyde solution and matrix stabilization by a mordating agent, and improve the sensitivity of the peroxidase-antiperoxidase (PAP) method for the immunocytochemical localization of several antigens. Procedure A is intended for the light-microscopic immunostaining of cellular elements containing high concentrations of antigen. Penetration of the immunoreagents is adequate without the use of detergents. Procedure B is particularly advantageous for the light-microscopic immunostaining of cellular elements that contain low concentrations of antigen, and for high-resolution microphotography. With procedure B, the tissue penetration of immunoreagents is more limited than with procedure A; however, neuronal cell bodies and dendrites are more easily penetrated by the immunoreagents than are axons. Neuronal cell bodies and dendrites thus become clearly detectable in the light-microscope, even when they are surrounded by numerous immunoreactive axon terminals, and especially after the blockage of axoplasmic transport by the topical injection of colchicine.     

Growth of measles virus in nervous tissues. III. Neurovirulence of SSPE virus in ferrets.

The Niigata-1 strain isolated from a patient with subacute sclerosing panencephalitis (SSPE) was inoculated intracerebrally into ferrets. Neurological signs developed in about 1 week in most of the animals. Histopathological examinations of the central nervous tissues revealed degenerative lesions in the parenchyma of the brains and inflammatory reactions predominantly in the meninges and choroid plexus. Virus antigen was demonstrated mainly in the nerve cells by immunofluorescent staining. The results indicated high affinity of the Niigata-1 strain to the nerve cells. In contrast, the Mantooth strain of SSPE virus in cell-free state did not exhibit neurovirulence in ferrets.     

A simple microradioisotopic assay for 5'-nucleotidase activity. Application to central nervous tissues

A radiochemical method for measurement of 5′-nucleotidase is described in which the product, [¹⁴C]adenosine passes through a small alumina column and is separated from substrate 5′-AMP, which is completely retained. Optimal conditions for measurement of 5′-nucleotidase in particulate preparations of brain and spinal cord are described.     

Identification and properties of phencyclidine-binding sites in nervous tissues

[3H]Phencyclidine (PCP) binds to a single class of noninteracting binding sites in rat brain membranes with an affinity Kd of 0.25 microM and a maximal binding capacity BM of 2.4 pmol/mg of membrane protein. PCP derivatives also interact with the muscarinic and mu-opiate receptors in rat brain membranes with affinities that are one or two orders of magnitude lower than those observed for the [3H]PCP-binding sites. Activities of 25 PCP derivatives in the rotarod assay are closely correlated to affinities of these molecules for the [3H]PCP-binding sites, but not for the muscarinic or mu-opiate receptors. Monohydroxylation of PCP generally decreases the affinity of PCP for the [3H]PCP- and muscarinic-binding sites and does not change the affinity for the mu-opiate receptor. The metaphenolic derivative of PCP does not follow these general rules; the affinities of this derivative for the [3H]PCP- and mu-opiate-binding sites are 8 and 430 times higher, respectively, than those of PCP itself. Voltage-clamp experiments with N1E 115 neuroblastoma cells show that PCP is an efficient blocker of both the K+ channel (EC50 = 2.6 microM) and the Na+ channel (EC50 = 9.2 microM).     

Characterization and comparison of lipids in different squid nervous tissues

We have studied the lipid composition of brain (optic and cerebral lobes), stellate ganglia and fin nerves of the squid. Cholesterol, phosphatidylethanolamine and phosphatidylcholine were the major lipids in these nervous tissues. Phosphatidylethanolamine contained about 3% of its amount in [corrected] plasmalogen form. Phosphatidylserine and -inositol, sphingomyelin and ceramide 2-aminoethylphosphonate were also present in significant amounts. In addition, cardiolipin and free fatty acids were detected in brain (each 2-3% of total lipids) and stellate ganglia (about 1% each), but not in fin nerves. Phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol from brain contained large amounts of polyunsaturated fatty acids, namely 20:4, 20:5 and 22:6 in the n-3 family. On the other hand, phosphatidylcholine, cardiolipin, and sphingomyelin, and ceramide 2-aminoethylphosphonate contained only saturated or monounsaturated C16-C18 fatty acids. The aldehyde moieties of ethanolamine plasmalogen were also C16-C18 saturated or monounsaturated. These lipid compositions are compared with those in other invertebrate nervous systems.     

Lipid metabolism of developing central nervous tissues in organotypic cultures

Lipid changes in rat brain grey matter were observed in a coculture system of innervating and target explants. The de novo biosynthesis of individual glycerolipids and the metabolism of fatty acids were investigated. Innervating grey matter cultures exhibited a substantial increase in neutral glycerolipid formation. Only slight modifications were observed in the fatty acid fraction. Target cells responded to innervation by a marked increase in phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine formation. In addition, the biosynthesis of arachidonate and docosahexaenoate was drastically enhanced. It is shown that neuritic bridges connecting the explants, rather than soluble factors, account for the effects observed. Putative mechanisms involved in changes of lipid metabolism are discussed.     

