bluuebunny/crossref_metadata_2025 huggingface dataset:
Permissive and non‐permissive reactive astrocytes: Immunofluorescence study with antibodies to the glial hyaluronate‐binding protein
Authors: Mansour H. , Asher R. , Dahl D. , Labkovsky B. , Perides G. , Bignami A.
Published: January 2005

Two distinct types of reactive astrocytes were studied in rat CNS. Reactive astrocytes secondary to penetrating trauma (anisomorphic gliosis) were induced by stab wounds to the brain. Reactive astrocytes secondary to Wallerian degeneration (isomorphic gliosis) were induced in spinal cord dorsal columns by dorsal rhizotomy proximal to dorsal root ganglia. Anisomorphic glial scars did not stain with antibodies to the glial hyaluronate‐binding protein (GHAP), a structural glycoprotein of white matter extracellular matrix. Conversely, isomorphic glial scars were still GHAP‐positive 3 months after dorsal root transection. Only after 5 months did GHAP immunoreactivity start to disappear from the isomorphic glial scar. Extensive dorsal rhizotomy was performed at the lumbar level to produce Wallerian degeneration of spinal cord dorsal columns. One month later, the rats were reoperated and two thoracic dorsal roots were implanted in the degenerated dorsal columns. The rats were examined 1 month after grafting. As expected, there was a dense anisomorphic glial scar at the site of surgery, while the dorsal columns above the graft showed isomorphic gliosis. Extensive axonal growth was observed in the dense glial scar surrounding the graft. Conversely, no axonal growth was observed in the degenerated dorsal columns undergoing isomorphic gliosis above the implant. The findings suggested that GHAP‐negative astrocytes responding to traumatic injury are permissive for axonal growth and that GHAP‐positive astrocytes responding to Wallerian degeneration are not permissive.
Modification of anoxic neuronal injury by human recombinant epidermal growth factor and its possible mechanism
Authors: Kinoshita A. , Yamada K. , Hayakawa T. , Kataoka K. , Mushiroi T. , Kohmura E. , Mogami H.
Published: January 2005

A primary culture of cortical neurons was established from a 17‐day rat embryo. Twenty‐four hours after plating, the culture medium was changed to a chemically defined one, and human recombinant EGF (rEGF) was added to the medium at concentrations of 1,10, and 100 ng/ml. Anoxic stress was exerted on the cultured neurons for 4 hr. Without anoxia, rEGF at a concentration of 10 ng/ml supported neuronal survival. When rEGF was not present in the culture medium, anoxic stress reduced neuronal density to one‐tenth that of the nonanoxic group. The rEGF improved neuronal recovery from anoxia significantly, with survival dependent on rEGF dosage. Monoclonal antibody for EGP receptors canceled the effect of rEGF on survival of neurons in a dose‐dependent manner. Therefore, rEGF functioned to support neuronal survival from anoxia through EGF receptors. Ontogeny of the EGF receptors was detected immunocytochemically with anti‐EGF receptor antibody. About 70% of the cultured neurons were stained by the anti‐EGF receptor antibody 3 days after change of medium, and immunoreactivity for EGF receptors was found to be located on the soma of neurons. Binding activity of EGF receptors was detected 5 days after change of medium with receptor binding assay and Scatchard analysis. High‐affinity binding sites were detected only in neurons cocultured with rEGF (10 ng/ml) for 5 days. The data indicate that rEGF in sufficient concentration induces high‐affinity sites of the EGF receptor and that EGF may have an antianoxic effect through the EGF receptors.
Role of nerve growth factor in oxidant‐antioxidant balance and neuronal injury. I. Stimulation of hydrogen peroxide resistance
Authors: Jackson G. R. , Apffel L. , Werrbach‐Perez K. , Perez‐Polo J. R.
Published: January 2005

The nerve growth factor protein (NGF) regulates neuronal cell death during the development of embryonic sensory and sympathetic neurons in the peripheral nervous system (PNS). NGF protects the rat pheochromocytoma line PC12, a useful model of NGF responsive peripheral neurons, from hydrogen peroxide, which interacts with ferrous iron to generate hydroxyl radicals. Exogenous catalase provides protection, whereas superoxide dismutase (SOD) has no effect on neuronal survival when PC12 cells are challenged with hydrogen peroxide. NGF treatment of PC12 cells increases the activity of catalase. NGF protection from hydrogen peroxide is partially abolished by aminotriazole (Az), a low molecular weight catalase inhibitor. Taken together, these data are consistent with the hypothesis that NGF protects from peroxidative events and consequent cell death via an induction of free radical detoxifying mechanisms, such as catalase activity.
Vasoactive intestinal peptide: A neurotrophic releasing agent and an astroglial mitogen
Authors: Brenneman Douglas E. , Nicol T. , Warren D. , Bowers L. M.
Published: January 2005

Vasoactive intestinal peptide (VIP) increases neuronal survival in dissociated spinal cord cultures during a critical period of development. In the present study, two mechanisms contributing to this action of VIP have been observed: (1) VIP was shown to be a secretagogue for neuron survival‐promoting activity; and (2) VIP was found to be an astroglial mitogen. A high molecular weight substance (>30 kDa), which increased neuronal survival in tetrodotoxin (TTX)‐treated spinal cord cultures, was detected in the medium from nonneuronal cells incubated for 1 hr with 0.1 nM VIP. In addition, 3H‐thymidine autoradiography and glial fibrillary acid protein (GFAP) immunocytochemistry were used to show that a 5 day treatment with (VIP) increased astroglial mitosis. This effect was specific for astroglia, as silver grain‐positive cells not exhibiting GFAP immunoreactivity did not increase in number after VIP treatment. The dual action of VIP may regulate glial‐derived trophic substances that are important for neuronal survival during the course of development.
Rescue of basal forebrain cholinergic neurons after implantation of genetically modified cells producing recombinant NGF
Authors: Strömberg I. , Wetmore C. J. , Ebendal T. , Ernfors P. , Persson H. , Olson L.
Published: January 2005

