bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuron‐specific splicing of C‐SRC cDNA RNA in human brain
Authors: Pyper J. M. , Bolen J. B.
Published: January 2005

The C‐SRC, C‐YES, and FYN genes encode three closely related tyrosine protein kinases that are expressed in human neural tissues. A unique form of the C‐SRC gene has been demonstrated to be expressed in avian and murine brain tissues as the result of alternative splicing between the third and fourth exons. This neuronal‐specific splicing event adds to the C‐SRC mRNA an 18 base pair exon capable of encoding the same six amino acids in both avian and murine neural tissues. The C–YES and FYN genes share with C‐SRC similar exon‐intron boundaries and a high degree of amino acid sequence homology in the 3/4 exon coding region. However, potential alternative splicing of the C‐YES and FYN genes in this region has not been previously investigated. In this study we have compared the expression of C‐SRC, C‐YES, and FYN RNAs in human lung, liver, brain, and placenta tissues and prepared cDNA clones spanning exons 3 and 4 for each of these genes from the different tissues. Sequence analysis of these cDNA clones revealed that the splicing patterns for the FYN and C‐YES genes were the same among the various tissues, whereas C‐SRC cDNAs isolated from brain contained 18 additional bases with the capacity to code for the same six amino acids present in the neural‐specific forms of avian and murine pp60c‐src.
Organization of oligodendroglial membrane sheets. I: Association of myelin basic protein and 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase with cytoskeleton
Authors: Dyer C. A. , Benjamins J. A.
Published: January 2005

Membrane sheets elaborated by cultured murine oligodendroglia provide a unique system for examining associations between myelin proteins and cytoskeletal elements. Interactions can be observed and manipulated more readily than in the multilamellar myelin membrane in vivo. Immunocytochemical staining of 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase (CNPase) shows that it is distributed diffusely in some regions of membrane sheets, but colocalized with tubulin in lacy networks and major veins in other regions. Staining with phalloidin also reveals two distributions of F‐actin: (1) small aggregates within the diffuse CNPase regions and (2) filaments colocalized with tubulin and CNPase in the lacy networks and veins. Application of colchicine at 10 μg/ml for 4 hr disrupts microtubular structures in the lacy network, while those in major veins remain intact. This suggests that microtubules in the lacy network are treadmilling more rapidly than those in the major veins. The distribution of CNPase and F‐actin is not altered under these conditions. In contrast, cytochalasin B disrupts F‐actin, microtubules, and CNPase in the lacy networks, indicating that cross‐linking between these three proteins is disrupted. Both colchicine and cytochalasin B cause fusion of myelin basic protein (MBP) domains in membrane sheets. This appears to be a consequence of disruption of microtubules in the lacy networks, which normally outline the MBP domains. In summary, these results provide evidence for (1) direct association of CNPase with F‐actin and tubulin in cytoskeletal structures and (2) organization of MBP into domains via association with microtubules in the lacy networks.
Glycerophosphorylcholine phosphocholine phosphodiesterase activity of rat brain myelin
Authors: Kanfer J. N. , McCartney D. G.
Published: January 2005

Myelin isolated from rat brain possessed the ability to release phosphorylcholine from glycerophosphoryl‐choline, and this activity was enriched 3.2‐fold over that of the original homogenate. This glycerophosphorylcholine phosphocholine phosphodiesterase activity had a pH optimum at 9.5, had a Km of 0.2 mM, and a Vmax of 150 nmoles/mg protein/hr. The enzyme had a specific requirement for Zn2+ with an optimum concentration at 0.25 mM. Maximum enzyme activity was at 50°C and an Arrhenius plot showed a breakpoint at 40°. p‐Nitrophenylphosphorylcholine was also hydrolyzed by purified myelin and was a competitive inhibitor of glycerophosphorylcholine phosphocholine phosphodiesterase activity with a Ki of 0.075 mM. Glycerolphosphorylethanolamine was hydrolyzed only 5% compared with GPC, but it was not an inhibitor.
Resistance to extrapyramidal effects of opiates in rats chronically treated with SCH 23390
Authors: De Montis G. M. , Devoto P. , Meloni D. , Porcella A. , Saba P. , Tagliamonte A.
Published: January 2005

Rats made tolerant to morphine show neither a change in brain opiate receptor number nor altered sensitivity to the inhibitory effect of opiates on striatal adenylate cyclase (AC) activity. Interestingly, SCH 23390, a selective blocker of D1 dopamine (DA) receptors which, given chronically to rats, induces a 32% increase in D1 receptor number and increases the Vmax of D1‐stimulated striatal AC, resulted in marked resistance to acute morphine effects. In particular, rats chronically treated with SCH 23390 failed to show muscular rigidity and increased striatal dihydroxyphenylacetic acid (DOPAC) concentration after morphine. Moreover, basal striatal AC activity in these animals had a significantly reduced sensitivity to opiate inhibition.

