bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of adult human astrocytes derived from explant culture
Authors: Estes M. L. , Ransohoff R. M. , McMahon J. T. , Jacobs B. S. , Barna B. P.
Published: January 2005

Four different human astrocytic cell lines established from either epilepsy surgical specimens or cerebral white matter obtained during thalamotomy for tremor in a patient with multiple sclerosis were characterized using morphologic analysis, ultrastructural attributes, growth characteristics, and immunocytochemical analysis. Immunocytochemical characterization of cultures indicated a mean of 84% of cells contained cytoplasmic glial fibrillary acidic protein (GFAP): to confirm that GFAP (+) cells also proliferated, bromodeoxyuridine (BrdU) uptake was measured in cell line. Our method of simplified explant culture allows establishment of astrocytic cell lines from a variety of pathologic substrates using limited amounts of human material.
Acetylcholine‐induced production of platelet‐activating factor by human fetal brain cells in culture
Authors: Sogos V. , Bussolino F. , Pilia E. , Torelli S. , Gremo F.
Published: January 2005

Platelet‐activating‐factor (PAF) is a potent, biologically active lipid mediator produced by several tissues, including brain. Its role in the central nervous system (CNS) is still unknown, even if its involvement in brain damage and neurotoxicity has been postulated. Its production by neural cells has been demonstrated in different species, but not in man. This paper provides evidence that PAF can be produced by human fetal neurons and/or glial cells in culture. Its synthesis dramatically increased upon stimulation with acetylcholine (ACh), and it was significantly lowered by the cholinergic receptor antagonist atropine. Almost no PAF was detected in the incubation medium, which indicated no release of PAF from cultured cells. Characterization of the cells in culture with specific monoclonal antibodies excluded the presence of endothelial cells or macrophages, which also produce PAF.
Folding and function of the myelin proteins from primary sequence data
Authors: Inouye H. , Kirschner D. A.
Published: January 2005

To explain how the myelin proteins are involved in the organization and function of the myelin sheath requires knowing their molecular structures. Except for P2 basic protein of PNS myelin, however, their structures are not yet known. As an aid to predicting their molecular folding and possible functions, we have developed a FORTRAN program to analyze the primary sequence data for proteins, and have applied this to the myelin proteins in particular, In this program, propensities for the secondary structure conformations as well as physical‐chemical parameters are assigned to the amino acids and the pattern of these parameters is examined by calculating their average values, autocorrelation functions and Fourier transforms. To compare two proteins, their sequences are aligned using a unitary scoring matrix, and homologies are searched by plotting a two‐dimensional map of the correlation coefficients.

Comparison of the corresponding myelin basic proteins (MBP) and P0 glycoproteins (P0) for rodent and shark showed that the conserved residues included most of the amino acids which were predicted to form the α or β conformations, while the altered residues were mainly in the hydrophilic and turn or coil regions. In both rodent and shark the putative extracellular domain of P0 glycoprotein displayed consecutive peaks of β propensity similar to that for the immunoglobulins, while the cytoplasmic domain showed α‐β‐α folding. To trace the immunoglobulin fold along the P0 sequence, we compared the β propensity curve of P0 with that of the immunoglobulin M603, whose three‐dimensional structure has been determined. We propose that the flat β‐sheets of P0 are orientated parallel to the membrane surface to facilitate their homotypic interaction in the extracellular space. An extra β‐fold in the extracellular domain of shark P0 compared with rodent P0 was found, and this may result in a greater attraction between the apposed extracellular surfaces and may account for a smaller extracellular space as measured by x‐ray diffraction. A computer search of the myelin protein sequences for functional motifs revealed sites for N‐glycosylation, phosphorylation, nucleotide binding, and certain enzyme activities. We note especially that there are potential nucleotide binding sites in proteolipid protein (PLP), MBP and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNP). This is consistent with the experimental observations that PLP acts like an ionophore or proton channel when reconstituted into planar lipid bilayers, MBP binds GTP, and CNP catalyzes in vitro the hydrolysis of 2′,3′ nucleotides into corresponding 2′‐nucleotides.
Expression of plasmolipin in oligodendrocytes
Authors: Fischer Itzhak , Cochary E. F. , Konola J. T. , Romano‐Clarke G.
Published: January 2005

Plasmolipin is a plasma membrane proteolipid which has recently been described as a component of myelin (Cochary et al.: Journal of Neurochemistry 55:602–610, 1990). The present study reports the expression and localization of plasmolipin in primary glial cultures and secondary oligooendrocyte cultures. Double‐label immunofluoresccnce showed that plasmolipin was expressed by galactoccrebroside (GC)‐positive oligodendrocytes, but was absent from astrocytes, characterized by their positive staining for glial fibrillary acidic protein (GFAP). At 1 week in culture plasmolipin staining was relatively weak in the cell body of some of the GC‐positive cells. During the following 3 weeks in culture plasmolipin staining of Oligodendrocytes gradually increased and was present in the cell body, its plasma membrane, and all the processes. However, the plasmclipin antibodies did not stain regions of the flat membrane sheets. Western blot analysis of homogenatcs from primary glial cultures showed that plasmolipin levels gradually increased during the first 5 weeks in culture. We conclude that the presence of plasmolipin in myelin is a result of its expression by Oligodendrocytes.
1,25‐Dihydroxyvitamin D3 is a potent inducer of nerve growth factor synthesis
Authors: Wion D. , Macgrogan D. , Neveu I. , Jehan F. , Houlgatte R. , Brachet P.
Published: January 2005