Mouse 3T3 fibroblasts were genetically modified by transfection with a mammalian expression vector containing the rat β‐nerve growth factor (NGF) gene. The transfected cell line, designated 3E, contains several hundred copies of the rat NGF gene and secretes high levels of biologically active NGF. Pieces of collagen gel containing the NGF‐secreting 3E cells were grafted to the brains of unilaterally fimbria‐fornixlesioned rats. Grafts of the genetically modified NGF‐producing cells rescued axotomized basal forebrain cholinergic neurons and significantly reduced cholinergic cell death in the medial septum as copared with rats treated with grafts of the parental 3T3 cells. Grafted fibroblast cells were detected, and rescue effects were noted up to 6 weeks after grafting. Local effects of NGF secreted by grafted cells were also seen at the gel‐brain border in the form of sprouting acetylcholinesterase immunoreactive host cortical fibers. We suggest that implantation of genetically modified cells producing NGF may have therapeutic applications in rescuing damaged central cholinergic neurons in senile dementia of the Alzheimer type as well as in providing trophic support for chromaffin tissue grafts in Parkinson's disease.
Role of the cAMP‐dependent protein kinase and protein kinase C in regulating the morphological differentiation of PC12 cells
Authors: Glowacka D. , Wagner J. A.
Published: January 2005

The cell line A126‐1B2 is a PC12‐derived mutant that is resistance to the toxic effects of dibutyryladenosine 3′:5′‐cyclic monophosphate (dbcAMP) and is deficient in adenosine 3′:5′‐cyclic monophosphate (cAMP)‐dependent protein kinase II (PKAII). This mutant formed neurites in response to nerve growth factor (NGF), but not in response to dbcAMP; and dbcAMP did not increase the rate of NGF‐dependent neurite formation. Thus, while PKAII is essential for process formation in response to agentsw that act through the cAMP‐dependent pathway, activation of PKAII is not essential for NGF‐dependent neurite formation. Unexpectedaly, NGF and phorbol 12‐myristate 13‐acetate (PMA; 10–1,000 nm) synergistically stimulated the formation of shortprocesses that were apparent within 30 min of NGF addition in 85% of these mutant dells. These processes were similar, but not identical, in appearance to the NGF‐dependent neurites that formed only after a period of 24–48 hr. This effect is dependent on the activation of protein kinase C (PKC) because an inactive phorbol ester was without effect. In contrast, therewas only a small effect of NGF and/or PMA on process formation in wiod type cells within the first few hours. The effect of PMA is not augmented by dbcAMP in the A126‐1B2 mutant cells. After several hours, PMA caused a concentration‐dependent decrease in cell adhesion; and higher concetrations of PMA resulted in a transient detachment of the cells and a loss of neurites. These experiments suggest a role for PKC in the regulation of process formation.
Myelin basic protein: Interaction with calmodulin and gangliosides
Authors: Chan K.‐F. J. , Robb N. D. , Chen W. H.
Published: January 2005

The structural characteristics of myelin basic protein (MBP) involved in protein‐protein and protein‐lipid interactions were investigated. Rabbit MBP could bind calmodulin (CaM) in the presence of Ca2+ to form a complex that remained undissociated in 8 M urea. However, no tight complex formation was observed when the divalent cation was absent. These results suggest that MBP may contain a hydrophobic domain similar to those in the other well‐characterized CaM‐binding proteins. The stoichiometry of calmodulin binding to MBP was approximately 1:1. Prior limited proteolysis of MBP with trypsin abolished the formation of the MBP‐CaM complex, indicating that the entire MBP polypeptide may be involved in the recognition of the hydrophobic clefts in CaM. MBP also formed tight complexes with gangliosides, but the presence of Ca2+ was not‐required. Binding of gangliosides to MBP‐CaM complex released CaM from the complex. The ganglioside‐binding sites in MBP were determined after trisecting the protein at two glutamic acid residues with Staphylococcus aureus V8 protease. Subsequent binding studies revealed that a 9.5‐kDa polypeptide, which may correspond to the NH2‐terminal domain (residues 1‐83) of MBP, had higher affinity for the binding of lucifer yellow CH‐labeled GM1 than did the other two polypeptides, of apparent molecular mass (Mr) 5,500 and 4,500, respectively. Among the various proteins in purified guinea pig brain myelin, synaptosomes, and synaptosomal membrances, MBP was found to have the highest affinity in binding lucifer yellow CH‐GM1.
Treatment with forskolin for 8 hours during the day increases melatonin synthesis in the Syrian hamster pineal gland in organ culture: The long lag period is required for RNA synthesis
Authors: Santana C. , Menendez‐Pelaez A. , Reiter R. J. , Guerrero J. M.
Published: January 2005

