Biomedical subjects

D Dahl

Publications and source records attributed to D Dahl.

At least 91 records · Page 5Linked to original sources

PubMed1987

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. The pattern of intermediate breakdown products depended on pH, with greater amounts of fragments detected at higher pH, and the patterns with the two enzymes were different. We showed that differences exist in cleavage sites and breakdown rates of the neurofilament proteins. The capacity of the cathepsin D present in the tissue to hydrolyze these substrates was high, even at pH close to neutral, and was greatly in excess of that needed for physiological neurofilament turnover.

Animals↗

PubMed1987

Axonal maturation in development--I. Characterization of monoclonal antibodies reacting with axon-specific neurofilament epitopes.

Monoclonal antibodies reacting with the high molecular weight neurofilament polypeptides (NF 150K and NF 200K) were obtained upon immunization with NF 150K and NF 200K isolated from bovine spinal cord by anion exchange chromatography. The five monoclonal antibodies obtained with NF 200K stained only axons. With three monoclonals the reactivity was abolished by digestion with phosphatase and by dilution of the supernatants in sodium potassium phosphate. The nine monoclonal antibodies obtained upon immunization with NF 150K stained both high molecular weight neurofilament polypeptides on immunoblots of bovine and rat spinal cord extracts with the exception of one monoclonal only reacting with the homologous antigen. The antibodies could be divided into two groups, axon-specific and conventional. Conventional antibodies decorated neurofilaments regardless of their location, i.e. axons, perikarya and dendrites. With all these antibodies the immunostaining was not affected by phosphatase digestion of neurofilament protein nor by dilution of the supernatants in sodium potassium phosphate. Axon-specific antibodies reacting with both NF 150K and NF 200K in rat spinal cord only stained the heterologous antigen (NF 200K) in rat optic nerve and sciatic nerve extracts. We suggest that some axon-specific neurofilament antibodies recognize neurofilament modifications other than phosphorylation; or, alternatively that they react with phosphorylated epitopes not accessible to phosphate or to exogenous phosphatases. Furthermore, we suggest that some neurofilament modifications do not occur uniformly throughout the nervous system.

Animals↗

PubMed1987

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. Another possibility was reactivity with phosphorylated epitopes inaccessible to exogenous phosphatases. Conventional neurofilament antibodies stained motor and sensory neurons in day 12 and day 13 rat embryos, respectively, as previously reported with neurofilament antisera. Immunoreactivity with axon-specific antibodies first appeared in motor and sensory axons at different times in development: day 13-14 (3 monoclonals); day 17 (6 monoclonals); day 21 (1 monoclonal); postnatal day 2 (1 monoclonal). There were no major differences between conventional and axon-specific antibodies as to the time of appearance of Purkinje cell baskets in postnatal rat cerebellum. With two exceptions all monoclonals first stained thin baskets on day 11. Immunoreactivity of Purkinje cell baskets with two monoclonals reacting with phosphorylated NF 200K first appeared on days 14 and 20. It is suggested that post-translational modifications may stabilize the neurofilaments, thus accounting for their late appearance by electron microscopy in development.

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PubMed1987

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. Only a small, proximal portion of the axons from these cells exhibited NF200D-LI. At later stages, however, this immunoreactive region extended to include progressively more distal parts. Spinal cord neurons first became NF200D-positive at day 9; however, many NF200D-negative neurons still remained in mature cultures. Also at these later stages, some axons were stained for less than their full length with the NF200D antibody. NF200P-LI was first apparent at day 17, in smooth and varicose axons and only where NF-LI was also present. In contrast, NF200P- and NF200D-LI were usually localized in mutually exclusive populations of axons and other fibers. In some, predominantly thick axons, however, the proximal segment was NF200D-positive, whereas the distal part exhibited solely NF200P-LI. In contrast to NF70 and NF150, the 200 kDa neurofilament is dilatory in its appearance in most neurons in culture. The development of the nonphosphoderivative precedes that of the phosphoderivative, and the respective ontogenies are specific for different neuronal types. Posttranslational phosphorylation of NF200 seems therefore to occur at a later stage of development than the induction of NF200 itself, while there is a wide variation in its rates of phosphorylation during passage down different axons.

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PubMed1986

Long-term potentiation in dentate gyrus: induction by asynchronous volleys in separate afferents.