On the other hand, decreased AC sensitivity to acetylcholine (ACh) inhibition observed in the striatum of rats chronically treated with DFP, an irreversible blocker of acetylcholinesterase, appeared to be secondary to the downregulation of muscarinic receptors and thus did not modify the opiate inhibitory capacity. It was concluded that although a potentiation of striatal AC impairs opiate action, such mechanism is not involved in morphine tolerance.
Cyclic adenosine monophosphate as a second messenger in horizontal cell uncoupling in the teleost retina
Authors: Laufer M. , Salas R. , Medina R. , Drujan B.
Published: January 2005

The reduction in the receptive field of horizontal cells of the teleost Eugerres plumieri observed upon dopamine (DA) superfusion is thought to be due to cell uncoupling. The possible mechanisms by which activation of DA receptors modify the electric coupling between horizontal cells were studied in the present work. It was found that the effect of DA in different preparations is mediated by a modification of intracellular concentration of cAMP and H+. The effects of intracellular injection of cAMP and H+ were studied in retinal horizontal cells of the teleost E. plumieri. A triple microelectrode was used to inject the ion iontophoretically, to pass current pulses, and to record voltages from the same cell, while a fourth microelectrode was used to record voltages from a neighboring cell in the same retinal layer. Responses evoked by light spots and annuli were evaluated simultaneously.

Coupling ratios between neighboring horizontal cells ranged from 0.22 to 0.45. The intercellular resistance (Rc), 0.5–3.5 × 106 ohms, and that of the remaining cell membrane resistance (Rm), 2.5–18 × 106 ohms, were calculated by means of a passive electrical model that has a hexagonal array.

The microinjection of H+ with injection current from +5 to +30 nA for 40 to 100 sec led to temporary and reversible light response reduction. The coupling ratio between two impaled cells was reduced by about 30%, and intercellular resistance (Rc) increment was 320% while cell membrane resistance (Rm) did not change consistently.

There was also a temporary and reversible Rm reduction (70–85%) and an Rc increment of 170–330% when cyclic adenosine monophosphate was iontophoretically injected with current from −30 to −40 nA for 50 to 170 sec. The coupling ratio between two impaled cells was reduced by about 40%, and light responses recorded from the injected cell showed a reduction in amplitude with the same time course as that of the resistive changes. The injection of Lucifer yellow into a horizontal cell under normal conditions always results in pronounced fluorescence for more distant cells; however, under constant injection of H+ or cAMP only the injected cell is fluorescent, which provides direct evidence of the reduction in the effectiveness of coupling between horizontal cells. The observed effects of intracellular H+ or cAMP injection correspond to the resistive changes in Rc and coupling ratio that occur in the horizontal cell network upon superfusion with a dopamine (DA) solution.
Survival of purified embryonic chick retinal ganglion cells in the presence of neurotrophic factors
Authors: Lehwalder D. , Jeffrey P. L. , Unsicker K.
Published: January 2005

In a search for neurotrophic factors (NTFs) regulating retinal ganglion cell (RGC) death in the chick embryo we have used purified and cultured RGCs. Purification of RGCs from embryonic day 10 was achieved by employing the “panning” method (Silverstein and Chun: Soc Neurosci Abstr 13:1054, 1987). The obtained neuron population consisted of 97% RGCs as demonstrated by retrograde labeling with a fluorescence dye. RGCs were cultured at low density in a chemically defined medium and shortterm survival (24 hr) was determined. In the absence of NTFs, less than 3% of the RGCs survived. In the presence of various crude or purified NTFs (eye, brain, and tectum extracts; glial‐conditioned medium; ciliary nerotrophic factor [CNTF]; nerve growth factor [NGF]) 31% to 52% of the RGCs were maintained. The effects of NGF and CNTF were not additive. Neither acidic nor basic fibroblast growth factor was able to maintain RGCs in culture. Our results, obtained with a culture system which allowed the analysis of direct trophic actions, suggest that NGF and CNTF may be NTFs for overlapping subpopulations of chick RGCs.
Aminoglycoside antibiotics impair calcium entry but not viability and motility in isolated cochlear outer hair cells
Authors: Dulon D. , Zajic G. , Aran J.‐M. , Schacht J.
Published: January 2005

Cochlear outer hair cells have been well established as primary targets of the ototoxic actions of aminoglycoside antibiotics. These cells, isolated from the guinea pig cochlea and maintained in short‐term culture, were used as a model for evaluating the acute effects of gentamicin on cell viability, depolarization‐induced transmembrane calcium flux, and depolarization‐induced motile responses.

On the basis of morphology and fluorochromasia, the presence of extracellular gentamicin as high as 5 mM did not affect the viability of the cells for up to 6 hr, the longest time tested. Viable cells showed binding of fluorescently tagged gentamicin to their base but excluded the drug from their cytoplasm. In response to [K+]‐depolarization, intracellular calcium levels (monitored with the fluorescent calcium‐sensitive dye fluo‐3) increased from a resting value of 218 ± 102 nM to 2,018 ± 1,077 nM concomitant with a cell shortening of 0.7% ± 1.3%. The depolarization‐induced calcium increase was apparently caused by calcium entry into the cell as it was inhibited by the calcium‐channel blocker methoxyverapamil and prevented in the absence of extracellular calcium. Both gentamicin and neomycin blocked the [K+]‐induced calcium increase at an IC50 of 50 μM. Despite the inhibition of calcium entry the ability of the outer hair cells to shorten under [K+]‐depolarization was not impaired; in fact, cell shortening was even more pronounced in the absence of calcium influx (2.6% ± 1.4%). This argues effectively against the existence of a calcium‐dependent actomyosin‐mediated component in [K+]‐induced shape changes.