The exposure of organ‐cultured pineal glands of Syrian hamsters to forskolin, an adenylate cyclase activator, caused marked increases in melatonin levels when glands were collected at the second half of the dark period and incubated for 4 h. However, when glands were collected at the beginning of the dark period and incubated with the same drug, a significant increase in melatonin content was observed only after 6–8 h. Likewise, when glnds were collected at the beginning of the light period, forskolin stimulated melatonin synthesis only after 6–8 h of incubation with the drug. These results support the existence of a relatively long lag period necessary for the induction of melatonin production in the Syrian hamster pineal gland. Experiments with actinomycin D indicate that RNA synthesis occurs during the lag period; thus, actinomycin D blocks the induction of melatonin synthesis by forskolin in glands collected at the beginning of the dark period and incubated for 8 h. In contrast, when pineal glands were collected from hamsters killed in the second half of the dark period, and incubated with forskolin for either 4 or 8 h, actinomycin D was unable to block the induction of melatonin production. These data suggest that RNA, presumably messenger RNA, which is necessary for increasing hamster pineal melatonin synthesis, is synthesized and accumulates during the first half of the night.
Expression of the oligodendrocyte marker 2′3′‐cyclic nucleotide 3′‐phosphodiesterase in non‐glial cells
Authors: Staugaitis S M , Bernier L. , Smith P R , Colman D. R.
Published: January 2005

The 46 kD isoform of the 2′3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPi) was expressed in HeLa cells by transfection of its cDNA clone. The distribution of this polypeptide as mapped by indirect immunofluorescence and conventional epifluorescence microscopy appeared diffuse and generally uniform throughout the cytoplasm. Confocal microscopic imaging and analysis of pseudocolores images confirnes this distribution but also revealed that there was a high concentration of CNPi meat the plasma membrane of the cell. This pattern is very similar to that observed by immunoelectronmicroscopy of myelinating oligodendrocytes (Trapp et al.: J Neurochem 51:859–868. 1988: Braum et al.: J Neurosci 8:3057–3066, 1988). These results suggest that CNP may interact with a membrane‐associated molecule that is not unique to oligofendrocytes.
Bundling and cross‐linking of intermediate. Filaments of the nervous system
Authors: Shaw G. , Hou Zhao‐Chen
Published: January 2005

Ultrastructural studies have shown that neurons and glia in the mammalian nervous system contain bundles of often hundreds or more individual intermediate filaments. The means by which these bundles are formed and maintained has not been examined. We describe a series of simple experiments indicating that intermediate filament bundles derived from neuronal and glial processes are extremely stable, being resistant to a variety of extreme conditions. Furthermore, a preliminary examination of the mechanism of bundling for both types of filaments rules out several models for the production and control of crosslinking. We conclude that the long processes of both neurons and glia are stabilized by bundled intermediate filaments exhibiting strong interfilament interactions. We present a reconcilication these findings with previous data suggesting that neurofilaments are not actively cross‐linked. We also describe a novel detergent‐insoluble annular cytoskeletal structure, which appears to constrict bundles of axonal neuro filaments locally.
Tyrosine hydroxylase gene expression in human ventral mesencephalon: Detection of tyrosine hydroxylase messenger RNA in neurites
Authors: Dumas S. , Javoy‐Agid F. , Hirsch E. , Agid Y. , Mallet J.
Published: January 2005

The distribution of both tyrosine hydroxylase (TH) antigen and messenger RNA was investigated in the ventral mesencephalon from human brain using a 35S‐labeled human TH cDNA probe and a polyclonal TH antibody. A hybridization signal was detected not only in the perikaryon but also in the neurites, most probably dendrites, of immunoreactive neurons. In cell bodies, the level of TH mRNA varies, revealing a regional heterogeneity. In the substantia nigra of patients with Parkinson's disease the number of labeled cells was markedly reduced as compared to controls.
Transferrin gene expression and secretion by rat brain cells in vitro
Authors: De los Monteros A. Espinosa , Kumar S. , Scully S. , Cole R. , de Vellis J.
Published: January 2005

We have previously shown by immunocytochemistry in rat primary glial cultures that transferrin (TF) is an early developmental marker for oligodendrocytes. The present work addresses the issue of Tf gene expression and synthesis by neural cells in vitro. For this purpose, we used rat embryonic neuronal cultures and newborn glial cultures of astrocytes and oligodendrocytes. Cultured fibroblasts and C6 glioma cells were used as negative controls. We found that Tf mRNA is present in oligodendrocytes, astrocytes, and neurons. However, oligodendrocytes and astrocytes, but not neurons, were shown to synthesize and secrete Tf. Neither fibroblasts nor C6 glioma cells expressed detectable amounts of Tf mRNA. Levels in astrocyte cultures appeared to be under hormonal control since hydrocortisone markedly reduced message levels. These results show that both astrocytes and oligodendrocytes can synthesize and secrete Tf under cell culture conditions. However, epigenetic factors, such as hydrocortisone, may repress the expression of Tf in astrocytes in vivo.
The relationship of expression of statin, the nuclear protein of nonproliferating cells, to the differentiation and cell cycle of astroglia in cultures and in situ
Authors: Fedoroff S. , Ahmed I. , Wang E.
Published: January 2005

Cells in the quiescent, nonproliferative state express a protein, statin, in their nuclei. When the cells re‐enter the cell cycle, statin disappears and another protein, cyclin, appears. We have examined mouse astroglia at various stages of differentiation in cultures and astroglia in adult mouse brains for the presence of statin. In cultures initiated from the neopallium of newborn mice, the glial fibrillary acidic protein (GFAP)+ stellate astrocytes were statin‐negative (statin−) but cyclin‐positive (cyclin+).In the same cultures, large flat cells (senescent cells) were statin+ but cyclin−. In frozen sections of the brains of adult mice and in brain smears, GFAP+ astrocytes were statin−. Neither stellate astrocytes grown in cultures for 30 or more days nor astrocytes in adult mouse brain were labeled when pulsed with bromodeoxyuridine (BudR). When astroglia were treated with dibutyryl cyclic adenosine monophosphate(dBcAMP), Large stellate cells that closely resemble reactive astrocytes in situ formed. These cells were all statin+ from 11–62 days in vitro; however, reactive astrocytes in mouse neopallium, 4–50 days after a stab wound, were statin−. In colony cultures, senescent cells became statin+, whereas stellate astrocytes and their precursor cells remained statin−. These observations indicate that normal astrocytes both in cultures and in situ retain the potential to divide and probably progress through the cell cycle at a very slow rate.
Low‐energy laser irradiation—a new measure for suppression of arachidonic acid metabolism in the optic nerve
Authors: Naveh N. , Bar‐Ilan A. , Rosner M. , Schwartz M. , Weissman C. , Belkin M.
Published: January 2005