Long-term potentiation (LTP), a long-lasting enhancement of synaptic efficacy, is considered a model for learning and memory. In anesthetized rats, activation of dentate granule cells by stimulating either the medial or lateral perforant pathway at frequencies of 100 to 400 Hz produced LTP of the stimulated pathway preferentially at 400 Hz. However, hippocampal pathways do not normally fire at this high rate. Stimuli at 200 Hz were then applied to either the medial or lateral pathway separately, to both pathways simultaneously, or to the two pathways asynchronously so that the composite stimulus applied to the granule cell dendrite was 400 Hz. LTP was produced preferentially in the asynchronous condition. Thus, lower frequency, physiological input volleys arriving asynchronously at medial and lateral synapses can induce LTP by activating a 400-Hz sensitive mechanism capable of integrating spatially separated granule cell inputs. This may reflect how LTP is normally produced in the dentate gyrus.

Animals↗

PubMed1986

Neurofilament proteins in fish: a study with monoclonal antibodies reacting with mammalian NF 150K and NF 200K.

Monoclonal antibodies were obtained upon immunization of mice with chicken brain antigen and with the two high molecular weight neurofilament proteins (NF 150K and NF 200K) isolated from bovine spinal cord by anion exchange chromatography. By the immunoblotting procedure, the antibodies selected for this study reacted with bovine NF 150K and NF 200K. By the same procedure the antibodies reacted with sea raven, goldfish, sea bass, shark, and trout spinal cord extracts. In goldfish and sea raven the antibodies stained a single band at approximately 150 kDa and 200 kDa, respectively. Two bands were stained in the shark, sea bass, and trout. In the shark and sea bass these bands were in the molecular weight range of mammalian NF 150K and NF 200K. In the trout the upper band was approximately 150 kDa and the lower band 130 kDa. Our findings suggest an early origin of NF 150K and NF 200K in vertebrate phylogeny as well as considerable divergence in several species.

Animals↗

PubMed1986

Influence of neurons of the parafascicular region on neuronal transmission from perforant pathway through dentate gyrus.

We have previously reported that activation of an ascending brainstem pathway by stimulation of the median raphe nucleus (MR) influences neuronal transmission from the perforant pathway through the dentate gyrus in a behaviorally dependent manner. In particular, stimulation of the MR markedly facilitated such transmission when applied during slow-wave sleep (SWS), but was ineffective when applied during the still-alert state (SAL). We present here evidence for a relay in this circuit located rostral to the MR in cells proximal to the fasciculus retroflexus (PF, parafascicular region). In contrast to stimulation of the MR, stimulation of the PF facilitates neuronal transmission from the perforant pathway through the dentate gyrus during both SWS and SAL indicating the presence of a gate at or proximal to the PF that is preferentially closed during SAL.

Animals↗

PubMed1986

Neurofilament phosphorylation in peripheral nerve regeneration.

A monoclonal antibody to the 200 kdalton neurofilament (NF) polypeptide selectively decorated axons in tissue sections. Neuronal perikarya and dendrites, including motor and sensory neurons reacting to axotomy, were not stained. Axonal staining was abolished by dilution of the monoclonal supernatants with phosphate buffer and by digestion of tissue sections with phosphatase, thus suggesting that the antibody reacted with a phosphorylated epitope. Conventional monoclonal and polyclonal antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites) were not affected by these procedures. Compared to conventional NF antibodies, staining with the axon-specific monoclonal antibody was a late event in peripheral nerve regeneration. One week after operation, the whole distal stump of crushed rat sciatic nerve was invaded by bundles of axons strongly reacting with conventional NF antibodies. Axon-specific NF immunoreactivity was confined to the proximal segment of the stump at this time and progressively extended distally in the following week. Furthermore, NF phosphorylation appeared to coincide with the return of a normal nerve structure as evidenced by the distribution of laminin immunoreactivity. Bundles of axons growing within columns of laminin-positive Schwann cells did not stain with the axon-specific NF antibody. Immunoreactivity with this antibody coincided with the return of a normal laminin pattern, i.e. selective decoration of the endoneurial basal membranes surrounding the axons.

Animals↗

PubMed1986

Short- and long-term consequences of intracranial injections of the excitotoxin, quinolinic acid, as evidenced by GFA immunohistochemistry of astrocytes.

