bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of huperzine A, a new cholinesterase inhibitor, on the central cholinergic system of the rat
Authors: Tang X.‐C. , De Sarno P. , Sugaya K. , Giacobini E.
Published: January 2005

The present study represents the first comprehensive investigation of the effect of huperzine A (HUP‐A), a new cholinesterase inhibitor (ChEI) isolated from a Lycopodium species, upon acetylcholinesterase (AChE) activity, acetylcholine (ACh) levels and release, and cholinergic receptors in rat brain following acute i.m. or i.p. administration. The study shows that HUP‐A can produce a long‐term inhibition of AChE activity in brain (up to 360 min) and an increase in the ACh levels up to 40% at 60 min. There is considerable regional variation in the degree of ACh elevation after HUP‐A with maximal values seen in frontal (125%) and parietal (105%) cortex and smaller increases (22–65%) in other brain regions. HUP‐A at concentrations of 10−6 to 10−4 M does not significantly alter the electrically evoked release of 3H‐ACh from cortical slices. With the exception of the highest concentrations (6 × 10−4 M) the displacement effect of HUP‐A for cholinergic ligands is stronger for 3H‐(−)nicotine than for 3H‐QNB. A parallel autoradiographic study in the mouse shows that 60 min after i.v. injection (183 μg/kg) the drug is present in all brain regions, but it is particularly concentrated in certain areas such as frontoparietal cortex, nucleus accumbens, hippocampal, and striatal cortex. Radioactivity is practically absent in the whole body at 12 hr. Our study suggests that this new ChEI has interesting cholinomimetic properties, and its effects satisfy more closely established criteria for an ideal ChEI for therapeutic use than previously tested compounds.
Cholinergic agonists selectively induce proliferative responses in the mature subpopulation of murine thymocytes
Authors: Rossi A. , Tria M. A. , Baschieri S. , Doria G. , Frasca D.
Published: January 2005

The incubation of murine thymocytes for 24 hr in the presence of cholinergic agonists induces selective proliferation of PNA− cells as measured by thymidine incorporation.

The response of PNA− cells is mediated by the activation of nicotinic receptors. This conclusion is suggested by the inability of the muscarinic agonist, oxotremorine, to induce cell proliferation and by the inhibition exerted by the nicotinic antagonist, d‐tubocurarine.

The presence of cholinergic axons in the murine thymus adds biological interest to the selective expression of functional cholinergic receptors on mature thymocytes.
Intracerebral transplantation of dissociated central nervous system tissue suspensions: Use of phaseolus vulgaris leucoagglutinin as a cell marker
Authors: Shigematsu K. , Kamo H. , Akiguchi I. , Kimura H.
Published: January 2005

Successfully transplanted neurons and their sprouting processes were demonstrated by Phaseolus vulgaris leucoagglutinin (PHA) marking. PHA is transported anterogradely and readily reveals the post‐transplantation growth of the neuronal processes. Suspensions of fetal central nervous tissue, prepared by dissociation of embryonic rat brain, were marked with PHA and then transplanted into the striatum of nonimmunosuppressed young adult rats. At various intervals thereafter (1 day, 2 days, 1 week, 2 weeks, 1 month, and 2 months), the animals were sacrificed for histological examination with PHA and tyrosine hydroxylase (TH) immunohistochemistry. Up to 2 weeks after transplantation, PHA immunohistochemistry was capable of demonstrating grafted neurons and their presumably regenerated neuronal processes. However, at periods 1 month or longer after transplantation, PHA immunohistochemistry was unreliable. Thus, the PHA marking method has limitations in terms of its retention period, when applied to the intracerebral transplantation of dissociated cell suspensions. Nevertheless, the method presented here can be utilized to study neuronal regeneration as well as the relationship between transplanted neurons and the host tissue.
Developmental profile of glutamine synthetase in lines of mice bred for ethanol sensitivity
Authors: Sakellaridis N. , Mangoura D. , Masserrano J. M. , Detsis V. , Leoni C. J. , Deitrich R. , Vernadakis A.
Published: January 2005

Glutamine synthetase (GS) activity was used as a marker to examine differences in astrocyte development in mice selectively bred for ethanol sensitivity: long sleep (LS), short sleep (SS), mild ethanol withdrawal (MEW), severe ethanol withdrawal (SEW) and control ethanol withdrawal (CEW). We found that (1) GS activity in MEW and SEW was higher than in LS and SS during the first 2 weeks of postnatal development, in the forebrain but not in the cerebellum; (2) lower GS activity was observed consistently in all areas examined with the SS mice as compared to the LS; (3) glutamine synthetase activity in MEW and SEW differed significantly from their controls (CEW) during the early developmental period regardless of the brain region examined; however, after 30 days of maturation, GS activity in SEW was higher than that in MEW and CEW in the forebrain. Astrocytes are known to contribute in the regulation of the neuronal microenvironment. Therefore, we interpret the differences we found in astrocytic function during early brain development among these lines of mice to account in part for the neuronal predisposition to ethanol sensitivity.
Increased density of perivascular adrenergic innervation in tibial and vagus nerves of spontaneously hypertensive rats
Authors: Koistinaho J. , Wadhwani K. C. , Rapoport S. I.
Published: January 2005