The results suggest the existence of voltage‐gated calcium channels in outer hair cells and that calcium influx through these channels is impaired by the aminoglycoside antibiotics neomycin and gentamicin. This action may be part of the acute ototoxic mechanism of these molecules. Furthermore, the results not only confirm the calcium independence of the depolarization‐induced motility but also suggest that calcium influx into outer hair cells opposes cell shortening.
Expression of synaptophysin and neuron‐specific enolase during neuronal differentiation in vitro: Effects of dimethyl sulfoxide
Authors: Schilling K. , Barco E. Blanco , Rhinehart D. , Pilgrim Ch.
Published: January 2005

Neural development in dissociated cell cultures of fetal rat brain can be expected to depend on synaptic interactions between cultured neurons. Therefore, an attempt was made to obtain a quantitative measure of the time course of synaptogenesis in such a culture system by assessing the level of the secretory vesicleassociated protein synaptophysin (p38). The developmental schedule of p38 was compared to that of neuron‐specific enolase (NSE), an established marker of neuronal differentiation.

Cultures were raised from dissociated 14 day‐old fetal rat diencephalon. In cultures grown for 1–2 days in vitro (DIV), p38‐immunoreactivity was preferentially located in neuronal perikarya. After 10–16 DLV, neurons in culture had formed a dense neuritic network, and almost all of the p38‐immunoreactivity occurred in the form of fine punctate deposits associated with neuronal processes that often outlined neuronal cell bodies in a basket‐like fashion. Electron‐microscopic immunocytochemistry proved the punctate deposits to be presynaptic elements, mostly in the form of axonal varicosities. Quantitative immunoblotting showed that levels of p38 increased from the start of cultivation to DIV 4, stayed fairly constant from DIV 4 to DIV 8, and rose again steeply to peak at DIV 12. In contrast, levels of NSE rose continuously up to DIV 12. After DIV 12, levels of both p38 and NSE fell again.

Treatment of cultures with dimethyl sulfoxide (DMSO), an agent known to induce differentiation in various normal and malignant cell types, resulted in a significant increase of p38 levels and in a decrease of NSE levels. The amount of p38 continued to increase beyond DIV 12, whereas NSE diminished after having reached a maximum at DIV 12.

Assessment of p38 levels as a measure to quantify synaptogenesis in culture has been validated through combined morphological observations and quantitative immunoblotting. The results show that expression of NSE and p38 during neuronal development follows different time schedules and that the two neuron‐specific proteins can be manipulated independently from each other. DMSO treatment appears to selectively promote synaptogenesis and/or maturation of specific synaptic properties.
Denervation and hyperinnervation in the nervous system of diabetic animals. I. The autonomic neuronal dystrophy of the gut
Authors: Di Giulio A. M. , Tenconi B. , La Croix R. , Mantegazza P. , Cattabeni F. , Gorio A.
Published: January 2005

Peripheral neuropathy is a correlate of experimental diabetes induced in rats by means of a single injection of alloxan. The autonomic and enteric innervation of the gut are profoundly affected in the small intestine of such animals. A complex process of denervation and hyperinnervation of the gut wall of diabetic animals is observed. It was previously reported that the cholinergic parasympathetic innervation of the intestine is markedly reduced. We have found that noradrenergic sympathetic axons hyperinnervate the duodenum of diabetic rats, whereas noradrenaline levels are significantly reduced in the jejunum. The putative enteric neurotransmitter dopamine is also present in higher levels in the duodenum. The intrinsic peptidergic neurons of the gut are deeply affected as well in diabetic rats. Substance P and met‐enkephalin content are remarkably reduced throughout the small intestine, whereas vasoactive intestinal polypeptide levels (VIP) are significantly increased in the duodenum. Indeed, immunocytochemical staining of the ileum did reveal hypertrophy of VIP‐positive axons in diabetic rats. The intrinsic serotoninergic innervation of the gut is apparently unaffected. Our results indicate that the changes of gut innervation observed in experimental diabetes are consistent with increased content and also likely with hyperinnervation by the neuronal systems involved in smooth muscle relaxation and decreased content and with denervation by those systems with smooth muscle contraction properties. Such a perturbed gut innervation may be responsible of the gastrointestinal dysfunctions that are among the most common complications of diabetes.
Phosphatidate phosphohydrolase in purified rat brain myelin
Authors: Vaswani K. K. , Ledeen R. W.
Published: January 2005

Highly purified myelin from rat brain stem has been shown to contain phosphatidate phosphohydrolase, an enzyme which converts phosphatidate to diacylglycerol. The high levels relative to cytosol and microsomes (17% and 22%, respectively) tended to preclude contamination by these fractions as the source of activity. Additional evidence came from study of repeated purification, mixing experiments, and washing of the myelin with salt and detergent. We conclude that this enzyme, in addition to being widely distributed in other subcellular fractions, is intrinsic to the myelin membrane. Through its activity it generates a key substrate for the cytidine (Kennedy) pathway which was previously shown to occur in this membrane.
Glucocorticoids modulate rat hypothalamic corticotrophin‐releasing factor release induced by interleukin‐1
Authors: Cambronero J. C. , Borrell José , Guaza C.
Published: January 2005

Continuous in vitro perifusion of rat hypothalami with interleukin‐1 β (IL‐1) increased corticotropinreleasing factor (CRF) secretion in a dose‐dependent manner, in the range of 1 to 5 U/ml. The stimulatory action of IL‐1 was significantly attenuated by dexamethasone, both by addition to the perifusion medium and by in vivo dexamethasone pretreatment. The data suggest that an immunoneuroendocrine interaction circuit, subjected to glucocorticoid negative feedback, has an essential role in maintaining organism homeostasis.
Glial cells of the oligodendrocyte lineage express proton‐activated Na+ channels
Authors: Sontheimer H. , Perouansky M. , Hoppe D. , Lux H. D. , Grantyn R. , Kettenmann H.
Published: January 2005