Astroglial reactions to intrastriatal and intrahypothalamic injections of the endogenous excitotoxin quinolinic acid (50 micrograms in 1 microliter) were studied in adult rats, using immunohistochemistry with antiserum to glial fibrillary acidic protein. Animals were sacrificed 6 h, 24 h, 3, 7 and 30 days or 1 year after the injection. Six and 24 h after quinolinic acid, the amount of glial fibrillary acidic protein-like immunoreactivity in the injected striatum was lower than in controls but returned to a normal level at 3 days. Not until 7 days was a clear striatal gliosis apparent, as evidenced by an increased density of glial fibrillary acidic protein-positive structures and brightly fluorescent, clearly hypertrophic cells. This gliosis was even more developed in animals sacrificed 30 days postoperatively. A weak astrocytic reaction was also observed in the ipsilateral corpus callosum at 6 h after quinolinic acid. By 3 days, a marked gliosis restricted to the injected hemisphere was present throughout corpus callosum and cortex cerebri. In animals sacrificed 30 days after quinolinic acid the extrastriatal astrocytic reaction was clearly diminished, although the striatal gliosis was still prominent. One year postinjection, no obvious gliosis could be observed in cortex cerebri or corpus callosum while striatal tissue, now markedly reduced in volume, was clearly gliotic. Using neurofilament antiserum, increased fluorescence intensity was noted in striatal nerve bundles during the first day after an intrastriatal quinolinic acid injection and persisted 1 year postoperatively. Controls were similarly injected with an equimolar amount of nicotinic acid, the non-excitatory, non-neurotoxic decarboxylation product of quinolinic acid. No changes in immunoreactivity of glial fibrillary acidic protein or neurofilament were found in these animals. In animals treated intrahypothalamically, a spherical central area almost devoid of glial fibrillary acidic protein-immunoreactivity was noted around the injection site 7 days after quinolinic acid administration. Around this area, gliosis was observed. Apart from a very restricted gliotic reaction around the needle tract, no astrocytic reaction was observed in nicotinic acid-injected control animals. We conclude that quinolinic acid causes both reversible and long-lasting gliosis when injected into the rat striatum. As a natural brain metabolite, quinolinic acid may constitute a particularly valuable tool for the elucidation of a possible role of glia in neurodegenerative disorders.

Animals↗

PubMed1986

Delayed phosphorylation of the largest neurofilament protein in rat optic nerve development.

Monoclonal antibodies selectively reacting with the high molecular weight neurofilament proteins (NF 150K and NF 200K) on immunoblots of bovine spinal cord extracts were obtained upon immunization of mice with chicken brain antigen and with highly purified NF 150K or NF 200K isolated from bovine spinal cord by anion exchange chromatography. Antibodies reacting with NF 200K or with both NF 150K and NF 200K were selected for this study. The antibodies were screened on immunoblots for reactivity with phosphorylated epitopes by dilution of the supernatants in sodium potassium phosphate as well as by treatment of nitrocellulose transfers with alkaline phosphatase. Abolishment of staining under these conditions was taken as evidence of reactivity with phosphorylated epitopes. With phosphate/phosphatase-sensitive antibodies, NF 200K immunoreactivity was a late event in rat optic nerve development. It was first observed at day 18 on immunoblots of sodium dodecyl sulfate extracts analyzed by gel electrophoresis. Conversely, with phosphate/phosphatase-insensitive antibodies, NF 200K immunoreactivity was already present on day 10, the earliest age in this study. With one monoclonal reacting with phosphorylated NF 150K and NF 200K, NF 150K immunoreactivity was already present on day 10. It is proposed that NF 200K expression precedes NF 200K phosphorylation in development.

Aging↗

PubMed1986

Heterogeneity of desmin, the muscle-type intermediate filament protein, in blood vessels and astrocytes.

Monoclonal antibodies were isolated from mice immunized with chicken gizzard desmin. Antibodies reacting with desmin on immunoblots and selectively decorating chicken and rat intestinal smooth muscle as well as the Z-line in striated muscle, were selected for this study. Based on their staining pattern on cryostat sections of chicken and rat cerebellum, spleen, kidney, aorta and femoral artery, monoclonal supernatants could be divided in three groups: (i) antibodies decorating astrocytes and vascular smooth muscle; (ii) antibodies decorating only vascular smooth muscle; (iii) antibodies decorating only astrocytes. Antibodies in group (i) and (iii) also stained GFA-negative Bergmann glia in chicken cerebellum. It is proposed that desmin may vary depending on the histological localization.

Animals↗

PubMed1986

Brain-specific hyaluronate-binding protein. A product of white matter astrocytes?