1,25‐dihydroxyvitamin D3(1,25‐(OH)2D3), a metabolically active form of vitamin D, is shown to increase in a dose‐dependent manner the cellular pool of NGF mRNA in murine L‐929 fibroblasts cultured in a serum‐free medium. This effect can be detected as early as 3 hours after 1,25‐(OH)2D3 addition and persists for at least 28 hours. It is accompanied by an enhancement of the amount of NGF‐protein secreted in the culture medium. Since the proto‐oncogene c‐fos appears involved in the regulation of the NGF gene (Mocchetti et al.: Proceedings of the National Academy of Sciences of the United States of America 86:3871‐895, 1989; Hengerer et at: Proceedings of the National Academy of Sciences of the United States of America87:3899‐3903, 1990), the effect of 1,25‐(OH)2D3on c‐fos expression was analysed and compared to that elicited by other inducers of the NGF gene, serum (Wion et al: FEBS‐ Letters 189:37‐41, 1985) and phorbol 12‐myristate 13‐acetate (PMA) (Wion et al: FEBS Letters 262:42‐44, 1990). Addition of serum or PMA to L‐929 cells was; rapidly followed by a transient activation of the c‐fos gene. In contrast,c‐fos transcripts remained undetected in the presence of 1,25‐(OH)3D3 The failure to find any evidence of c‐fos expression suggests that 1,25‐(OH)2D3could enhance the pool of NGF mRNA by a mechanism independent of the c‐fos pathway. The effective concentration range, as low as 10−10 M raises the possibility that 1,25‐(OH)2D3 could represent one of the serum factors that might contribute to the regulation of the NGF gene in vitro and possibly in vivo. This suggests a possible therapeutic interest for 1,25‐(OH)2D3in neurodegenerative diseases.
Co‐activation of insulin‐like growth factor‐I receptors and protein kinase C results in parasympathetic neuronal survival
Authors: Crouch M. F. , Hendry I. A.
Published: January 2005

We have studied the interaction between several growth factors to promote parasympathetic neuronal survival. Neither insulin nor insulin‐like growth factor‐I (IGF‐I) had any effect on the survival of embryonic day 8 chick ciliary neurons in culture. Similarly, the protein kinase C activator phorbol dibutyrate (PdBu) had only a minor survival‐promoting activity. In combination with PdBu, however, IGF‐I or insulin, at concentrations sufficient to act through the IGF‐I receptor, were highly synergistic. In a similar fashion, acidic fibroblast growth factor (aFGF)‐induced neuronal survival was greatly enhanced by PdBu, as well as by insulin or IGF‐I. When added alone, aFGF‐induced cell survival required the presence of 1% serum. However, addition of aFGF, IGF‐I, or insulin with PdBu under serum‐free conditions replaced the serum requirement. That is, these agonist combinations could apparently induce the second messenger requirement for ciliary neuronal survival. Therefore, IGF‐I must now be included in the list of candidate molecules responsible for directing parasympathetic nerve formation. The synergy between agonists observed in these experiments highlights the possibility that combinations of growth factors, rather than sole molecules, may dictate parasympathetic nervous system development in vivo.
Increases in transforming growth factor‐β mRNA in hippocampus during response to entorhinal cortex lesions in intact and adrenalectomized rats
Authors: Nichols N. R. , Laping N. J. , Day J. R. , Finch C. E.
Published: January 2005

Transforming growth factor‐β mRNA was detected with a rat TGF‐β1 coding sequence probe as a 2.5 kb band by RNA blot hybridization of total RNA from the adult male rat hippocampus. Following electrolytic lesions of the entorhinal cortex that cause hippocampal deaffer entation and synaptic remodeling, TGF‐β mRNA increases 5‐fold in ipsflateral hippocampus when compared with intact controls. This increase was independent of prior adrenalectomy or corticosterone‐replacement. These data demonstrate that TGF‐β gene expression increases in response to hippocampal deafferentation.
Complex expression pattern of tenascin during innervation of the posterior limb buds of the developing chicken
Authors: Martini R. , Schachner M.
Published: January 2005

The histological localization of the extracellular matrix glycoprotein tenasein was studied during the formation of peripheral nerves in the developing chick hindlimb (embryonic stages 21.5 to 30) by light and electron microscopic immunological methods to obtain insights into the molecule's functional role in the pathway formation by motor and sensory nerves. At stages 21.5 and 23, nerve roots and plexus were surrounded by high tenascin‐immunorenctivity, whereas the not yet innervated limb bud was not imiminoreactive. During inncrvation of the limb bud at stages 24.5 and 25, tenascin was detectable at the limb bud base and restricted in its expression to the proximal nerve regions. The nerve tips did not contact areas of elevated tenascin‐immunoreactivity. At stages 26 to 28 the dorsal and ventral trunks of the crural and sciatic nerves were surrounded by tenascin‐immunoreactivity, which was localized between Schwann and mesenchymal cells. The tips of the growing nerve had now reached the tenascin‐positive interface between bone and muscle anlagen. This interface was contacted tangentially rather than penetrated by the nerve tips. The medial and lateral femoral cutaneous nerves were surrounded by high and weak tenascin‐immunoreactivity, respectively. In both nerves, tenascin‐immunoreactivity was absent where the nerves branched extensively to innervate the skin. The cutaneous nerves diverging from the sciatic nerve were of very low tenascin‐immunoreactivity or tenascin‐negative at all developmental stages tested. At stages 29 and 30, muscle nerves, having just entered the ten‐ascin‐negative muscles, exhibited strong immunoreactivity, whereas the more proximally situated trunks of the sciatic nerve were weakly and discontinuously labeled, particularly at sites where smaller nerves were branching off. Since the cutaneous branches of the sciatic nerve were always of low tenascin‐immunoreactivity, the question was raised whether tenascin expression in the sciatic nerve depended on the presence of motor axons. Spinal cords of stage 19 or 20 embryos were therefore removed and tenascin expression was investigated at stages 26 and 27. Some of the residual nerves were weakly tenascin‐immunoreaetive, whereas others were tenascin‐negative. Our observations suggest that tenascin is not involved in the initial guidance of peripheral nerves to their targets. Rather, neuron‐induced tenascin appears to stabilize the proximal nerve trunks during a transient time period, possibly by preventing axons and Schwann cells from intermingling with the surrounding mesenchyme, thus, contributing to nerve fiber compaction. Conversely, nerve branching may be elicited by reduced levels of tenascin. Furthermore, tenascin may divert growth cones from the developing bone tissue and direct muscle afferents to their appropriate targets.
Stimulation of energy metabolism by α‐adrenergic agonists in primary cultures of astrocytes
Authors: Subbarao K. V. , Hertz L.
Published: January 2005