Neurons and oligodendrocytes, but not type I astrocytes and Schwann cells, generate large Na+ currents in response to a step increase of [H+]. Proton‐activated Na+ channels are the first cationic channels expressed in neuronal precursor cells from the mammalian brain. Glial precursor cells cultured from mouse brain are also capable of generating Na+ currents in response to step acidification (INa(H)). With further development along the oligodendrocyte lineage, this property is retained, whereas voltage‐activated Na+ and K+ currents disappear. Comparing INa(H) of oligodendrocytes with INa(H) of their precursor cells did not reveal a difference in current amplitude, suggesting a higher density of INa(H) channels on the (smaller) precursor cells. The properties of INa(H) in glial precursor cells and oligodendrocytes are similar to those of neurons, with respect to activation conditions, time course, and the effect of extracellular Ca2+ concentrations. The results are consistent with previous observations which showed that oligodendrocytes partially preserve their chemically activated, but completely lose their voltage‐activated, ion channels.
Binding properties of liposomes containing the myelin‐associated glycoprotein MAG to neural cell cultures
Authors: Sadoul R. , Fahrig T. , Bartsch U. , Schachner M.
Published: January 2005

The myelin‐associated glycoprotein MAG is a neural cell adhesion molecule which belongs to the immunoglobulin superfamily and the carbohydrate based L2/HNK‐1 family of adhesion molecules. In this study we further characterize the adhesive properties of MAG. MAG incorporated into liposomes bound to cultured peripheral and central nervous system neurons known to be myelinated in vivo. Expression of the neuronal MAG receptor(s) on spinal cord neurons increased with time in culture and correlated with the time of active myelination of these neurons in vivo. MAG bound only poorly if at all to cerebellar neurons which are not myelinated in vivo and not to cultured oligodendrocytes or Schwann cells. A low level of MAG binding to astrocytes or fibroblast‐like cells that was MAG antibody molecules L1 and N‐CAM, two other members of the immunoglobulin superfamily, were not found to be the neuronal receptors for MAG. RGD containing peptides did not inhibit binding of MAG‐liposomes to neurons. The soluble form of MAG which contains most, if not all, of the extracellular domain of the molecule and binds to collagen, did not interfere with the binding of MAG‐liposomes to neurons. Conversely, MAG‐liposomes did not bind to collagen, suggesting the extracellular domain of the molecule.
Dual‐label retrograde transport: CNS innervation of the mouse thymus distinct from other mediastinum viscera
Authors: Tollefson L. , Bulloch K.
Published: January 2005

Two different retrogradely transported tracers were identified and used to distinguish between the innervation of the thymus gland and the innervation of adjacent tissues residing within the mouse mediastinum. To determine the best procedure for peripheral dual‐labeled retrograde transport, several different tracers, vehicles, and injection protocols were developed and/or tested. In the periphery, it proved ineffective to use Rhodamine latex microspheres (Luma‐Fluor) as a retrograde transport agent. Horseradish peroxidase (HRP) suspended in the water‐soluble carrier KY Jelly and Fluro‐gold (FG) in an aqueous solution was the most effective combination of retrograde transport tracers. Equivalent doses of each of these tracers were determined, and injections of HRP or FG into the thymus and the esophagus produced distinct and separate patterns of labeled neurons in the spinal cord or brain stem. Throughout these CNS areas, some neurons contained both tracers suggesting that (1) these areas are innervated by neurons that give rise to collaterals to these tissues, (2) that the retrograde tracers are transported by nerves that innervate the lymphatics of the mediastinum, and/or (3) that some diffusion of the tracers occurs from the test organs and tissues to other organs and tissues of the mediastinum.
Expression of transfected genes by differentiated, postmitotic neurons and photoreceptors in primary cell cultures
Authors: Werner M. , Madreperla S. , Lieberman P. , Adler R.
Published: January 2005

We report here that differentiated, primary, postmitotic neurons and photoreceptors in cultures obtained from embryonic chicks can express foreign genes after transfection by the calcium phosphate method. A variety of viral promoters were tested by using either β‐galactosidase or chloramphenicol acetyltransferase as reporter genes. Histochemical and immunocytochemical analysis showed β‐galactosidase expression by both neurons and photoreceptors. As commonly observed with dividing cells, transfection efficiencies showed inter‐experimental variability, with efficiencies ranging from 2% to 20% using the same plasmid. On the other hand, intra‐experimental variability between replicate dishes was much smaller. Analysis using the highly sensitive enzymatic assay for chloramphenicol acetyl transferase (CAT) showed that all of the promoter/enhancers‐CAT constructs tested, with the exception of a construct containing the Maloney sarcoma virus promoter, led to the expression of detectable activity when transfected into cultured retinal cells. The calcium phosphate treatment used for cell transactions did not show detectable effects on overall cell survival, although it caused selective decreases in some metabolic activities of the cells. The studies demonstrate that it is possible to obtain expression of genes transfected into primary, postmitotic neuronal cells.
Purification of an endogenous 68 kD inhibitor of calcium‐activated neutral proteinase (CANP) from bovine brain: Immunoblot identification and characterization
Authors: Banik N. L. , Chakrabarti A. K. , Hogan E. L.
Published: January 2005