Helium‐neon lowenergy laser (LEL) irradiation, known for its therapeutic effects in arthritis, wound healing, and muscular strain, was applied to eyes of rats that had sustained and optic nerve crush injury. Crush injury to the optic nerve resulted in a longlasting in vitro elevation of prostaglandin E2(PGE2) production (1.8‐3.9‐fold above control values during the 12 day study period). LEL irradiation per se had no effect in vitro on PGE2 production by the optic nerve; however, LEL irradiation of eyes with crushed optic nerve inhibited the enhanced production of PGE2 and leukotriene B4 in vitro by 55% and 75% during the late and immediate phase after trauma, respectively. Our findings of the suppressive effect of LEL irradiation on arachidonic acid metabolism are reminiscent of the actions of steroidal and nonsteroidal anti‐inflammatory drugs, and might indicate, for the first time, the possible biochemical mechanisms associated with the clinical anti‐inflammatory effects of LEL irradiation.
GD3 ganglioside is prevalent in fully differentiated neurons from rat retina
Authors: Daniotti J. L. , Landa C. A. , Gravotta D. , Maccioni H. J. F.
Published: January 2005

Adult mammalian retinas contain unusually high amounts of GD3, a ganglioside of the lactosylceramide series. In this respect, they differ from adult avian retina and other regions of the adult avian and mammalian brain, where GD3 is a minor ganglioside and gangliosides of the gangliotetraosylceramide series (GM1, GD1a, GD1b, GT1b) are the predominant ones. We compare here the ganglioside patterns of rat, human, horse, and guinea pig retinas, which are known to differ in the degree of vascularization and astrocytic cell content. All these retinas showed a prevalence of pathway “b” gangliosides over pathway “a” gangliosides but showed no correlation between GD3 content and the degree of vascularization and astrocytic cell content. Immunostaining of rat retina sections showed the presence of GD3 in the inner and outer plexiform layers and also in the ganglion cell and inner nuclear layers. About 60% of the cells dissociated from rat retina showed immuno‐co‐localization of GD3 and the neuronal marker class III β tubulin isotype or cholera toxin binding. All morphologically identifiable glial Muller cells coexpress GD3 and gangliotetraosylgangliosides. GD3 was a minor ganglioside among these axonally transported by ganglion cells in rats and guinea pigs, suggesting that it is either not synthesized by ganglion cells or, if so, it is restricted to the cell soma and/or dendritic tree.

Our results demonstrate that, unlike neurons from avian retina and other regions of avian and mammalian brain, neurons from mammalian retina not only contain gangliosides of the gangliotetraosylceramide series but also keep a prevalence of gangliosides of the lactosylceramide series (GD3) when they are fully differentiated.
Involvement of dihydropyridine‐sensitive calcium channels in nerve growth factor‐dependent neurite outgrowth by sympathetic neurons
Authors: Rogers M. , Hendry I.
Published: January 2005

We have used a number of pharmacological manipulations of calcium influx to alteeer the nerve growth factor (NGF)‐elicited neurite outgrowth response of SCG neurons. Our results indecate that influx of extracellular calcium is critical to sympathetic SCG neurite outgrowth. Effective blockade of this process was produced by the inorganic calcium channel blockers Cd2+ (with an IC50 of 48μM), Co2+ (129μM), and Ni2+ (180 μM). More specifically, there is a significant contribution from dihydropyridine‐sensitive L‐type calcium channels to NGF‐activated neurite outgrowth, as evidenced by the significant inhibition of neurite outgrowth by diltiazem (IC50 of 17μM) and nifedipine (3μM). Further, increases in calcium influx can elicit an enhanced neurite outgrowth response, as shown by the calcium channel agonist Bay K 8644 wich potentiated neurite outgrowth by up to 40%.
Tellurium‐induced neuropathy: A model for reversible reductions in myelin protein gene expression
Authors: Toews A. D. , Lee S. Y. , Popko B. , Morell P.
Published: January 2005

Inclusion of 1.1% tellurium in the diet of developing rats causes a highly synchronous primary demyelination of peripheral nerves, which is followed closely by a period of rapid remyelination. The demyelination is related to the inhibition of squalene epoxidase activity, which results in a block in cholesterol synthesis and accumulation of squalene. We now report that the demyelination resulting from this limiting of the supply of an intrinsic component of myelin (cholesterol) leads to repression of the expression of mRNA for myelin‐specific proteins. Tellurium exposure resulted in an increase in total RNA (largely rRNA) in sciatic nerve, which could not be accounted for by cellular proliferation; these increased levels of rRNA may be a reactive response of Schwann cells to toxic insult and may relate to the higher levels of protein synthesis required during remyelination. In contrast, steadystate levels of mRNA, determined by Northern blot analysis, for Po and myelin basic protein were markedly decreased (levels after 5 days of tellurium exposure were only 10‐15% of control levels as a fraction of total RNA and 25‐35% of control levels when the increased levels of total RNA were taken into account). Message levels increased during the subsequent period of remylination and reached near‐normal levels 30 days after beginning tellurium exposure. Although message levels for the myelin‐associated glycoprotein showed a similar temporal pattern, levels did not decrease as greatly and subsequently increased sooner than did levels for Po and myelin basic protein. The coordinate alterations in message levels for myelin proteins indicate that Schwann cells can down‐regulate and then up‐regulate the synthesis of myelin in response to alterations in the supply of membrane components.
Iron‐enriched oligodendrocytes: A reexamination of their spatial distribution
Authors: LeVine S. M. , Macklin W. B.
Published: January 2005

