Vimentin-GFAP transition in primary dissociated cultures of rat embryo spinal cord.

Primary dissociated cultures derived from 15-day-old rat embryo spinal cord with or without dorsal root ganglia (DRG) were grown on polylysine, Primaria and laminin substrates. On polylysine and Primaria substrates, spinal cord neurons formed aggregates connected by bundles of neurites in a distinctive pattern similar to that observed in cultures derived from embryonal rat brain and neonatal rat cerebellum. After 2 days in culture, the number of cells stained with GFAP antibodies progressively increased within the vimentin-positive monolayer surrounding the neuronal aggregates.

Human fetal spinal cord xenografts survive in the eye of athymic nude rat hosts.

Human fetal spinal cord tissue was recovered from elective abortions and grafted to the anterior chamber of the eye of adult athymic nude rats. The transplants slowly became vascularized from the host iris during the first months. There was a clear cut stage-dependent survival and growth along a more "human" time-table.

Effects of glycosaminoglycans and proteinase inhibitors on astroglia-induced detachment of cultured rat cerebellar neurons.

Neurons in mixed primary embryonic CNS cultures degenerate secondary to their detachment from the substratum. The present study demonstrates that in primary cultures of postnatal cerebellum, detachment of neurons can be prevented by antiproliferative drugs which inhibit the growth of astroglia. Several types of proteinase inhibitors did not affect the process of detachment.

Axonal regeneration in old multiple sclerosis plaques. Immunohistochemical study with monoclonal antibodies to phosphorylated and non-phosphorylated neurofilament proteins.

Cryostat sections of two old plaques removed at autopsy from the spinal cord of a 62-year-old man with multiple sclerosis of 24-year duration were studied by indirect immunofluorescence with antibodies to neurofilament proteins, glial fibrillary acidic protein (GFAP), glial hyaluronate-binding protein (GHAP), vimentin and laminin. The neurofilament monoclonal antibodies used in this study reacted with phosphorylated epitopes of the two large polypeptides of the neurofilament triplet (NF 150K, NF 200K). As previously reported [Dahl D, Labkovsky B, Bignami A (1989) Brain Res Bull 22:225-232], the neurofilament antibodies either stained axons in the distal stump of transected sciatic nerve in the early stages of regeneration or late in the process, i.

Glial hyaluronate-binding protein in Wallerian degeneration of dog spinal cord.

Wallerian degeneration of spinal cord dorsal columns was produced in three dogs by unilateral extradural dorsal rhizotomy at the lower thoracic level. The spinal cord was studied 1 month, 2 months, and 3 months after surgery. Transverse cryostat sections at the site rhizotomy and at the mid-thoracic level were stained by indirect immunofluorescence with antibodies to the glial fibrillary acidic protein (GFAP) and to the glial hyaluronate-binding protein (GHAP).

Structural similarity of hyaluronate binding proteins in brain and cartilage.

A glial hyaluronate-binding protein (GHAP) was isolated from human brain white matter by affinity chromatography on immobilized hyaluronate. The 60 kDa protein appeared remarkably homogeneous by reversed-phase high pressure liquid chromatography analysis. Four cyanogen bromide peptides and 10 tryptic peptides were characterized by amino acid sequence, a total of 12 sequences since overlaps were found between 2 cyanogen bromide and 2 tryptic peptide sequences.

Astroglia-induced detachment of central neurons but astroglia-dependent growth of peripheral neurons in rat embryonic spinal cord primary cultures.

In mixed primary cultures, intrinsic neurons from embryonic mammalian brains degenerate secondary to their detachment from the substratum and this is caused by the under-growing co-cultured astroglia. In the present study we sought to find out whether or not peripheral neurons, sensory and motor neurons which reside and/or only project outside the CNS respectively, interact with astroglia similarly as their central counterparts do. Mixed primary cultures prepared from dissociated embryonic rat spinal cord and dorsal root ganglia were examined by phase and immunofluorescence microscopy after labeling with antibodies to neurofilaments (neuronal markers) and to glial fibrillary acidic protein and vimentin (astroglia markers).

Early and late appearance of neurofilament phosphorylated epitopes in rat nervous system development: in vivo and in vitro study with monoclonal antibodies.

Neurofilament phosphorylation in rat nervous system development was studied by indirect immunofluorescence with monoclonal antibodies reacting with phosphorylated epitopes in tissue sections and in primary dissociated cultures. The antibodies either decorated neurofilaments shortly after their appearance or after a considerable delay (from 4 to 9 days in vivo and from 12 to 27 days in vitro), thus suggesting the existence of at least two classes of phosphorylated epitopes. With most antibodies there was a good correlation between in vivo and in vitro findings as to the early or late appearance of phosphorylated epitopes.