Adrenergic innervation of epiperineurial arterioles and of the endoneurium of the tibial and vagus nerves of spontaneously hypertensive rats (SHR) and normotensive controls. (WKY) was studied by glyoxylic acid‐induced fluorescence and paraformaldehyde‐induced fluorescence methods. Adrenergic perivascular innervation of epiperineurial arterioles in both nerves was denser in SHR than in controls. Mean density in the endoneurium also was higher in SHR in both nerve types. These results suggest that adrenergic perivascular innervation, which may influence nerve blood flow, becomes greater in density in peripheral nerves during chronic hypertension.
Progressive restriction in the distribution of the Hox‐1.3 homeodomain protein during embryogenesis
Authors: Tani M. , Odenwald W. F. , Lazzarini R. A. , Friedrich V. L.
Published: January 2005

Expression of the murine homeobox containing gene Hox‐1.3 was analyzed in mouse embryos using polyclonal antisera to peptides predicted from cDNA and genomic sequences. At the earliest stage examined, 7.5 days gestation, cell nuclei throughout the three embryonic germ layers and in extraembryonic structures derived from the fertilized ovum were strongly immunoreactive. Rostro‐caudal gradients or other patterns of regional differentiation in levels of expression could not be seen. Surrounding maternal tissue showed only weak immunoreactivity. At 8.5 days gestation, immunoreactivity was present in all embryonic structures including neural tube, somites and lateral plate mesoderm, ectoderm and endoderm. Immunoreactivity was progressively restricted thereafter. At 17 days gestation, strong immunoreactivity was largely restricted to the nervous system, both central and peripheral. Spinal cord was well stained, with a dramatic reduction in intensity near the junction of spinal cord and brain. In addition to this overall pattern, enhanced immunoreactivity appeared in limited populations of newly‐formed neuroblasts of spinal cord and brain, suggesting that Hox‐1.3 might serve to regulate the development of specific types of neurons following cessation of precursor cell mitosis.
Heat stress increases delivery of a unique sub‐population of proteins conveyed by fast axonal transport
Authors: Stone G. C. , Dougher M. M.
Published: January 2005

The effect of heat stress on protein synthesis and fast axonal transport was examined in vitro in bullfrog dorsal root ganglion (DRG) and associated spinal/sciatic nerve. Qualitative and quantitative changes of individual 35S‐methionine‐labelled proteins were determined following DRG labelling and fast transport in respective nerves via two‐dimensional gel electrophoresis/autoradiography. Elevation of temperature from 18°C to 33°C for up to 6 hr resulted in a marked increase in synthesis of five individual DRG species of approximately 74,000 daltons that comigrate with heat shock proteins (HSPs). A quantitative comparison of species within this subset revealed two subgroups differentially affected by stress. The three most basic proteins were induced to approximately 1300% of unstressed controls after 6 hr of stress, while the two most acidic species demonstrated an increase to only 300% of controls over the same period. The relative abundance of 25 additional DRG proteins were uneffected by heat stress. Of 70 35S‐labelled fast‐transported proteins similarly analyzed, 15, comprising 5 families, were consistently transported at >150% of controls following up to 6 hr of heat stress. Over this period all 15 proteins shared a similar profile of abundance relative to non‐induced proteins. Transport was elevated to the greatest extent after 2 hr of stress, declined after 3 hr, and tended to rebound at later times. The remaining 55 fast‐transported protein spots analyzed were unaffected. An increased delivery of this unique sub‐population of 15 fast‐transported proteins suggests a possible involvement in early cellular events that mediate heat stress in the nervous system.
Reduction of glutamine synthetase specific activity in cultured astroglia by ferrous chloride
Authors: Tiffany‐Castiglioni E. , Roberts J. A. , Sheeler‐Gough L. V.
Published: January 2005