Previous histochemical studies of iron in the brain demonstrated iron enrichment in oligodendrocytes, but the iron‐enriched oligodendrocytes had a spatially restricted distribution that excluded major white matter tracts. In this study we used techniques designed to permeabilize tissue, combined with iron histochemistry, and we observed iron‐enriched oligodendrocytes in regions previously thought to lack such cells. Our data suggest that most, if not all, oligodendrocytes are enriched in iron. We suggest that iron functions in the formation and/or maintenance of the myelin sheet and may play a role in the pathology of myelin diseases.
Lurcher purkinje cells express glutamic acid decarboxylase and calbindin mRNAs
Authors: Wuenschell C. W. , Messer A. , Tobin A. J.
Published: January 2005

Purkinje neurons in immature Lurcher (Lc/ +) mice are destined to die as a result of a defect intrinsic to the dying cells. We have used in situ hybridization to determine whether the Lc allele interferes with the normal program of gene expression in the doomed Purkinje cells. In P21 mice, degeneration of Purkinje cells is well underway, but the surviving Purkinje cells continue to express the mRNAs for both glutamate decarboxylase and calbindin D28k, two proteins whose expression is characteristic of normal Purkinje neurons. We conclude that the Lc allele probably does not interfere with the developmental program but acts to cause cell death in already differentiated Purkinje neurons.
Properties of acetylcholinesterase in axolemma‐enriched fractions isolated from bovine splenic nerve
Authors: Hannesson H. H. , DeVries G. H.
Published: January 2005

The properties of acetylcholinesterase (AChE) in axolemma‐enriched fractions (AEF) from bovine splenic nerve were investigated to see if they differed in any way from those of the AChE in diaphragm muscle. The axolemmal enzyme had a low Km for acetylthiocholine (ca. 90 μM), exhibited substrate inhibition, and had a well‐defined optimum of substrate concentration of 1mM. The rate of hydrolysis of substrate decreased with increasing acyl chain length (acetyl‐>propionyl‐>butyryl‐). The AChE inhibitors eserine and hexamethonium were competitive inhibitors of the membrane‐bound enzyme, whereas lidocaine was a noncompetitive inhibitor; these results were comparable to the effect of these inhibitors on diaphragm muscle AChE. The axolemmal enzyme was more efficiently solubilized and more stable in nonionic detergents such as Triton X‐100 and Tween 20 than charged detergents such as lysolecithin and zwitterionic detergents. These results indicate that the AChE present in bovine splenic nerve AEF is identical to the previously characterized AChE from other sources.
Differential effect of μ, δ, and κ ligands on G protein α subunits in cultured brain cells
Authors: Vogel Zvi , Barg J. , Attali B. , Simantov R.
Published: January 2005

Rat and guinea pig fetal brain cell cultures and immunoblotting techniques were used to study the effect of receptor selective opioids on the level of the membrane‐bound α1 and α0 GTP binding protein subunits. Incubation of rat hindbrain cultures with the μ selective peptide DAGO decreased the amount of both α proteins. The reduction observed was equivalent to 36% in α0 and 41% in α1. On the other hand, incubation of rat forebrain cultures with this peptide had an opposite effect, increasing the α0 and α1 levels by 66% and 68%, respectively. This differential effect of the peptide on the G proteins at the two brain areas may reflect the selective interaction at the receptor level; DAGO induced a fast and effective receptor down‐regulation (50% decrease in Bmax) in hindbrain but not in forebrain cultures. Moreover, δ and μ selective ligands differed in their effect, as indicated by the finding that the δ selective peptide DPDPE increased the amount of both α proteins in hindbrain cultures by 40%. Similar experiments conducted with guinea pig brain aggregate cultures indicated that the κ selective agonist U50, 488 decreased the amount of the membrane bound α1 protein subunit by 56%. The results thus indicate that opioid agonists, interacting selectively with the three types of opioid receptors, induce a complex repertoire of changes in the immunoreactive levels of the membrane‐bound α GTP binding protein subunits in various CNS structures.
Axonal dependency of the postnatal upregulation in neurofilament expression
Authors: Schwartz M. L. , Shneidman P. S. , Bruce J. , Schlaepfer W. W.
Published: January 2005