The distribution of glial fibrillary acidic (GFA) protein and hyaluronectin, a hyaluronate-binding protein isolated from human brain, was compared in brain, spinal cord and optic nerves of pigs and dogs by indirect immunofluorescence with monoclonal antibodies. In spinal cord white matter the localization of the two proteins was similar, both antigens forming a mesh surrounding myelinated axons. A similar distribution of the two proteins was also observed in the periventricular glia as well as in the glia limitans of spinal cord and optic nerves. Cerebral white matter was hyaluronectin-positive, but the GFA-positive stellate astrocytes did not stain with hyaluronectin antibodies in this location. Hyaluronectin antibodies did not stain grey matter, the granular layer of the cerebellum excepted. The astrocytes identified with GFA antibodies in hyaluronectin-negative grey matter were: the fibrous astrocytes forming the glia limitans on the surface of the cerebral hemispheres; the protoplasmic astrocytes of cerebral isocortex and basal ganglia; the fibrous astrocytes of cerebral allocortex (hippocampus); Bergmann radial glia in the molecular layer of the cerebellar cortex; and fibrous astrocytes of spinal cord anterior and posterior horns. It is concluded that the hyaluronectin fraction reacting with the monoclonal antibodies is a brain-specific protein probably produced by white matter astrocytes. We propose to call this fraction brain-specific hyaluronectin, to be distinguished from other fractions reacting with polyclonal antibodies and with different localizations.

Animals↗

PubMed1986

Neurofilament phosphorylation in development. A sign of axonal maturation?

Monoclonal antibodies to the 200K neurofilament (NF) protein selectively decorated axons in tissue sections. Dilution of the antibodies in phosphate buffer and digestion with phosphatase abolished the stain. With conventional monoclonal and polyclonal NF antibodies, i.e. antibodies decorating NF regardless of their location (axons, perikarya and dendrites), the staining was not affected by this treatment. With all antibodies, axon-specific and conventional, the staining was abolished by trypsin digestion. Subsequent digestion with phosphatase did not restore the staining. Compared with conventional NF antibodies, staining with axon-specific anti-NF 200K was a late phenomenon in chick embryo development. NF 200K immunoreactivity was first observed in peripheral nerves and in the anterior columns of the spinal cord on day 10. Sensory ganglia and optic nerve fibers were negative. With conventional NF antibodies these structures were stained on days 4 and 5, respectively. In the following days of development the study was confined to the retina, optic nerves, cranial peripheral nerves and sensory ganglia. Up to day 16, bundles of thin peripheral nerve fibers, strongly decorated by conventional NF antibodies, did not stain with anti-NF 200K in double labelling experiments. Nerve bundles emerging from the ganglia were also negative, although some thick nerve fibers within the ganglia were stained. NF 200K immunoreactivity was first observed on day 17 in the optic nerve and in the layer of optic nerve fibers. At this time, staining was confined to the bundle emerging from the temporal side of the retina. In newborn chicken, only few fibers stained with anti-NF 200K in the nasal bundle, while the temporal bundle was well stained. It is suggested that the NF 200K antibodies reacted with a phosphorylated epitope in the axon, and that NF phosphorylation is a late event in ontogenesis probably related to axonal maturation.

Animals↗

PubMed1986

Growth and development of intraocular fetal cortex cerebri grafts in rats of different ages.

Cortex cerebri pieces from fetal donors were homologously and bilaterally grafted to the anterior chamber of the eye of 1.5-, 3.0- and 7.5-month-old rats. Repeated stereoscopic in vivo measurements revealed that the grafts in the young group grew to a size twice as large as those in the older two groups. The degree of gliosis was studied immunohistochemically using antibodies against glial fibrillary acidic protein. Both grafts to young and to older hosts were clearly gliotic as compared to normal cerebral cortex. However, the gliosis was significantly more pronounced in grafts to 3.0- and 7.5-month-old hosts as compared to grafts to 1.5-month-old hosts. The vascular component was evaluated using antibodies against laminin. We found laminin immunofluorescence to be an excellent marker of brain tissue vascularization, particularly at the capillary level, revealing the entire capillary tree and endothelial budding. The density of the vascular plexus and the average thickness of the capillaries of cortex cerebri grafted to the youngest recipients was remarkably similar to normal cerebral cortex. In marked contrast, grafts to the older hosts had a clearly pathological vascular network characterized by few, thick-walled blood vessels and very few normal-looking capillaries. We conclude that host age factors profoundly influence development and growth of intraocular brain tissue grafts.