Immature and mature rat astroglia in culture were assayed for glutamine synthetase (GS) activity after a single exposure to the epileptogen FeCl2. Cells were cultured with both standard and elevated extracellular potassium or glutamate (Glu) concentration. FeCl2 reduced GS activity below control levels, whereas high Glu increased GS activity. However, stimulation by high Glu was significantly attenuated in cultures given both FeCl2 and high Glu, indicating that cells treated with FeCl2 were not able to respond as effectively to increased extracellular glutamate by increasing their GS activity. The significance of these findings is that glial regulation of the neuronal environment may be impaired, based on the proposed importance of GS in ammonia detoxification in the brain.
Denervation and hyperinnervation in the nervous system of diabetic animals: III. Functional alterations of G proteins in diabetic encephalopathy
Authors: Abbracchio M. P. , Di Luca M. , Di Giulio A. M. , Cattabeni F. , Tenconi B. , Gorio A.
Published: January 2005

G protein‐mediated effects on cAMP production were evaluated in the corpus striatum of diabetic rats 5 and 14 weeks after alloxan injection by measuring both D1‐receptor‐induced stimulation and D2‐receptormediated inhibition of adenylate‐cyclase activity. At 5 weeks of diabetes, no obvious alterations of G protein functions were detected. Both dopamine‐stimulated adenylate cyclase and bromocriptine‐induced inhibition of enzyme activity were indeed similar in control and diabetic animals. Fourteen weeks after alloxan injection, profound alterations were observed. Dopamine‐stimulated cAMP production was markedly increased in diabetic rats, whereas bromocriptine ability to reduce cAMP formation was almost abolished at this late stage of diabetes. Hypoactivity of Gi/Go proteins was also confirmed by the reduced ability of the GTP non‐hydrolyzable analog GTP‐γ‐S to inhibit forskolin‐stimulation of adenylate cyclase. These results show an apparent functional imbalance between Gs and Gi/Go‐mediated transduction mechanisms, with an increased efficacy of Gs activity likely due to the loss of Gi/Go inhibitory functions. Concomitantly with such transductional alteration detected in chronic diabetes, we observed a marked increase of the striatal content of met‐enkephalin, which is known to utilize Gi/Go proteins for inhibition of adenylate cyclase. The measurement of other transmitters (vaso‐active intestinal peptide, substance P, serotonin, noradrenaline, and dopamine) did not reveal any difference with respect to controls. The observed transductional defect in diabetic animals and the increased content and/or hyperinnervation by the metenkephalinergic system could be correlated as mutual compensatory mechanisms.
Platelet‐activating factor stimulates a Fos/Jun/AP‐1 transcriptional signaling system in human neuroblastoma cells
Authors: Squinto S. P. , Block A. L. , Braquet P. , Bazan N. G.
Published: January 2005

Platelet‐activating factor (PAF) elicits a rapid and transient activation of the proto‐oncogenes c‐fos and c‐jun in SH‐SY5Y neuroblastoma cells, but only to a minor extent in Molt‐4 T‐lymphocytes. This effect is inhibited by pretreatment of cells with the PAF antagonist BN 52021, suggesting the involvement of a specific receptor. Moreover, PAF treatment can activate gene expression through an AP‐1 element, and we propose that genomic trans‐activation may occur in target genes containing a functional AP‐1 transcription sequence. These results may further under standing of the molecular mechanisms by which PAF contributes to long‐term phenotypic changes in the nervous system.
Regulation of cAMP levels by protein kinase C in C6 rat glioma cells
Authors: Bressler J. P. , Tinsely P.
Published: January 2005

Cultures of rat C6 rat glioma cells exhibit a diminished response to isoproterenol and forskolin after being treated with phorbol 12,13‐dibutyrate (PDbU). An IC50 for PDbU of 38±5 nM and 62±8 nM was observed in the isoproternol and forskolin response, respectively. Similarly, C6 cultures exhibited a diminished response to isoproternol and forskolin after an overnight incubation with phospholipase C. We previously demonstrated that this treatment will increase diacylglycerol levels in these cells (Bressler: J Neurochem 48:181–186, 1987). An IC50 for phospholipase C of 6.0±0.1 × 10 −1 and 7.0±0.1 × 10−1 units/ml was observed for the isoproterenol and forskolin response, respectively. A kinetic analysis suggests that the site of PDbU‐mediated inhibition to beta‐adrenergic and forskolin stimulation was different. Degradation of cAMP was a contributory factor since elevated cAMP levels decreased faster in PDbU treated cells than in nontreated cells. In addition, PDbU treated cells exhibited a significantly higher level of phosphodiesterase activity. We conclude that activation of protein kinase C and subsequent stimulation of phosphodiesterase activity contributes to the inhibition of the beta‐adrenergic and forskolin mediated increase in cAMP levels in intact C6 rat glioma cells. The consequences of lower cAMP levels in sustaining differentiated function in the C6 rat glioma cell line will be discussed.
Effect of low‐dose lead acetate exposure on the metabolism of nucleic acids and lipids in cerebellum and hippocampus of rat during postnatal development
Authors: Agodi A. , Viola M. , Alberghina M. , Stella A. M. Giuffrida
Published: January 2005