Neurofilament phosphorylation in axons and perikarya: immunofluorescence study of the rat spinal cord and dorsal root ganglia with monoclonal antibodies.

Rat dorsal root ganglia and spinal cord were stained with 12 monoclonal antibodies reacting with phosphorylated epitopes of two neurofilament proteins (NF 150K and NF 200K). Three monoclonal antibodies were axon-specific in both locations; neuronal perikarya were not stained. Nine monoclonal antibodies stained a subpopulation of neurofilament-positive sensory neurons, as indicated by double labeling experiments with polyclonal antibodies reacting with phosphorylated and dephosphorylated forms of the neurofilament protein triplet.

Central administration of cholecystokinin potentiates evoked potential amplitude in the hippocampal dentate gyrus.

Cholecystokinin and related ligands were injected into a lateral ventricle and recordings made of the field potential in the hippocampal dentate gyrus evoked by stimulation of the medial entorhinal cortex. With the exception of the unsulfated octapeptide, these compounds induced an increase in population action potential amplitude. The unsulfated octapeptide was found to inhibit the action of its sulfated congener, and alone induced a decrease in response amplitude.

Hyaluronate-binding protein produced by white matter astrocytes: an axonal growth repellent?

Cells dissociated from 6-day rat cerebellum were seeded on glass coverslips coated with polylysine on one half and hyaluronectin on the other. Cell attachment and neurite extension were drastically reduced on hyaluronectin-coated surfaces.

2,5-Hexanedione-induced accumulations of neurofilament-immunoreactive material throughout the rat autonomic nervous system.

In rats intoxicated with 2,5-hexanedione, nerve fibres supplying virtually all visceral organs showed large numbers of densely immunoreactive accumulations of neurofilament-like material, of fusiform, elongated, smoothly tapering morphology. In the gut, round to oval, morphologically different lesions were also present, and abnormal neurofilament-immunoreactive accumulations were revealed in oesophageal terminal end-plates. An extensive damage to autonomic nerve fibres, which are largely non-myelinated, was thus revealed in 2,5-hexanedione intoxication.

Expression of brain-specific hyaluronectin (BHN), a hyaluronate-binding protein, in dog postnatal development.

Monoclonal antibodies reacting with the brain-specific form of hyaluronectin, a hyaluronate-binding protein, were used in conjunction with antibodies to the glial fibrillary acidic protein (GFAP), the subunit of astrocyte-specific intermediate filaments, to study the postnatal development of spinal cord and cerebral white matter in the dog. As previously reported, the distributions of brain-specific hyaluronectin (BHN) and GFAP in adult dog spinal cord white matter were similar. Both antigens formed a mesh surrounding individual myelinated axons.

Immunological study of a neurofilament protein variant (S150) with polyclonal and monoclonal antibodies.

Ten polyclonal neurofilament antibodies were tested for domain specificity with immunoblots of chymotrypsin digests of a neurofilament protein of 150 kDa (NF 150K). In contrast to most monoclonal antibodies previously reported, the five polyclonal antibodies which showed domain specificity reacted with the 40 kDa alpha-helical rod domain of the molecule. (With one exception, monoclonal antibodies reacted with the 100 kDa carboxy-terminal peripheral domain).

Neurofilament-like and glial fibrillary acidic protein-like immunoreactivities in rat and guinea-pig sympathetic ganglia in situ and after perturbation.

The presence of neurofilament (NF)-like and glial fibrillary acidic protein (GFAP)-like immunoreactivities was studied in sympathetic ganglia of adult rats and guinea pigs during normal conditions and after perturbation. In the superior cervical ganglion (SCG) of normal rats, many ganglion cells and nerve fibers show NF immunoreactivity. Some of these nerve fibers disappear after preganglionic decentralization of SCG; this indicates the presence of a mixture of pre- and postganglionic NF-positive nerves in the ganglion.

Temporal and topographic relationships between the phosphorylated and nonphosphorylated epitopes of the 200 kDa neurofilament protein during development in vitro.