A coordinated up‐regulation in the expression of all three neurofilament (NF) proteins occurs during postnatal development in the rat (Schlaepfer and Bruce, J Neurosci Res [in press], 1990a). In the present study, sciatic nerves were transected in neonatal rats in order to determine the effects of axotomy on the postnatal upregulation of NF expression in neurons of rat dorsal root ganglia (DRG). Left sciatic nerves were transected at postnatal day 3 (P3), 6 (P6), 8 (P8), or 10 (P10). mRNA and protein levels of the light (NF‐L), mid‐sized (NF‐M), and heavy (NF‐H) NF proteins were compared in L4 and L5 DRGs from the transected (left) vs. control (right) sides of the same animals at varying intervals after transection. When nerves were transected at P10, mRNA levels of all three NF proteins declined markedly in the parent DRG neurons, thereby completely interrupting the postnatal upregulation of NF expression. P10 transections also led to widespread chromatolytic changes in axotomized neurons, indistinguishable from those that occur in adult DRG following sciatic nerve transection (Goldstein et al., J Neurosci 7:1586–1594, 1987). Nerve transections at earlier (e.g., P3) neonatal timepoints also led to a decrease of NF expression, but to a lesser extent than that which resulted from a P10 transection. Also, P3 transections caused only minimal chromatolytic changes in the axotomized neurons. Thus, the postnatal upregulation of NF expression is dependent upon axonal continuity and the extent of axonal dependency increases during early postnatal development. These findings support the hypothesis that the postnatal upregulation of NF expression, the axotomy‐induced downregulation of NF expression and the chromatolytic reaction to nerve transection are all dependent upon or responsive to axonal‐ or target cell‐derived signals that are acquired during postnatal maturation.
Rat facial motoneurons express increased levels of calcitonin gene‐related peptide mRNA in response to axotomy
Authors: Haas C. A. , Streit W. J. , Kreutzbeg G. W.
Published: January 2005

The expression andAbteilung localization of the mRNA encoding the calcitonin gene‐related peptide (CGRP) were analyzed in the rat facial nucleus after axotomy by Northern blot analysis and by in situ hybridization histochemistry (ISH) using a synthetic 32P‐labeled oligonucleotide probe. Northern blot analysis revealed the presence of the 1.2 kb CGRP mRNA in RNA extracted from the facial nucleus. This mRNA species was strongly increased after axotomy of the facial nerve. By ISH increased levels of CGRP mRNA were observed as soon as 16 hr after axotomy compared with the unoperated nucleus. CGRP mRNA could be localized in more than 50% of the motoneurons. Three populations of motoneurons with no, moderate, or strong labeling for CGRP mRNA could be distinguished. Peak expression of CGRP mRNA during the first 48 hr was followed by a decline to moderate levels at day 4 after lesion, and to almost basal levels at days 7 and 9. These data demonstrate that axotomy of the facial nerve leads to an early and strong induction of CGRP gene expression in motoneurons of the facial nucleus.
Regulation of β‐nerve growth factor expression by inflammatory mediators in hippocampal cultures
Authors: Friedman W. J. , Lärkfors L. , Ayer‐LeLievre C. , Ebendal T. , Olson L. , Persson H.
Published: January 2005

Substances which regulate expression of nerve growth factor (NGF) were examined in embryonic rat: hippocampal cultures containing both neurons and glial cells. Both cell types expressed NGF mRNA when cultivated in vitro. Lipopolysaccharide, an activator of macrophages, elicited a significant increase in NGF mRNA. Interleukin‐1β evoked a similar increase in NGF mRNA which was accompanied by a rise in NGF protein. The II‐1‐induced increase was partially blocked by indomethacin, suggesting that prostaglandins might mediate this effect. Treatment of the cultures directly with prostaglandin E2 resulted in elevated levels of both NGF mRNA and protein. Thus, agents which promote inflammatory activity appear to increase NGF expression. Moreover, a suppressor of inflammation, dexamethasone, decreased NGF expression. Our observations indicate that a variety of immunomodulators regulate NGF expression in the hippocampus.
Gene delivery to glioma cells in rat brain by grafting of a retrovirus packaging cell line
Authors: Short M. P. , Choi B. C. , Lee J. K. , Malick A. , Breakefield X. O. , Martuza R. L.
Published: January 2005

Retrovirus vectors only integrate into the genome of dividing cells and can thus be used to selectively infect tumor cells in the adult rat brain. Gene delivery was assessed by using the retrovirus BAG vector, which bears the Escherichia coli lacZ gene under the MoMLV LTR promoter‐enhancer element, and by histochemical staining for bacterial beta‐galactosidase activity. Direct injection of this vector (90–900 cfu) into the adult rat brain, with or without prior inoculation of C6 glioma cells (2 × 105 cells) resulted in labeling of only a few cells as assessed 1 week later. When the psi 2‐BAG packaging line was grafted into the brain, labeled psi 2‐BAG cells could be found after 1 day, but not after 5 days, following grafting, suggesting that the grafted cells had been rejected and that no endogenous cells had integrated released vector, or that expression of lacZ had been turned off. In contrast, when the psi 2‐BAG packaging line was grafted into a brain region, which had been inoculated previously with rat C6 glioma cells (2 × 105 cells), beta‐galactosidase labeling of these tumor cells, identified by immunocytochemistry for glial fibriliary acidic protein and S100, could be demonstrated 10 days later. Thus, grafting of retrovirus packaging lines into adult brain provides a means to infect tumor cells in situ. The grafted packaging cells may continue to release retrovirus particles for an extended period, thus infecting more cells at the stage of division appropriate for viral integration, as compared to inoculation of the virus alone. Grafting of retrovirus packaging cell lines could be used to selectively deliver “killer” or “suppressor” genes to tumor cells in the brain to curtail their growth.
Preferential uptake and metabolism of docosahexaenoic acid in membrane phospholipids from rod and cone photoreceptor cells of human and monkey retinas
Authors: de Turco E. B. Rodriguez , Gordon W. C. , Peyman G. A. , Bazan N. G.
Published: January 2005

The uptake, metabolism, and cellular distribution of [3H]docosahexaenoic acid (DHA) in human and monkey retinas were studied with biochemical and autoradiographic techniques. In specimens from two human retina biopsies, incubated for 4 hr or 6 hr with [3H]docosahexaenoic acid (110 nM), 80% of the esterified [3H]fatty acid was recovered in phospholipids and the remainder in triacylglycerols and diacylglycerols. The distribution of [3H]DHA in individual phospholipids (PL) was similar in both retinas, with phosphatidylcholine (PC) and phosphatidylethanolamine (PE) accounting for most of the label. A similar labeling profile was observed in glycerolipids from monkey retina, and after 1 hr of incubation, high labeling of phosphatidic acid (PA, 11%) and phosphatidylinositol (PI, 20%) was observed.