Noradrenaline effects on glucose oxidation were studled in primary cultures of astrocytes. CO2 formation from labeled glucose was enhanced in the presence of; noradrenaline. The stimulatory effect by noradrenaline was exerted both on lactate formation (∼20%) I and on tricarboxylic acid activity (CO2 production from glutamate) (∼40%). The effect was, at least partly, exerted on the alpha‐ketoglutarate dehydrogenase step. The EC50 value for noradrenaline on lactate formation was significantly lower (60 nM) than that on oxidative metabolism (1,900 nM). Studies with specific adrenergic agonists and antagonists showed that various receptor subtypes are involved. Thus, the effect on lactate formation wasmediated exclusively by stimulation of an alpha1 receptor whereas oxidative metabolism was enhanced by both alpha1 and alpha2 receptor stimulation. No effects were exerted by beta receptor agonists or antagonists.
Morphology and antibody recognition of synthetic β‐amyloid peptides
Authors: Fraser P. E. , Duffy L. K. , O'Malley M. B. , Nguyen J. , Inouye H. , Kirschner D. A.
Published: January 2005

To elucidate the relationship between amyloid fibril formation in Alzheimer disease (AD) and the primary structure of the β‐amyloid protein (β‐AP), we investigated the ability of peptides sharing sequences with β‐AP to form fibrils in vitro and to recognize anti‐β‐amyloid antisera. The peptides, which were synthesized using a FMOC solid phase procedure and purified by HPLC, consisted of residues 6–25 from the putativeaqueous domain, residues 22–35, which overlaps the putative aqueous and transmembrane domains, and residues 1–38 and 1–40 representing nearly the full length of β‐AP. Electron microscopy of negative‐stained or thin‐sectioned preparations revealed that the peptides assembled into fibrils having different morphologies, some of which resembled in situ AD amyloid. Peptide 6–25 fibrils had diameters of 50–80 å and occasionally showed a central groove suggestive of constituent filaments. Cross sections of the fibril showed a penta‐ or hexameric arrangement ofglobular subunits with diameters of 25–30 å. Peptide 22–35 fibrils were helical, with a pitch of 1,100 å and a width of 120 A at its greatest and 50–60 å at its narrowest. The fibrils formed by peptides 1–38 and 1–40 were 70–90 å in diameter. When the peptide assemblies were singly oriented by sedimentation or doubly oriented in a magnetic field, their X‐ray diffraction patterns all showed reflections typical of a cross‐β pleated sheet conformation. The patterns differed mainly in their small‐angle equatorial intensity, which arises from the packing of fibrils having different widths. Antiserum raised to either native amyloid or to synthetic peptide β‐(l–28) was highly reactive in an inhibition‐ELISA assay to β‐(6–25) and β‐(1–38), but not to β‐(22–35), and immunostained β‐(1–40) on Western blots. These studies show that the β‐(6–25), β‐(l–38) and β‐(1–40) peptides can assemble into cross‐β fibrils that retain epitopes characteristic of AD amyloid.
Fetal rat septal cells adhere to and extend processes on basement membrane, laminin, and a synthetic peptide from the laminin A chain sequence
Authors: Jucker M. , Kleinman H. K. , Ingram D. K.
Published: January 2005

Responses of rat embryonic septal cells to reconstituted basement membrane, laminin, and laminin A chain‐derived synthetic peptides were studied in culture. Dissociated fetal E16/17 septal cells were grown for three days on differently coated plastic substrata. Reconstituted basement membrane (Ma‐trigel), laminin, and a 19‐amino acid synthetic peptide CSRARKQAASIKVAVSADR‐NH2 (PA22‐2) from the laminin A chain sequence mediated cell‐substratum adhesion and promoted neurite outgrowth. In contrast, cells did not attach to or form processes on uncoated plastic or on plastic substrata coated with synthetic, laminin‐derived control peptides. Polyethylenimine (PEI) supported the adhesion and survival of fetal septal cells; however, when lami‐nin was added to the medium during cell plating or 18 hr afterward, a dose‐dependent increase was observed in neurite outgrowth of cells attached to this substratum. Cells grown for 6 days on PEI in the presence of laminin showed a determined increase in the number of cholinergic neurons as marked by acetylcholinesterase staining. These data suggest that the subpopulation of cholinergic septal neurons present in the septal cells studied here were also responding to laminin. The results of this in vitro study suggest potential uses for basement membrane, laminin, or synthetic peptides, sucha as PA22‐2, in fetal septal grafts to enhance regeneration in the damaged septo‐hippocampal system.
Myelin/oligodendrocyte‐specific protein: A novel surface membrane protein that associates with microtubules
Authors: Dyer C. A. , Hickey W. F. , Geisert E. E.
Published: January 2005