A calcium‐activated neutral proteinase (CANP)‐specific endogenous inhibitor (calpastatin) was purified from bovine brain by successive column chromatography. The purified inhibitor exhibited a major band on sodium dodecylsulfate polyacrylamide gel electrophoresis with an approximate molecular weight of 68 kD. The polyclonal antisera raised to the inhibitor strongly reacted with the 68 kD protein band. Two lightly stained bands approximately 55–68 kD and 120–130 kD were also recognized by the inhibitor antiserum. The inhibitor specifically inhibited CANP activity and the half‐maximal inhibition was found with 75 ng of calpastatin per 1 μg of CANP in a final volume of 125 μl. Cathepsin B and papain were not inhibited by the inhibitor, while trypsin and chymotrypsin were inhibited to some extent. The inhibitor formed a complex with CANP and the inactive complex was dissociated into active fractions of enzyme and calpastatin in the presence of EGTA.
Effects of atrial and brain natriuretic peptides upon cyclic GMP levels, potassium transport, and receptor binding in rat astrocytes
Authors: Beaumont K. , Tan P. K.
Published: January 2005

The ability of atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) to alter cyclic GMP levels and NaKCl cotransport in rat neocortical astrocytes was determined. At concentrations of 10−9–10−M, rat ANP99–126(rANF), rat ANP102–126 (auriculin B), and rat ANP103–126 (atriopeptin III) stimulated 6‐ to 100‐fold increases in cyclic GMP levels. Porcine BNP (pBNP) and rat BNP (rBNP) were 20%–90% as effective as rANF over most of this concentration range, although 10−6M pBNP produced a greater effect than rANF. NaKCl cotransport as measured by bumetanide‐sensitive 86Rb + influx was not altered by exposure of astrocytes to 10−6M rANF, pBNP, or rBNP. Both pBNP and rBNP, as well as rat ANP103–123 (atriopeptin I) and des[gln18, ser19, gly20, leu21, gly22] ANF4−‐NH2 (C‐ANF4–23) strongly competed for specific 125I‐rANF binding sites in astrocyte membranes with affinities ranging from 0.03 to 0.4 nM, suggesting that virtually all binding sites measured at subnanomolar concentrations of 125I‐rANF were of the ANP‐C (ANF‐R2) receptor subtype. These receptors are thought to serve a clearance function (Maack et al.: Science 238:675–678, 1987) and may be linked to a guanylate cyclase activity that is chemically and pharmacologically distinct from that coupled to ANP‐A (ANF‐R1) receptors (Féthiere et al.:Mol Pharmacol35:584–592, 1989). ANP receptors on astrocytes may function in limiting the access of ANP and BNP to neurons involved in body fluid and cardiovascular regulation.
Characterization of HNK‐1 bearing glycoproteins in human peripheral nerve myelin
Authors: Van Den Berg L. H. , Sadiq S. A. , Thomas F. P. , Latov Norman
Published: January 2005

The HNK‐1 carbohydrate epitope, which is shared by several members of the immunoglobulin gene superfamily, is also the target epitope for IgM anti‐MAG autoantibodies in patients with demyelinating neuropathy. By Western blot analysis, there are 7 HNK‐1 immunoreactive glycoproteins in human peripheral nerve myelin, two of which have previously been identified as the myelin associated glycoprotein (MAG) and the Po glycoprotein. In this study, the remaining HNK‐1 bearing glycoprotein bands were characterized by immunoblot and NH2‐terminal sequence analysis, and were all identified as degradation products or aggregates of the Po glycoprotein. MAG and Po are therefore the only HNK‐1 bearing glycoproteins in human peripheral nerve myelin.
125I‐β‐nerve growth factor binding is reduced in rat brain after stress exposure
Authors: Taglialatela G. , Angelucci L. , Ramacci M. T. , Foreman P. J. , Perez‐Polo J. R.
Published: January 2005

In the central nervous system (CNS), the presence of nerve growth factor (NGF) and its receptor, NGFR, in cholinergic neurons has been demonstrated. In this study we report that, after exposure to stress, there was a reduction in total binding of NGF in the hippocampus and basal forebrain of 3.5‐month‐old rats without significant changes in the frontal cortex or cerebellum. Chronic treatment with acetyl‐l‐carnitine (ALCAR), that prevents some age‐related impairments of CNS, for 1.5 months, decreased NGF binding in hippocampus and basal forebrain but abolished the stress‐related reduction of NGF binding observed in the hippocampus of untreated rats.
Role of nerve growth factor in oxidant‐antioxidant balance and neuronal injury. II. A conditioning lesion paradigm
Authors: Jackson G. R. , Werrbach‐Perez K. , Perez‐Polo J. R.
Published: January 2005

The PC12 rat pheochromocytoma cell line is a nerve growth factor (NGF) responsive line that is protected by NGF from the toxic effects of hydrogen peroxidation. In part, NGF protection of PC12 cells acts through a shift in oxidant‐antioxidant metabolism by the enhancement of catalase activity. When PC12 cells are used in a conditioning lesion paradigm to study the effects of an initial sublethal peroxidative insult on subsequent responses to injury, a low dose conditioning lesion protects even in the absence of NGF. The magnitude of the protective effect exerted by the conditioning lesion, however, is augmented in the presence of NGF, since a significant cytoprotective effect is observed over a wider range of H2O2 concentrations. Neuronal injury due to treatment with a high dose of H2O2 (5 mM) has a cytotoxic effect that cannot be prevented by NGF treatment and is, in itself, not conditioning in nature. This in vitro model system lends itself to the development of explanations regarding the salutory effects of conditioning lesions at the molecular level.
A small subpopulation of progesterone receptor‐containing neurons in the guinea pig arcuate nucleus projects to the median eminence
Authors: Poulain P. , Warembourg M. , Jolivet A.
Published: January 2005