In both human and monkey retinas, a preferential uptake of [3H]DHA by photoreceptor cells was revealed by autoradiography. Cone photoreceptors showed a slightly higher density of silver grains in their inner segments than did rod photoreceptors. Photoreceptors accounted for 59% and 79% of the total [3H]DHA taken up by the human and monkey retinas, respectively, the remainder being distributed throughout the neural retina. In conclusion, this study shows for the first time that in human and monkey retinas, DHA is taken up with a high degree of selectivity by photoreceptor cells, and then becomes esterified mainly into phospholipids that will be subsequently utilized for the synthesis of new disc membranes.
Age‐related decreases in muscarinic cholinergic receptor binding in the human brain measured with positron emission tomography (PET)
Authors: Dewey S. L. , Volkow N. D. , Logan J. , MacGregor R. R. , Fowler J. S. , Schlyer D. J. , Bendriem B.
Published: January 2005

Muscarinic cholinergic Ml and M2 receptors in young and aged adult male volunteers were studied using [N‐11C‐methyl]‐benztropine, a specific muscarinic cholinergic receptor ligand, and high resolution positron emission tomography (PET). A regionally specific pattern of decreased binding was observed in aged volunteers. Using two separate methods of data analysis, thalamic, hippocampal and cerebellar regions showed no decreases in the apparent specific binding of [N‐11C‐methyl]‐benztropine while frontal, parietal, temporal and occipital cortices as well as the corpus striatum showed age related changes in binding that declined (in 82 yrs old subject) to about 50 % of the value obtained from the youngest volunteer (19 yrs). These data suggest that regions high in muscarinic receptor density, the corpus striatum and the cortical mantle, show a greater rate of decline than those areas that have a relatively low number of muscarinic receptors. Furthermore, this study demonstrates the usefulness of PET and [N‐11C‐methyl]‐benztropine for assessing age related regional changes in muscarinic cholinergic receptor binding in the living human brain.
Cellular distribution of transferrin, ferritin, and iron in normal and aged human brains
Authors: Connor J. R. , Menzies S. L. , Martin S. M. St. , Mufson E. J.
Published: January 2005

The iron transport protein, transferrin, and the iron storage protein ferritin were examined immunohistochemically along with iron in a number of brain regions from normal and aged humans. Two age groups were examined: a middle‐aged group (28–49 years), and an older group (60‐90 years). Transferrin, ferritin, and iron are found throughout all brain regions examined, predominantly in the perikaryal cytoplasm of cells that are small and round, fitting the description of oligodendrocytes. These cells are present in the optic nerve and in both the gray and white matter of the cerebral cortex, cerebellum, and olfactory bulb in both age groups. Ferritin is also found in microglial cells in the gray matter of most of these brain regions. In the subcortical regions examined (corpus striatum, hippocampus, amygdala), in addition to oligodendrocytes, astrocytes can frequently be observed that contain transferrin, ferritin, and iron. There is an age‐related alteration in cell labeling: astrocytes in both gray and white matter contained transferrin in the oldest age group, whereas in the younger group the subcortical transferrin immunoreactivity was confined mostly to oligodendrocytes. Ferritin in the subcortical brain regions is also present in astrocytes but is primarily confined to those in the gray matter, even in the oldest age group. Iron is found predominantly in oligodendrocytes, although a few iron‐positive astrocytes and microglia can be identified. These results indicate that (1) normally oligodendrocytes contain much of the iron and iron‐binding proteins found in the brain; and (2) an increase in age is associated with altered cellular distribution of iron‐binding proteins, but the altered distribution is specific to glial cells. These results suggest glial cells may have previously undescribed functions related to metal regulation and sequestration.
Morphologic heterogeneity of human adult astrocytes in culture: Correlation with HLA‐DR expression
Authors: Yong V. W. , Yong F. P. , Olivier A. , Robitaille Y. , Antel J. P.
Published: January 2005

Human adult astrocytes derived from brain surgical resections showed marked morphologic heterogeneity when cultured in vitro, ranging from a flat, fibroblast‐like appearance to process‐bearing cells with little soma cytoplasm. The majority of cells were intermediate in morphology, bearing a prominent cytoplasmic cell body with processes radiating from them. The morphologic heterogeneity was more extensive than that of adult rat astrocytes, and was not correlated with the extent of attendant gliosis in the surgical specimens, or the site of the surgical resection. None of the human astrocytes expressed A2B5, thus preventing their classification on basis of lineage into type 1 or type 2 astrocytes. However, functional differences appear to exist between subpopulations of human astrocytes, since the proportion of process‐bearing human astrocytes that expressed HLA‐DR in vitro was significantly greater than that found for flat astrocytes.
Active polysomes in the axoplasm of the squid giant axon
Authors: Giuditta Antonio , Menichini E. , Capano C. Perrone , Langella M. , Martin R. , Castigli E. , Kaplan B. B.
Published: January 2005