Galectin-1 plays essential roles in adult mammalian nervous tissues. Roles of oxidized galectin-1

Previous data have suggested that galectin-1 is expressed widely in nervous tissues at embryonic stages but becomes restricted mainly to peripheral nervous tissues with maturation. Though the expression is intense in adult mammalian peripheral neurons, there had been no report about functions of galectin-1 there. Recently we discovered a factor that enhanced peripheral axonal regeneration. The factor was identified as oxidized galectin-1 with three intramolecular disulfide bonds and showed no lectin activity. Oxidized recombinant human galectin-1 (rhGAL-1/Ox) showed the same nerve growth promoting activity at very low concentrations (pg/ml). rhGAL-1/Ox at similarly low concentration was also effective in in vivo experiments of axonal regeneration. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited the axonal regeneration in vivo as well as in vitro. Since galectin-1 is expressed in the regenerating sciatic nerves as well as in both sensory neurons and motor neurons, these results suggest that galectin-1 is secreted into the extracellular space to be oxidized, and then, in its oxidized form, to regulate initial repair after axotomy. The administration of oxidized galectin-1 effectively promoted functional recovery after sciatic nerve injury in vivo. Oxidized galectin-1, hence, appears to play an important role in promoting axonal regeneration, working as a kind of cytokine, not as a lectin. Recent reports indicated additional roles of cytosolic galectin-1 in neural diseases, such as ALS. Furthermore galectin-1 has been proved to be a downstream target of DeltaFosB. In hippocampus of rat brain, expression of DeltaFosB is induced immediately after ischemia-reperfusion, suggesting that galectin-1 may also play important roles in central nervous system after injury.     

Galectin-1 plays essential roles in adult mammalian nervous tissues. Roles of oxidized galectin-1

Previous data have suggested that galectin-1 is expressed widely in nervous tissues at embryonic stages but becomes restricted mainly to peripheral nervous tissues with maturation. Though the expression is intense in adult mammalian peripheral neurons, there had been no report about functions of galectin-1 there. Recently we discovered a factor that enhanced peripheral axonal regeneration. The factor was identified as oxidized galectin-1 with three intramolecular disulfide bonds and showed no lectin activity. Oxidized recombinant human galectin-1 (rhGAL-1/Ox) showed the same nerve growth promoting activity at very low concentrations (pg/ml). rhGAL-1/Ox at similarly low concentration was also effective in in vivo experiments of axonal regeneration. Moreover, the application of functional anti-rhGAL-1 antibody strongly inhibited the axonal regeneration in vivo as well as in vitro. Since galectin-1 is expressed in the regenerating sciatic nerves as well as in both sensory neurons and motor neurons, these results suggest that galectin-1 is secreted into the extracellular space to be oxidized, and then, in its oxidized form, to regulate initial repair after axotomy. The administration of oxidized galectin-1 effectively promoted functional recovery after sciatic nerve injury in vivo. Oxidized galectin-1, hence, appears to play an important role in promoting axonal regeneration, working as a kind of cytokine, not as a lectin. Recent reports indicated additional roles of cytosolic galectin-1 in neural diseases, such as ALS. Furthermore galectin-1 has been proved to be a downstream target of ΔFosB. In hippocampus of rat brain, expression of ΔFosB is induced immediately after ischemia-reperfusion, suggesting that galectin-1 may also play important roles in central nervous system after injury. Published in 2004.     

Excess of taurine supplementation in rat: effects on GABA-related amino acids in developing nervous tissues.

The effects of taurine supplementation on GABA-related amino acid homeostasis in developing nervous tissues of suckling rats were studied. In the first two weeks of postnatal growth, cerebral cortex and cerebellum appear more accessible to taurine supplementation in comparison to retina; in addition, different changes in excitatory/inhibitory amino acids were observed. After the 5th day of life, in the retina and cerebellum of taurine-supplemented pups a decrease in GABA levels was found; in contrast, in cerebral cortex GABA content significantly increased throughout 20 days of postnatal growth. In all nervous tissues studied (except for cerebellum) glutamine concentration increased at the 5th day; then in cerebellum and in retina, but not in cerebral cortex, a significant decrease until the 20th day occurred. Furthermore, in cerebellum and retina taurine supplementation decreased glutamate levels, in comparison to controls, at the 10th and until the 20th day of postnatal life, respectively, whereas in cerebral cortex an increase in glutamate level was observed only at the 5th day. In conclusion, taurine supplementation, in excess to the usual amount from the mother's milk, affected the glutamate compartments in various cell types. The changes in GABA-related amino acid concentrations in cerebral cortex, cerebellum, and retina may depend on the different pattern of the metabolic processes at different maturative stages.