Only a few proteins are known to be exclusively expressed in central nervous system (CNS) myelin. A novel surface membrane protein expressed only in CNS myelin and oligodendrocytes of higher vertebrates has been identified by a monoclonal antibody. This CNS myelin/oligodendrocyte‐specific protein, IMOSP, has a molecular weight of 48 kDa and a pI of : Approximately 6.7. In the presence of the monoclonal antibody, MOSP remains on the surface of cultured oligodendrocytes but becomes associated with cytoplasmic microtubules. Our results suggest that MOSP plays an important role in membrane/cytoskeleton interactions during the formation and maintenance of CNS myelin. MOSP also may play a critical role in the pathogenesis of diseases of CNS myelin.
Characterization of ramified microglia in tissue culture: Pinocytosis and motility
Authors: Ward S. A. , Ransom P. A. , Booth P. L. , Thomas W. Eric
Published: January 2005

Functional properties of ramified microglia were investigated in primary cultures of rat cerebral cortical celts. These microglia could be readily identified in both fixed and living cultures through previously established features. Based on their destruction by 5 mM L‐leucine methyl ester, a high level of intrinsic endocytotic activity was established. When cultures were incubated with fluorescent latex beads to assess phagocytosis, little or no such activity was exhibited by ramified cells. However, when cultures were incubated with dyes or other soluble tracer compounds, these cells always exhibited labeling. This labeling was selective for ramified microglia in the cultures and was demonstrated using a variety of compounds, including trypan blue, lucifer yellow, horseradish peroxidase (HRP), and India ink. Intracellular label could be observed in vesicular structures; this localization corresponded to an active cellular process. Also, cellular labeling was inhibited by the presence of colchicine. These features supported the inference that the labeling was attributable to pinocytosis, and this process appeared to account for the vast majority of endocytotic activity in the ramified microglia. Possible physiological significance of this pinocytotic activity was indicated by the accumulation of various neurotransmitters/modulators: γ‐aminobutyric acid and vasoactive intestinal polypeptide (VIP). Ramified cells in these cultures have been previously noted to exhibit a constant and rapid pattern of motility, which was consistently observed here through time‐lapse video recording; pinocytosis and rapid motility were shown to concur in individual cells. Based on their high intrinsic pinocytotic activity and pattern of cellular motility, the ramified microglia specifically are suggested to serve a constitutive function of fluid cleansing within the interstitial spaces of brain tissue.
The neurotrophic factor, n‐hexacosanol, reduces the neuronal damage induced by the neurotoxin, kainic acid
Authors: Borg Jacques
Published: January 2005

The long‐chain fatty alcohol, n‐hexacosanol, has been shown to possess neurotrophic properties in vitro on rat CNS cultures (Borg et al., 1987) and to promote the survival of septal cholinergic neurons after experimental axotomy (Borg et al., 1990). Long‐chain alcohols have also been shown to be synthesized and metabolised by rat brain during development (Bishop and Hayra, 1981; Natarajan et al., 1984). The present study was undertaken in order to find out if a nonproteic neurotrophic factor like n‐hexacosanol may be able to reduce the neuronal damages induced by the excitatory amino acid, kainic acid. When administered chronically by intraperitoneal injection, hexacosanol (1 mg/kg) protected the pyramidal neurons of the hippocampus from the neurotoxic degeneration induced by an intracerebroventricular infusion of kainic acid in rats; the extent of the damage was limited to a small part of the CA3 region. Mor‐phometric analysis showed that 72% of the neurons that would have died following kainic acid injection were spared by hexacosanol. Moreover the increased locomotor activity induced by the neurotoxin was also inhibited by hexacosanol and the behavioral effect was statistically correlated to the extent of neuronal loss. The present study suggests a possible role for nonproteic neurotrophic compounds against neurotoxic damages on central neurons. Moreover the peripheral administration of hexacosanol may lead to a significant breakthrough in the treatment of excito‐toxin‐related human diseases.
Non‐competitive antagonists of N‐methyl‐D‐aspartate prevent spontaneous neuronal death in primary cultures of embryonic rat cortex
Authors: Drian M.‐J. , Kamenka J.‐M. , Pirat J.‐L. , Privat A.
Published: January 2005

Primary cultures of embryonic rat cerebral cortex were treated after 17 days in vitro for 10 min with a single dose of the non‐competitive antagonists of N‐methyl‐D‐aspartate (NMDA) receptor MK 801, TCP, and GK 11. They were then maintained in vitro for 31, 59, or 73 days, and then processed for the immu‐nocytochemical detection of neuron‐specific enolase (NSE). Immunoreactive cells were counted in treated and control cultures, and it was found that, except at 31 days, treated cultures contained far more NSE immunoreactive cells than controls. Moreover, this effect was dose‐dependent, since with both TCP and GK 11 neuron survival was significantly higher with, respectively, 20 μM and 5 μM than with the lowest concentration of 2.5 μM. We tentatively conclude that spontaneous neuron death occurring in primary cultures in vitro is at least partly related to the NMDA‐associated Ca++ property of the molecules we used is to block this channel. The relevance of this mechanism of cell death in vitro to neuronal death in vivo is discussed.
Migration patterns of donor astrocytes after reciprocal striatum–cerebellum transplantation into newborn hosts
Authors: Jacque C. , Suard I. , Collins P. , Baumann N.
Published: January 2005