In female guinea pigs, a combination of retrograde tracing and immunofluorescence for progesterone receptors (PR) was applied to determine if PR‐immunoreactive (PR‐IR) neurons in the arcuate nucleus (AR) send their axons directly to the median eminence (ME). Axonal projections to the ME were studied by different techniques using fluorescent dyes. From 31 adult animals, ovariectomized and primed by estradiol, small deposits of Lucifer Yellow (LY) were made on the cut surface of the ME, either by direct application of LY crystals or by iontophoresis. These techniques were carried out on excised mediobasal hypothalamus maintained in vitro and allowed visualization of AR perikarya projecting to the ME after dye diffusion in the severed axons. In another group of ten immature animals primed by estradiol, Granular Blue (GB) was injected in the jugular vein. Blood‐borne GB was taken up in the ME by intact nerve endings and retrogradely transported to the perikarya of origin. PR‐IR neurons and perikarya filled with LY or retrogradely labeled by GB were intermingled with each other throughout the restrocaudal extent of the AR. Double‐labeled cells, displaying PR immunoreactivity and dye labeling, were observed consistently, but their number was small. This result demonstrates that some AR neurons sending axonal projections to the ME are target cells for progesterone. As the majority of PR‐IR neurons in the AR do not project to ME, it is suggested that most PR‐IR neurons present in this nucleus form local circuit projections or project to distant areas of the central nervous system.
Interleukin‐2 enhances chick and rat sympathetic, but not sensory, neurite outgrowth
Authors: Haugen P. Keely , Letourneau P. C.
Published: January 2005

Interactions between the immune and nervous systems could be important for normal development and function of both. To determine if a lymphokine, interleukin‐2 (IL‐2), represents a link between these two systems, sympathetic and sensory neurons from embryonic chick and neonatal rat were cultured in media containing human recombinant IL‐2. In chick sympathetic chain and rat superior cervical ganglia cultures, IL‐2 enhanced the number of neurons with neurites and the lenght of those neurites significantly over control cultures. Sensory neurons from chick and rat dorsal root ganglia were not affected by culture in IL‐2. Sympathetic neuron response to IL‐2 was concentration‐dependent, with an optimum around 2‐0.2 U/ml (100‐10 pM). Immunofluorescence with an anti‐IL‐2 receptor antibody demonstrated specific staining of sympathetic neurons, but not sensory neurons, implying that sympathetic neurons may have a receptor for IL‐2 on their surface.
Amino acid mdification of proteins in regenerating sciatic nerves of rats
Authors: Chakraborty G. , Yu M. , Luo D. , Sturman J. A. , Ingoglia N. A.
Published: January 2005

Recent experiments have shown that Arg, Lys, and Leu can be incorporated posttranslationally into proteins of regenerating sciatic nerves of rats. The presentexperiments investigate a mixture of 15 radioactie amino acids to determine if additonalo amino acids can be cojugated posttranslationally to protein of regenerating nerves. Proteins of regenerating sciatic nerves or rats were able to incorporate Arg, Lys, pRO, Val, Ala, Phe, and Ser in relatively large amounts and Asp, Glu, Thr, Gly Ile, His, and Tur in relatively low or undetectable amounts, in the most advanced potion of the regenerating nerves. Two‐Dimensional SDS PAGE showed incorporation of the amono acid mixture into distinct radioactive peaks with moleculer weights in the 80–90 kD, 22–46 kD, and 17 kD ranges with isoelectric point between 5.0 and 7.9. Most of the amono acids were incorporated into proteins in all of the molecular weight ranges. But Ser was incorporated in highest amounts in the 17 kD range. In some cases results indicated that single proteins were modified by several amino acids.

While we do not yet know which amino acids modify specidic nerve proteins or the function of the modifications in nerve regenration, these studies demonstrate the participation of some but not all amono acids in posttranslational modification reactions and the selective modification of specific groups of nerve preoteins by these amono acids.
Identification of GTP‐ binding proteins in myelin and oligodendrocyte membranes
Authors: Braun P. E. , Horvath E. , Yong V. W. , Bernier L.
Published: January 2005

Myelin membranes purified from mouse and rat brain are associated with alpha subunits of four signal transducing guanosine triphosphate (GTP)‐ binding proteins: Go, Gi, Gs, and ras. Four low‐molecular‐weight (Mr) GTP‐binding proteins are also present, as demonstrated by the binding of GTP to proteins immobilized in nitrocellulose. This latter group is more prominent at early stages of myelination and remains associated with isolated myelin membranes despite repetitive cycles of purification. At least one nonmyelin subcellular membrane fraction possesses the same proteins. The total membrane fraction of cultured oligodendrocytes is associated with both groups of GTP‐binding proteins. None of the well‐known myelin proteins bound GTP by the procedure described.
Opioids influence neurotransmitter phenotypic expression in chick embryonic neuronal cultures
Authors: Vernadakis A. , Kentroti S.
Published: January 2005