Animals↗

PubMed1986

Astrocyte responses to dopaminergic denervations by 6-hydroxydopamine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine as evidenced by glial fibrillary acidic protein immunohistochemistry.

Astrocytic responses to dopaminergic denervation by two widely used dopamine neurotoxins, 6-hydroxydopamine (6-OHDA) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) were monitored using immunofluorescence with antibodies against glial fibrillary acidic protein (GFA) while neurofilament (NF) antibodies were used to monitor neuronal disturbances. Following stereotaxic injection of 6-OHDA into the nigrostriatal dopamine bundle in rats, an increased amount of GFA-immunoreactivity in striatum was detectable after 24 hours and remained after one month. Retrograde degeneration of nigral neurons led to gliosis in the cell body area. At the site of injection, astrocytes were destroyed and NF-immunoreactivity increased. New astrocytes invaded the injection area during the first month after injection. MPTP given systemically to mice in a dose that causes marked dopaminergic denervation of striatum also caused marked increases of GFA-immunoreactivity in striatum. These changes were larger in C57 BL/6 mice, known to be more sensitive to MPTP, than in N.M.R.I. mice, which are less sensitive to MPTP. The glial responses to MPTP-induced dopaminergic denervation did not occur when the dopamine neurotoxic effects were prevented by pretreatment with nomifensine or pargyline. It is concluded that dopaminergic denervation by neurotoxins causes rapid and profound changes in striatal astrocytes characterized by increased GFA-immunoreactivity. These changes remained up to a month after denervation and should be taken into account when functional consequences of dopaminergic denervations are discussed.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

PubMed1986

Maintenance of neuritic arborization in mitotic neuroblasts identified by neurofilament immunoreactivity. A scanning electron microscope study of primary cell cultures from embryonic rat cerebral hemispheres.

The morphology of mitotic cells identified as neuronal precursors by neurofilament immunoreactivity was studied by scanning electron microscopy in primary dissociated cultures of fetal rat cerebral hemispheres. It is shown that neurofilament-positive neuroblasts maintain their neuritic arborization during cell division suggesting a cytoskeletal function for the neurofilament.

Animals↗

PubMed1986

Astroglial development in microencephalic rat brain after fetal methylazoxymethanol treatment.

Treatment of pregnant rats on gestation day 15 with methylazoxymethanol (MAM) leads to a marked microencephaly in the offspring with a considerable atrophy in cerebral cortex, hippocampus and striatum. The development of the astrocytic populations in these atrophic regions was studied by means of immunohistochemistry using an antiserum against glial fibrillary acidic protein (GFA). The distribution and density of GFA-positive structures were not notably altered in the parietal cortex, hippocampal formation and striatum after prenatal MAM-treatment as compared to control. Also the individual astrocytes were morphologically similar in experimental and control animals in all regions analyzed. We suggest that an adjustment of the astrocytic development has occurred in response to the changed neuronal environment. Alternatively, MAM-treatment may affect neuronal and glial precursor cells leading to a seemingly normal astrocytic cell density.

Animals↗

PubMed1986

Brain-specific hyaluronate-binding protein: an immunohistological study with monoclonal antibodies of human and bovine central nervous system.

Hyaluronectin is a protein isolated from acid extracts of human brain by affinity chromatography on immobilized hyaluronate. With polyclonal antibodies, it was immunohistologically localized in the rat at the nodes of Ranvier of central and peripheral myelinated fibers and in mesenchymal tissues. Compared to adult rat, hyaluronectin-immunoreactive material was more abundant in embryonal rat brain and mesenchyma. We report a different localization in human and bovine tissues with monoclonal antibodies reacting with human hyaluronectin by NaDodSO4/PAGE and immunoblotting but not staining rat tissues by immunohistology. In human and calf the antigen reacting with hyaluronectin monoclonal antibodies was brain specific, while several peripheral tissues were stained by the polyclonal antibodies. In human and bovine central nervous system monoclonal antibodies stained white matter and tissues formed predominantly by glial fibers (e.g., subependymal glia). In white matter hyaluronectin-immunoreactive material formed a delicate mesh surrounding individual myelinated fibers, a pattern compatible with the distribution of fine astroglial processes in this location. Gray matter did not stain with monoclonal antibodies, the granular layer of the cerebellum excepted. The findings suggest that human hyaluronectin is heterogeneous and comprises at least two fractions. The main fraction is a brain-specific protein, probably produced by white matter astrocytes. Another fraction cross-reacting with rat is more abundant in embryonal tissues, including mesenchyma and brain.

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