Fragments of striatum or cerebellum from E 25 rabbit embryo were implanted into either the striatum or the mesencephalon of newborn mice. Implanted rabbit astrocytes were selectively identified by mono‐donal antibodies to the GFAP which are unable to combine with mouse GFAP. Previous investigations had shown that xenogenic astrocytes have the capacto migrate in host CNS. The purpose of this study was to compare the patterns of migration of transplant‐derived astroglial cells according to the topographic origin of the transplant and location of the grafting site. We found that the migration pattern of the grafted cells from any of both selected sites of implantation was independent from the topographic origin of the transplant. The routes as well as the distances of migration were similar after homo‐ or heterotopic transplantation. We conclude that astroglial cells or their precursors do not express information which would direct them to move specifically toward a defined region in the host brain according to the region of origin in the donor.
Monoclonal antibodies to the heavy neurofilament subunit (NF‐H) of torpedo cholinergic neurons
Authors: Faigon M. , Hadas E. , Alroy G. , Chapman J. , Auerbach J. M. , Michaelson D. M.
Published: January 2005

Previous studies from our laboratory suggest that Alzheimer'disease sera contain a repertoire of antibodies to the heavy neurofilament subunit (NF‐H) and that a subpopulation of these antibodies bind specifically to epitopes highly enriched in NF‐H isolated from the purely cholinergic electromotor neurons of Torpedo. In the present study, we prepared and characterized monoclonal antibodies (MAbs) that bind to epitopes specifically enriched in Torpedo cholinergic neurons. This was performed by a differential enzyme‐linked immunosorbent assay (ELISA) in which MAbs were selected that bind to epitopes much more abundant in the NF‐H protein of Torpedo cholinergic neurons than in NF‐H from the chemically heterogeneous Torpedo spinal cord. This yielded four MAbs, three of which (TC4, TC8, and TC21) were found to be specific to NF‐H and one (TC15) that reacts with both NF‐H and the medium‐size neurofilament sub‐unit NF‐M. Dephosphorylation abolishes the binding of MAbs TC4 and TC15 to Torpedo cholinergic NF‐H, partially reduces that of MAb TC21 and has no effect on the binding of MAb TC8. This suggests that the antigenic sites specific to Torpedo cholinergic NF‐H contain phosphoryiated as well as non phosphory‐lated epitopes. All the MAbs cross‐react with rat brain NF‐H.
Kinetics of dopamine and noradrenaline transport in synaptosomes from cerebellum, striatum and frontal cortex of normal and reeler mice
Authors: Efthimiopoulos S. , Giompres P. , Valcana T.
Published: January 2005

Recent evidence indicates that the cerebellum has a dopaminergic system. In order to elucidate further the dopaminergic system in the cerebellum, we investigated the transport of dopamine (DA) in synaptosomal preparations of normal and reeler mice. For comparative purposes we also studied DA transport in synaptosomal preparations from striatum and frontal cortex and compared DA transport to nor‐adrenaline (NA) transport.

[3H]‐DA transport into cerebellar synaptosomes was found to be a Na+‐dependent, two component system–a high affinity, low capacity and a low affinity, high capacity. In striatum [33H]‐DA is transported by a similar high but different low affinity component. Maximal velocities of both transport components in the striatum were higher than the corresponding ones in the cerebellum. In the frontal cortex we also observed two [3H]‐DA transport components with affinities significantly lower than those in cerebellum and striatum.

[3H]‐NA transport into synaptosomes, prepared from the three brain regions studied, showed two transport components with similar Kt and Vmax values, except for the high affinity component in striatum whose affinity is lower.

In reeler mice [3H]‐DA transport was different from normal only in the cerebellum where the maximal velocity for both transport components was significantly higher (2 x). In contrast, no significant difference was observed in the transport of [3H]‐NA.

The accumulated [3H]‐DA from cerebellar slices was found to be releasable by K+ stimulation, in a Ca++‐dependent manner, and most of the released radioactivity was in the form of [3H]‐DA.

These results indicate that in the cerebellum there is a low‐density dopaminergic system which is distinct from the corresponding noradrenergic system.
Epidermal growth factor and transforming growth factor α induce c‐fos gene expression in retinal muller cells in vivo
Authors: Sagar S. M. , Edwards R. H. , Sharp F. R.
Published: January 2005

Epidermal growth factor (EGF) and transforming growth factor a (TGFα) are peptides that act at a common receptor and are mitogenic for immature astrocytes and trophic for developing brain neurons in vitro. However, a role for these growth factors in the mature nervous system has not been established. To investigate the actions of EGF and TGFα in the adult central nervous system (CNS) in vivo, the growth factors were injected into the vitreous cavity of adult male rabbits. After varying intervals, the retinas were examined for c‐fos mRNA by Northern blot hybridization or Fos (and Fos‐related antigen) protein by immunocytochemistry. EGF induction of c‐fos mRNA occurs within 30 min and persists more than 4 hr. Fos nuclear immunostaining is induced selectively in nuclei of Muller cells by both EGF and TGFα. Fos‐like immunoreactivity appears within 1 hr and persists more than 9 hr after EGF injection. These observations demonstrate that mature retinal Muller cells respond to exogenously applied EGF and TGFα in vivo, although the effect of the growth factors is not necessarily direct. The expression of c‐fos and other immediate early genes provides a short‐ term marker that can be used to investigate the role of growth factors in normal retinal physiology and responses to injury.
Hypothyroidism selectively reduces the rate and amount of transport for specific SCb proteins in the hyt/hyt mouse optic nerve
Authors: Stein S. A. , McIntire D. D. , Kirkpatrick L. L. , Adams P. M. , Brady S. T.
Published: January 2005