There is considerable interest in the role of endogenous opioid peptides in neural growth and differentiation. In this study we used neuron‐enriched cultures derived from 3‐day‐old chick embryos to test the effects of endogeneous enkephalins on neurotransmitter phenotypic expression. Cultures were grown in serum‐free chemically defined medium and were treated with either Met‐enkephalin antiserum (anti‐Met) to immunoneutralize enkephalins, or with naloxone, a universal opioid receptor antagonist, to block receptor‐mediated actions of released endogenous opioids. The enzyme activities of choline acetyl‐transferase (ChAT) and tyrosine hydroxylase (TH) were used as markers for cholinergic and catecholaminergic phenotypic expression, respectively. We found that cultures treated with anti‐Met or naloxone exhibited strikingly different neuronal growth patterns as compared to controls. In addition, ChAT activity was enhanced by anti‐Met, and TH activity by both anti‐Met and naloxone. These findings lend support to the possibility that neuropeptides may be co‐localized with neurotransmitters and that peptides released into the microenvironment affect neuronal phenotypic expression by differential receptor subtypes.
Reversibility of ganglioside effects on astrocyte morphology
Authors: Dekker A. , Manthorpe M. , Varon S.
Published: January 2005

The B‐subunit of cholera toxin (BCT) induces a morphological change in cultured rat cerebral astrocytes from flat (epithelioid) to stellate (process‐bearing). This stellation is reversed by the gangliosides GM1 and GD1a at concentrations of 10 μM or higher. Upon changing to a ganglioside‐free medium, the flat astrocyted reacquire the stellate morphology within 3 hr, indicating that the antistellation effect of gangliosides is reversible. The possibility that this reversibility was due to a loss of exogenously acquired gangliosides from the cell membrane can be ruled out since pretreatment with GM1, but not GD1a, which does not bind BCT, results in an increased responsiveness to BCT, which was identical whether measured immediately after withdrawal of the ganglioside or 3 hr later. Asialo‐GM1, which neither binds BCT nor reverses BCT‐induced stellation by itself, prevents the return to stellation after withdrawal of the gangliosides. These data suggest that while gangliosides remain associated with the cell, their effect on astrocytes can change from opposing to permitting the stellate morphology.
Toxicity of 6‐hydroxydopamine and dopamine for dopaminergic neurons in culture
Authors: Michel P. P. , Hefti F.
Published: January 2005

Toxicity of 6‐hydroxydopamine (6‐OHDA) and dopamine were studied in cultures of dissociated fetal rat mesencephalic cells. To assess survival and function of dopaminergic cells we quantified the number of tyrosine hydroxylase‐positive cells and measured dopamine uptake. Non‐dopaminergic cells were monitored by counting the number of cells visible with phase‐contrast microscopy and measuring GABA uptake. 6‐OHDA, in contrast to MPP +, which selectively destroyed dopaminergic neurons, was found to be a non‐selective neurotoxin in this culture system. Between 10 and 100 μM, dopaminergic and non‐dopaminergic cells were destroyed. At concentrations frequently used to lesion catecholaminergic neurons in vivo, 6‐OHDA resulted in structural fixation and loss of viability of dopaminergic and non‐dopaminergic cells. Dopamine produced the same actions at slightly higher concentrations. One hundred to 300 μM was toxic for all cell types, and concentrations above 300 μM resulted in fixation. The findings suggest that 6‐OHDA cannot be considered a selective toxin for catecholaminergic neurons in vitro. The demonstrated toxicity of dopamine tends to support speculations that processes related to dopamine metabolism may play a role in the pathogenesis of Parkinson's disease.
Expression of glial fibrillary acidic protein and vimentin in mouse lens epithelial cells during development in vivo and during proliferation and differentiation in vitro: Comparison with the developmental appearance of GFAP in the mouse central nervous system
Authors: Boyer S. , Maunoury R. , Gomès D. , de Néchaud B. , Hill A. M. , Dupouey P.
Published: January 2005

Analysis of glial fibrillary acidic protein (GFAP) and vimentin in mouse lens epithelial cells (MLEC) during ontogenesis revealed a two‐step developmental expression similar to that observed in astrocytcs. Vimentin was first immunostained at E11 corresponding with the closure of the lens vesicle, whereas GFAP was detected only after a further 7 days (E18); this protein appeared simultaneously in the mouse lens and CNS. In the latter case, it was present in the hypothalamic tanycytes and spinal cord. This similarity in the timing of appearance of GFAP in the non‐neural MLEC and in fetal astrocytes suggests a common mechanism for its expression in tissues of different embryological origin. However, it has previously been observed that, in contrast to the situation in astrocytes, GFAP disappears from differentiating MLEC in vivo. We have shown that in vitro this protein also disappears rapidly from MLEC in the presence of fetal calf serum (FCS). However, the use of mouse serum instead of FCS inhibited the migration of MLEC out of the explant, and in these cells GFAP persisted.
Immunohistochemical localisation of nerve growth factor in a subpopulation of chick spinal ganglion neurons
Authors: Keller A. , Williams R. , Vahaviolos J. , Auffray C. , Rush R. A.
Published: January 2005

We have previously isolated and sequenced the chicken nerve growth factor (NGF) gene and now, from the deduced amino acid sequence, selected and produced peptides suitable for use as antigens. Antisera raised against these peptides inhibit the biological activity of a partially purified preparation of native chicken NGF and, when used in immunocytochemical studies, allow the visualisation of sensory neurons accumulating endogenous NGF. Immunoreactive cells form a distinct population of small neurons which may correspond to the well‐described neurons generated in the dorsomedial area of spinal ganglia. We conclude that two subpopulations of neurons exist within dorsal root ganglia, whose requirements for, and use of, NGF may be quite distinct.
Patterns of cerebral cortex mRNA expression
Authors: Bernal J. , Godbout M. , Hasel K. W. , Travis G. H. , Sutcliffe J. G.
Published: January 2005