Axons and axon terminals are widely believed to lack the capacity to synthesize proteins, relying instead on the delivery of proteins made in the perikaryon. In agreement with this view, axoplasmic proteins synthesized by the isolated giant axon of the squid are believed to derive entirely from periaxonal glial cells. However, squid axoplusm is known to contain the requisite components of an extra‐mitocliondrial protein synthetic system, including protdn factors, tRNAs, rRNAs, and a heterogeneous family of mRNAs. Hence, the giant axon could, in principle, maintain an endogenous protein synthetic capacity. Here, we report that the squid giant axon also contains active polysomes and niRNA, which hybridizes to a riboprobe encoding murine neurofilament protein. Taken together, these findings provide direct evidence that proteins (including the putative neuron‐specific neurofilament protein) are also synthesized de novo in the axonal compartment.
Increased PKA and PKC activities accompany neuronal differentiation of NT2/D1 cells
Authors: Abraham I. , Sampson K. E. , Powers E. A. , Mayo J. K. , Ruff V. A. , Leach K. L.
Published: January 2005

After retinoic acid treatment, a large percentage of cells of the human embryonal carcinoma cell line NT2/D1 differentiate into neuronal cells. We demonstrate here that the differentiated cells, but not the undifferentiated cells, contain high levels of neurofilament mRNA. We have also measured mRNA, protein, and activity levels of two kinases, cAMP‐dependent protein kinase (PKA) and protein kinase C (PKC), in order to explore the role of protein kinases in the establishment of the differentiated state. RNA levels for the catalytic (Cα and Cβ) subunits of PKA increased after differentiation. Total PKA activity levels increased 7‐fold in the differentiated cells. Parallel with this, a rise in the level of catalytic subunit protein occurred. A 12‐fold induction of Type 2 (β) PKC mRNA levels was observed after neuronal differentiation. Increases in PKC activity and in Type 2 (β) and Type 3 (α) PKC protein levels also accompa nied differentiation. These changes in PKA‐ and PKC‐specific RNA levels and enzyme activity may be necessary for production and maintenance of the differentiated state in these cells.
Microglial cells are a component of the perivascular glia limitans
Authors: Lassmann H. , Zimprich F. , Vass K. , Hickey W. F.
Published: January 2005

The ultrastructural relation between microglial cells and cerebral blood vessels was studied in rat brains by immune electron microscopy using antibodies against the common leukocyte antigen (Oxl), the complement receptor 3 (Ox42), and against class I and class II histocompatibility antigens (MHC antigens; Ox3, Ox6, Oxl8, and I1‐69). Microglial cell processes were found incorporated between the astrocytic foot processes of the glia limitans in 4–13% of cerebral microvessels. After intravenous injection of γ‐interferon, either alone or in combination with tumor necrosis factor, these microglial cell processes expressed classes I and II MHC antigens. Studies in (Lewis × DA)F1‐DA bone marrow chimeras demonstrated that these cell processes belonged to resident microglia. This study suggests that microglial cells may play an important role in antigen recognition at the blood‐brain barrier.
Expression of IGF‐I and insulin receptor genes in the rat central nervous system: A developmental, regional, and cellular analysis
Authors: Baron‐Van Evercooren A. , Olichon‐Berthe C. , Kowalski A. , Visciano G. , Van Obberghen E.
Published: January 2005

Using a solution‐hybridization assay and specific oligonucleotidic probes, we have studied IGF‐I and insulin receptor mRNAs in the rat central nervous system during development. The expression of mRNAs was maximal at embryonic day 15 and 20 for IGF‐I receptors, and at embryonic day 20 and the day of birth for insulin receptors. After birth, the expression of both receptor transcripts decreased and reached minimal levels in the adult. At the time at which these transcripts were maximally expressed (embryonic day 20), the regional analysis indicated that IGF‐I receptor transcripts were widely distributed in the brain. In contrast, insulin receptor transcripts were restricted to certain areas in which they were coexpressed with the IGF‐I receptor transcripts. We next analyzed which cells at embryonic day 20 expressed those receptor transcripts. Late embryonic neurons, astrocytes, and neonatal progenitors of oligodendrocytes synthesized both IGF‐I and insulin receptor mRNAs after a short time in culture. However, astrocytes expressed preferentially IGF‐I receptor transcripts, while young progenitors for oligodendrocytes expressed high levels of insulin receptor transcripts. As a whole, our data indicate that during rat CNS development expression of IGF‐I and insulin receptors appears to be stage‐and cell‐specific. The differences observed between the expression of both receptors might point to a specific, but coordinative role of IGF‐I and insulin and their receptors during that time.
T‐lymphocyte entry into the central nervous system
Authors: Hickey W. F. , Hsu B. L. , Kimura H.
Published: January 2005

The entry of T‐lymphocytes into the parenchyma of the central nervous system is a critical early feature in the pathogenesis of many experimental and spontaneously occurring immune‐mediated illnesses. The physiological mechanisms controlling this entry have not been elucidated. This study reports that T‐cell entry into the rat CNS appears to be primarily dependent upon the activation state of the lymphocytes; T‐lymphoblasts enter the CNS (and all other tissues examined) in an apparently random manner while T cells not in blast phase are excluded. Antigen specificity, MHC compatibility, T‐cell phenotypce and T‐cell receptor gene usage do not appear related to the ability of cells to enter. This study demonstrates that when T‐lymphoblasts are introduced into the circulation they rapidly appear in the CNS tissue. Their concentration in the CNS reaches a peak between 9 and 12 hr, and lymphocytes which have entered, exit within 1 to 2 days. Cells capable of reacting with a CNS antigen remain in the tissue or cyclically reenter to initiate inflammation if they are able to recognize their antigen in the correct MHC context. This observation also appears to pertain to the entry of activated T cells into many other tissues, although their concentrations in these non‐CNS sites was not quantitatied.