Thyroid hormone significantly affects molecular and neuroanatomical properties of the developing nervous system. Altered connectivity in hypothyroidism may reflect reductions in process growth, alterations in process maintenance, or changes in synaptogenesis or synaptic maintenance. These events are dependent on microtubules, neurofilaments, microfilaments, and associated molecular components. Reductions in delivery of microtubules and neurofilaments to the distal axon by slow component a (SCa) of axonal transport may contribute to the neuroanatomical abnormalities of hypothyroidism (Stein et al., J Neurosci Res 28:121–133, 1991). However, hypothyroidism might also affect the axon and synaptic connections by altering slow component b (SCb), which includes actin microfilaments and proteins that contribute to synaptic function, i.e., clathrin, HSC70 (clathrin uncoating ATPase), spectrin, and calmodulin. To determine the effect of hypothyroidism on SCb proteins, slow axonal transport was analyzed in optic nerves of hyt/hyt hypothyroid mice, which have severe primary hypothyroidism, and euthyroid control mice. Clathrin, spectrin, HSC70, and actin showed significant reductions in transport velocity in hyt/hyt optic nerves relative to euthyroid nerves, but the transport rate for calmodulin was less affected. However, the amount of calmodulin was significantly elevated in hyt/hyt nerve over euthyroid nerves. Hypothyroidism selectively reduces transport of SCb proteins, which are thought to play significant roles in synaptic function and in the growth cone. The effects of hypothyroidism on microtubules and neurofilaments combined with actions on SCb suggest that changes in neuronal function associated with reduced thyroid hormone during development and maturity (i.e., alterations in neuronal connectivity, nerve conduction, and synaptic function) may be mediated in part by effects on slow axonal transport.
Regional variation in the abundance of axonal cytoskeletal proteins
Authors: Watson D.
Published: January 2005

The relative abundance of several axonal cytoskeletal proteins was determined by immunoassay at various sites in the peripheral and central nervous systems of adult rats. Within the peripheral nervous system, the ratio of tubulin to neurofilaments was greatest for nerves composed of unmyelinated axons and least for nerves with large myelinated axons. MAP1 protein was more prominent in unmyelinated fibers; conversely tau proteins were relatively more abundant in large myelinated axons. An immunochemical index of neurofilament phosphorylation was less for unmyelinated fibers than for myelinated ones.

In the fimbria‐fornix, pyramidal tract, and superior cerebellar peduncle, similar trends were observed: small axons had more MAP1, less tau, and a greater ratio of tubulin to neurofilament proteins. The phosphorylation index was greatest for the superior cerebellar peduncle, the tract with the largest axons. The immunochemical index of neurofilament phosphorylation was greater for the optic nerve than for axonal tracts in the brain proper. These results suggest that development of large myelinated axons is associated with greater neurofilament content, neurofilament phosphorylation, and with greater abundance of tau proteins in the CNS and the PNS; however, quantitative aspects of these relationships differ in the PNS and the CNS.
Induction of tyrosine hydroxylase in the rat substantia nigra by local injection of forskolin
Authors: Leviel V. , Guibert B. , Mallet J. , Faucon‐Biguet N.
Published: January 2005

Forskolin (FSK) was locally injected into the substantia nigra (SN) of anesthetised rats. The day after injection (24 and 36 hr), tyrosine hydroxylase (TH) activity increased locally in this structure but remained unmodified in the ipsilateral caudate nucleus (CN). The amount of messenger RNA for TH (TH‐mRNA) was also increased in the SN 24 hr after the injection. However, TH protein content was modified neither locally in the SN nor in the ipsilateral CN. In addition, the decrease of the ratio between dopamine and its first metabolite in the CN and the SN suggested a decreased activity of the dopaminergic nigral cells. The absence of increase of the protein synthesis in spite of the fact that TH‐gene transcription was initiated could be the consequence of the inhibition of dopaminergic cells by the drug. These results confirm that, in vivo, TH induction is cAMP‐dependent and demonstrate that the TH‐gene activity is not strictly coupled to the activity of dopaminergic cells in the SN.
Adenosine‐dependent regulation of cyclic AMP accumulation in primary cultures of rat astrocytes and neurons
Authors: Murphy M. G. , Moak C. M. , Byczko Z. , MacDonald W. F.
Published: January 2005