The ontogeny of the triplet of neurofilament proteins (NF), and the phosphorylated and nonphosphorylated derivatives of the 200 kDa neurofilament subunit (NF200P, NF200D) have been investigated in dissociated cultures prepared from gestational day 13 mouse spinal cord and dorsal root ganglia (DRG), using immunocytochemical methods. Neurofilament-like immunoreactivity (NF-LI), as detected with antiserum, occurred in the somata and processes of all neurons from day 1 in culture, and reached a maximum density and intensity at days 16-20. The first labeling of neurons by NF200D antibodies occurred at day 3, and was confined to DRG cells.

Systemically administered cholecystokinin affects an evoked potential in the hippocampal dentate gyrus.

Cholecystokinin was administered systemically while recordings were taken of the evoked action potential in the hippocampal dentate gyrus to stimulation of the medial entorhinal cortex. The sulphated octapeptide and t. BOC CCK-4, but not the unsulphated octapeptide or tetrapeptide, increased the amplitude of the evoked action potential in a dose-dependent manner, whereas the unsulphated octapeptide decreased it.

Buffers and H2O2 reduce neuronal death and/or enhance differentiation of neurons and astrocytes in dissociated mouse brain cultures.

Tissue of the mammalian central nervous system (CNS) undergoes complex and uncoordinated pathological responses upon injury. Efforts to develop pharmacological approaches to achieve functionally meaningful regeneration largely have been unsuccessful. Assuming that anoxia and drop in tissue H are initiating factors in most pathological sequences consequent to CNS injury, we studied the effects of increasing the buffering and oxygenating capacities of the medium on dissociated embryo brain cultures.

The breakdown of the individual neurofilament proteins by cathepsin D.

In a continuing study of proteolysis of CNS proteins by CNS enzymes, neurofilament proteins (210 K, 155 K, 70 K) and desmin were separated, and the breakdown of individual proteins by purified brain cathepsin D was measured and compared to breakdown by plasma thrombin. With both cathepsin D and thrombin, the rate of breakdown of the 70 K protein was the highest, followed by the 155 K, and that of the 210 K was the lowest. With each substrate cathepsin D breakdown was the highest at pH 3; small but significant breakdown could be seen at pH 6.

Maturation of a large neurofilament protein (NF 150K) in rat postnatal development.

The mammalian neurofilament is made of three neuron-specific proteins with approximate molecular weights of 70 kilodaltons (kDa) (NF 70K), 150 kDa (NF 150K), and 200 kDa (NF 200K) by SDS-PAGE. As previously reported in the rat by Strocchi et al (J Neurochem 39:1132-1141, 1982) and Nixon et al (J Cell Biol 94:150-58, 1982), NF 150K comprises three molecular weight variants with the same isoelectric point. A fourth lower molecular weight and slightly less acidic variant was identified by monoclonal and polyclonal antibodies reacting with the alpha-helical middle domain of NF 150K.

Glial fibrillary acidic protein (GFAP)-like immunoreactivity in normal and transected rat olfactory nerve.

Normal and transected rat olfactory nerves were stained immunohistochemically using a monoclonal antibody previously shown to selectively detect GFAP-like immunoreactivity in central astrocytes but not in peripheral Schwann cells. Low levels of "central" type GFAP were found in the olfactory nerves, presumably in ensheathing cells. The levels of GFAP increased dramatically after nerve transection.

Axonal maturation in development--II. Immunofluorescence study of rat spinal cord and cerebellum with axon-specific neurofilament antibodies.

Neurofilament monoclonal antibodies derived from mice immunized with chicken brain antigen or purified bovine NF 150K and NF 200K either stained only axons or they stained neuronal perikarya, dendrites and axons. Antibodies in the second group were called conventional because they decorated tissue sections like the neurofibrillary methods of traditional histology. Axon-specific antibodies either reacted with phosphorylated epitopes or they were phosphate/phosphatase insensitive thus suggesting reactivity with post-translational modifications other than phosphorylation.

Sustained seizures cause circumscribed cerebral changes in glial fibrillary acidic protein, neurofilament and laminin immunofluorescence.

Sustained experimental seizures in rats have previously been shown to cause an extensive necrosis in pars reticulata of substantia nigra (SNPR) and globus pallidus (GP). In the present paper we have studied the effects of hexafluorodiethyl ether-induced seizures on the immunoreactivity seen with antibodies directed against glial fibrillary acidic protein, GFA, used to visualize astrocytes, antibodies to the glycoprotein laminin as a marker for blood vessel walls and neurofilament (NF) antibodies to monitor neuronal disturbances. Already 12 h after a 20-min seizure period a reduction in GFA immunofluorescence intensity was observed in SNPR.