A pool of 163 clones, isolated by screening 60,000 members of a Macaca fascicularis cerebral cortex cDNA library with a cortex‐minus‐cerebellum subtracted probe prepared by the phenol enhancement method, was analyzed by Northern blot hybridization studies. One hundred fifty‐three of these clones corresponded to 22 RNAs whose abundance was at least 2‐fold higher in cerebral cortex poly (A)+ RNA samples than in samples of cerebellar poly(A)+ Seven of these RNAs, represented by 131 clones, were undetectable in cerebellum. Only 10 of the 163 clones proved to be false positives. The abundance of several of these cortex‐enriched RNAs was altered in Alzheimer's disease brains. Several RNAs that were present in cerebral cortex but undetectable in cerebellum were generally enriched in telencephalon, although none was restricted to the cortex. One of the cortex enriched RNAs, whose nucleotide sequence is presented, encoded monkey preprocholecystokinin. Overall, this study provides insights into the powers and limitations of subtractive hybridization and into the patterns of gene expression in the central nervous system.
Isolation, characterization, and substrate properties of the external limiting membrane from the avian embryonic optic tectum
Authors: Kröger S. , Niehörster L.
Published: January 2005

The external limiting membrane of the avian embryonic optic tectum is isolated by mechanically separating the neuronal mesencephalon from the overlying mesenchymal tissue. The preparation consists of a basal lamina which is covered on its neural side by endfeet of neuroepithelial cells and has attached to it on its meningeal side a collageneous stroma, containing blood vessels. The external limiting membrane can be flat‐mounted on a piece of nitrocellulose filter as mechanical support. It covers an area between 0.3 and 1 the cm2, depending on the age of me donor embryo. The endfeet can be removed together with all cellular components of the meninges by treatment with 2% Triton‐X‐100 or with distilled water. The basal lamina itself is approximately 80 nm thick and consists of two laminae rarae and a central lamina densa. Immunohistochemical staining reveals that the basal lamina in the embryo, after isolation and after detergent extraction of the isolated preparation, contains type IV collagen, nidogen, laminin, and low density heparan sulfate proteoglycan as do other basement membranes. Antibodies against the neural cell adhesion molecule (N‐CAM), chondroitin sulfate proteoglycan, and fibronectin fail to stain the external limiting membrane, but these proteins were clearly identified in the blood vessel‐containing meninges or in the optic tectum.

The flat‐mounted external limiting membrane preparation was used as substrate to culture several different neural tissues of central and peripheral origin. Explants of neural crest cells, dorsal root ganglia, and sympathetic ganglia can be cultured on the external limiting membrane. All explants grow well on the basal lamina preparations whether the endfeet are attached or detergent‐extracted prior to explantation; however, neurite outgrowth from sympathetic ganglia is reduced in the presence of the endfeet. Although the endfoot‐lined external limiting membrane represents at least part of the immediate environment encountered by retinal axons as they invade the optic tectum and despite its excellent properties as a substrate for retinal axons in vitro, cues guiding the orientation of axons were not detected in the flat‐mounted preparation.
Prolonged ongoing discharges of sensory nerves as recorded in isolated nerves in the rat
Authors: Baik‐Han E. J. , Kim K. J. , Chung Jin Mo
Published: January 2005

Whether or not injury to a mammalian sensory nerve produces prolonged discharges is a controversial issue. Because of this controversy and its potential ramifications in both experimental and clinical conditions, we examined discharges in sectioned sensory nerves of the rat. In anesthetized rats, either a dorsal root or a saphenous nerve was isolated by sectioning both proximally and distally. Multi‐unit recordings from these isolated nerves showed low levels of prolonged ongoing discharges often lasting for at least 2 hr. Furthermore, results from short‐term (1–2 days) survival surgeries indicated that prolonged ongoing discharges could last for days. Sectioning the ventral root produced discharges for only a short period. Various pieces of evidence suggested that the sources of impulse generation are multiple, occurring in the middle of an uninjured axon at a site away from the injury, as well as at the injured site. There is circumstantial evidence which suggests that these prolonged discharges are produced in physiological conditions or at least under normal experimental conditions.
Novel NGF‐induced proteins in PC12 cells: Immunological evidence for their presence in brain nerve endings using a single monoclonal antibody
Authors: Kohtz D. Stave , Puszkin S.
Published: January 2005

A monoclonal antibody, S‐11D9, detected a group of novel polypeptides whose expression was induced 6 hr after incubation of PC12 cells with nerve growth factor. The antigens also were visualized by immune precipitation and Western blotting in synaptic vesicles, clathrin‐coated vesicles, and synaptic plasma membrane prepared from bovine brain. A large polypeptide was detected on the synaptic plasma membrane; an intermediate size protein was visualized on the plasma membrane and on both synaptic and clathrin‐coated vesicles, while a smaller molecule was found only on brain Golgi‐enriched membrane preparations. Immunofluorescence labeling with S‐11D9 on nerve growth factor‐stimulated PC12 cells showed the antigens distributed in the cytoplasm and concentrated in discrete areas on the tip of most neurites. The particular distribution of these proteins in vesicles and synaptic plasma membrane and the finding that the different antigens share a common epitope recognized by a single monoclonal antibody open the possibility that these molecules are related markers for organelle transport pathways in nerve cells.