The regulation of intracellular cyclic AMP (cAMP) formation by adenosine (Ado) and its analogues has been examined in primary cultures of rat‐brain astrocytes and neurons. In the presence of the phosphodiesterase inhibitor, Ro 20‐1724, basal levels of cAMP ranged from 40–120 pmol/mg protein in both cell types. Levels were not altered by treating the cells with Ado deaminase, which suggested that they did not produce appreciable amounts of endogenous Ado under standard culture conditions. In the astrocytes, μM quantities of agonists increased cAMP up to 30‐fold higher than basal values; the relative potencies were typical of an A2 Ado receptor (NECA > Ado > R‐PIA). Neuron‐enriched cultures exhibited a maximum fourfold increase in cAMP in response to NECA; this was decreased a further eightfold when the cultures had prolonged exposure to the antimitotic agent, c‐Ara, to eliminate >98% of the nonneuronal cells. Low (nM) amounts of the Ado agonists inhibited cAMP formation in both cell types. In the astrocytes, the order of potency of inhibition of isoproterenol‐stimulated cAMP formation was typical of an A1 receptor (R‐PIA > Ado > NECA); maximum inhibition was 55–65%. Isoproterenol did not increase cAMP in the neuronal cultures. However, forskolin‐stimulated formation was effectively (∼50%) inhibited by A1 Ado agonists; inhibition was not affected by prolonged treatment with c‐Ara. From this study we tentatively concluded that rat astrocytes and neurons both contain inhibitory A1 Ado receptors, but that the stimulatory “A2” subtype is localized mainly on astrocytes.
Nicotinic and muscarinic subtypes in the human brain: Changes with aging and dementia
Authors: Nordberg A. , Alafuzoff I. , Winblad B.
Published: January 2005

Different effects of normal aging on muscarinic and nicotinic receptor subtypes were observed in postmortem brain tissue from different regions of the human brain. A significant decrease in Ml and M2 receptors was found in cerebral cortex, while the Ml and especially the M2 receptors increased with age in the thalamus. A similar pattern of changes was also observed when using (−)3H‐nicotine as ligandfor nicotinic receptors in the cortex and thalamus. No significant changes in nicotinic receptor binding were observed with age in the cortex or thalamus, when using 3H‐acetylcholine as ligand. Nicotinic and muscarinic receptors in the brain are not equally affected in dementia disorders. A marked loss of high affinity nicotinic receptors was observed in cortical tissue from patients with Alzheimer's disease and with multi‐infaret dementia (MID). The muscarinic receptors were (both Ml and M2) increased in Alzheimer cortical tissue while they were decreased in MID.
Collagen IV is present in the developing CNS during Drosophila neurogenesis
Authors: Mirre C. , Le Parco Y. , Knibiehler B.
Published: January 2005

By means of immunocytochemistry with a battery of specific antibodies, we describe the expression of the collagen IV chain produced by the gene DCgl during the two phases of Drosophilaneurogenesis. DgC1 was not expressed in neuronal tissues as shown by in situ hybridization, but the onset of its expression in cells of mesodermal origin was concomitant with the appearance of collagen IV on early axon pathways and peripheral nerve roots in the embryonic developing CNS. A similar situation was found during imaginal CNS development at metamorphosis, when collagen IV immunoreactivity was detected on centrifugal and centripetal nerve pathways, and specially on retinula axons that develop from the eye imaginal disc towards the lamina anlage in the brain optic lobe. Our results strongly suggest that collagen IV could be involved, together with other informative molecules of basement membranes, in a dynamic process of cell‐matrix interactions during the establishment of initial axon pathways and neurite outgrowth in vivo.
Analysis of promoter activity and 5′ genomic structure of the neural cell adhesion molecule L1
Authors: Kohl A. , Giese K. P. , Mohajeri M. H. , Montag D. , Moos M. , Schachner M.
Published: January 2005

To gain insight into the molecular mechanisms underlying the regulation of expression of the neural cell adhesion molecule L1 and into the exon–intron structure of the L1 gene, a genomic clone from the mouse was characterized. The clone was identified by screening an EMBL3 library with an L1‐specific cDNA probe and comprises approximately 15 kb, in which the first 2,206 nucleotides of the coding region are included. Of the 5 of 6 immunoglobulin (Ig)‐like domains sequenced, all are encoded by 2 exons, with the first exon being smaller than the second. The exon encoding the signal peptide is separated from a mini‐exon containing 15 bp by a large intron, approximately 2.6 kb in length, whereas the other introns are smaller, with the coding information for the Ig‐like domains 3–5 clustered in a 1,643‐bp‐long fragment with introns only 110–217 bp in length. The 5′ upstream region of the clone comprises 5 kb, with the first 112 bp lying upstream to the coding sequence and containing a start site for transcription. No consensus sequence for a TATA box was found. Consensus DNA sequences for the binding of the gene products of Hox 1.3, engrailed and bicoid, are localized upstream to the transcription start site. A 1,262‐bp fragment containing part of the first exon showed promoter activity in neuroblastoma cells, but hardly in L cells and not in CHO cells, indicating that this fragment is sufficient for neural cell directed promoter activity. © 1992 Wiley‐Liss, Inc.
Novel stage in the oligodendrocyte lineage defined by reactivity of progenitors with R‐mAb prior to O1 anti‐galactocerebroside
Authors: Bansal R. , Pfeiffer S. E.
Published: January 2005

The developmentally regulated appearance of surface immuno‐reactivity of proligodendroblasts [oligodendrocyte progenitors reacting with monoclonal antibodies A007 and O4, but not anti‐galactocerebroside (GalC), i.e., A007/O4+ GalC−] to monoclonal antibodies R‐mAb and O1 was studied both in culture and in vivo. In both cases staining with R‐mAb shortly preceded that with O1; that is, a transient population of R‐mAb+ O1− cells was observed. R‐mAb− O1+ cells were not detected. Differential staining with R‐mAb and O1 was also noted at the subcellular level. In younger cultures in which R‐mAb+ cells were first acquiring O1 immunoreactivity, many of these cells were stained by O1 only on the cell bodies and proximal portions of the processes, whereas in contrast R‐mAb stained the whole cell, including the distal portions of the processes. Only in older, more mature R‐mAb+ cells did O1 also stain the distal portions of processes. The expression of reactivity to R‐mAb and O1 was compared to the proliferative capacity of the cells. Proliferation [assessed by bromodeoxyuridine (BrdU) incorporation] of both R‐mAb+ and O1+ cells was negligible both in culture and in vivo. However, treatment of cells in culture with 10 ng/ml basic fibroblast growth factor resulted in an enhancement of proliferation of the R‐mAb+ cells. Within the proliferating R‐mAb+BrdU+ population, 80% of the cells were O1− (i.e., anti‐galactocerebroside negative). These events occur during a critical period of development when A007/O4+ proligodendroblasts begin to become post‐mitotic and express surface galactocerebroside. The data demonstrate that the use of R‐mAb as an “anti‐GalC” must be interpreted with caution, and indicate the utility of dual staining with R‐mAb and O1 to (1) further subdivide the oligodendrocyte lineage, thus identifying an additional, GalC− developmental compartment, and (2) observe the distribution of R‐mAb and O1 immunoreactivity at a subcellular level. © 1992 Wiley‐Liss, Inc.
Immunocompetent cell markers in human fetal astrocytes and neurons in culture
Authors: Ennas M. G. , Cocchia D. , Silvetti E. , Sogos V. , Riva A. , Torelli S. , Gremo Fulvia
Published: January 2005

During the past few years, evidence has accumulated that interaction with peripheral immune cells as well as immunoregolatory functions in the central nervous system (CNS) can be played by several types of brain resident cells. Since very little information is available in man, however, we investigated the presence of markers so far considered typical of immunocompetent cells in in vitro cultures of human fetal brain. Immunocytochemistry at the light, scanning, and transmission electron microscopic levels revealed positivity for a very restricted range of macrophage antigens in astrocytes, which, however, were incapable of phagocytosis. In particular, expression of the major histocompatibility complex–class II antigen HLADR was observed in the cytoplasm and on the cell surface of GFA‐P+ astrocytes and increased with time in culture and cell passages. Among the T‐lymphocyte markers tested, Thy.1 and CD4 were positive. Both neurons and astrocytes carried Thy.1 from early cell passages. Noteworthy was the presence of CD4, which serves as the receptor for AIDS virus, in neurons from the first 2 weeks, whereas astrocytes became positive after only 4–6 weeks. Even if most staining was in the cytoplasm, some was exposed on cell surface. Astrocytes were found positive for the B‐lymphocyte marker CD21, the cellular receptor for Epstein‐Barr virus, whereas CD24 was detected in both neurons and astrocytes. Both antigens are related to B‐cell proliferation. Results are in favour of the hypothesis of human brain cells being actively involved in CNS immunological events. © 1992 Wiley‐Liss, Inc.
Endogenous aFGF expression and cellular changes after a demyelinating lesion in the spinal cord of adult normal mice: Immunohistochemical study
Authors: Tourbah A. , Baron‐van Evercooren A. , Oliver L. , Raulais D. , Jeanny J. C. , Gumpel M.
Published: January 2005

Fibroblast growth factors (FGFs) are known to act on glial cells in vitro. At the present time, their involvement in the remyelinating process of the adult central nervous system (CNS) is still unknown. In the present study, using immunohistochemistry (IHC), we investigated the evolution in time and space of acidic FGF (aFGF) expression and CNS cell changes occurring after a chemically induced demyelinating lesion. In a first early period, aFGF immunostaining was shown to decrease around the demyelinated area. A dramatic increase was then observed and was accompanied by an increase of cell density around and inside the lesion. This was correlated with the beginning of remyelination. Late after demyelination, while remyelination was still in progress, aFGF immunostaining of the lesion and unlesioned spinal cord were comparable. A role of aFGF in remyelination is proposed. © 1992 Wiley‐Liss, Inc.
Differential expression of carboxyl terminal derivatives of amyloid precursor protein among cell lines
Authors: Wolozin B. , Bacic M. , Merrill M. J. , Lesch K. P. , Chen C. , Lebovics R. S. , Sunderland T.
Published: January 2005

Understanding the pathway for amyloid precursor protein (APP) catabolism has become an important line of investigation. APP is a ubiquitous membrane bound protein that is rapidly cleaved at the membrane, yielding a secreted protein identical to protease nexin II and an internalized 11.5 kDa 100 residue C terminal derivative (CTD). The levels of CTDs in a variety of cell lines have been examined and were found to differ. Cell types associated with the pathology of Alzheimer's disease (AD), such as olfactory neuroblasts (ON) and cortical vascular endothelial cells, have higher levels of CTDs than lymphoblasts and melanoma cells. The mechanism of CTD catabolism appears to involve the lysosome because blockade of lysosomal but not endosomal or mitochondrial function results in increased levels of CTDs. Under these conditions, production of larger, amyloidogenic CTDs is also seen. In cells possessing higher levels of CTDs we find that the mechanism for production of amyloidogenic CTDs may involve the internalization of intact full‐length APP. Thus, inhibition of the lysosomal system appears capable of generating amyloidogenic peptides. The amount of amyloidogenic peptides appears to vary among cell lines. Such variation may shed light on why amyloid accumulates around specific cell types such as vascular endothelial cells, neurons, and glia. Finally, disfunction of the lysosomal system may play a role in the pathogenesis of Alzheimer's disease. © 1992 Wiley‐Liss, Inc.