Postnatal exposure (from the second day after birth to 30 days) of rat pups to low levels of lead acetate (50 mg/kg body weight/day), administered by gastric intubation, yielded a maximum blood level of 76.1μg/100 ml, at day 15 of age. Cerebellar and hippocampal lead contents were 8.67μg/100 mg and 11.7μg/100 mg, respectively, at day 30 of age. This lead exposure has been shown to elicit little change in some biochemical parameters in cerebellum and hippocampus. At the three ages investigated (5,15, and 30 days after birth) there were no alterations of body weight; brain, cerebellum, and hippocampus wet weight; and DNA, RNA, protein and phospholipid content, either in total tissue or in mitochondria. A similar invariance following lead exposure was observed in mitochondrial succinate dehydrogenase and cytochrome oxidase activities. After intraperitoneal administration, the incorporation of [methyl‐14C]thymidine into DNA and [5,6‐3H]uridine into RNA of cerebellum and hippocampus showed a significant decrease only at day 5, reaching the control value at 15 and 30 days of age. After intraperitoneal injection, [2‐3H]glycerol incorporation into total lipids and phospholipids of cerebellum and hippocampus also showed no significant changes in Pb‐treated pups compared to controls at all three postnatal ages.

We concluded that subclinical lead administration exerts its effect by slowing cell proliferation in the very early growth phase of the brain. It is likely that a metabolic compensative response to subtoxic effect of lead acetate may be brought about in cerebellum and hippocampus during critical phases of nervous system development between days 15 and 30.
Molecular cloning of cDNAs that encode the chicken Po protein: Evidence for early expression in avians
Authors: Barbu M.
Published: January 2005

As the major transbilayer glycoprotein of peripheral myelin, Po is believed to play a prominent role in the formation of this structure by Schwann cells. The amino acid sequence of the chicken Po molecule, deduced from the nucleotide sequence of cDNA clones, is reported here. Comparison with the mammalian molecule reveals an extensive overall homology, thus underlining the importance both of the cytoplasmic and of the extracellular domains of this protein in the stablishment and preservation of peripheral myelin structure. Unexpectedly, an avian Po cDNA probe was found to hybridise with several mRNA species, in exclusively in peripheral nerve and in proportions that varied according to the developmental stag. The expression of these transcripts was detected significantly earlier than that of po mRNA in nammals.
Developmental expression of glial fibrillary acidic protein mRNA in the rat brain analyzed by in situ hybridization
Authors: Landry C. F. , Ivy G. O. , Brown I. R.
Published: January 2005

Glial fibrillary acidic protein (GFAP) accumulates in astrocytes during development. We have characterized the increase in GFAP mRNA during development of the rat brain by using Northern blotting and in situ hybridization histochemistry and have found a caudal to rostral gradient of expression, consistent with overall brain maturation. GFAP mRNA was first obseved at embryonic day 16 (E16) in the glial limitans of the ventral hindbrain. During brain development message levels increased rostrally and by postnatal day 5 (P5) the entire glial limitans showed a positive signal which persisted into adulthood. GFAP mRNA was also found to accumulate in a caudal to rostral direction within the Purkinje cell layer of cerebllum beginning shortly after birth. By P5 the entire layer was positive and signal in this region could be localized to Bergmann glia by P15. A transient elevation in GFAP mRNA was apparent during the second postnatal week in cerebellum and cerebrum. Using in situ hybridization, a peak in message levels was observed at P15 and could be localized primarily to the deep white matter of cerebellum, to the corpus callosum, and to certain hippocampal fiber tracts. The pattern of GFAP expression in these regions is consistent with the differentiation of interfascicular glia and the appearance of type‐2 astrocytes during the initial events of myelination. GFAP mRNA levels in white matter were greatly reduced in the adult. The pronounced regional differences in GFAP mRNA expression during development may reflect the differentiation of subpopulations of astrocytes.
Alterations in glutamine synthetase activity by FeCl2‐induced focal and kindled amygdaloid seizures
Authors: Tiffany‐Castiglioni E. C. , Peterson S. L. , Castiglioni A. J.
Published: January 2005

The purpose of this study was to determine the effects of two experimental models of chronic epilepsy in rats on the activity of the astroglial enzyme glutamine synthetase. FeCl2‐induced cortial lesions that increased the sensitivity to pentylenetetrazol‐induced generalized seizures also significantly increased glutamine synthetase activity in the homotopic site of the contralateral cortex. A decrease in cortical glutamine synthetase was found in the cortex ipsilateral to the stimulated amygdala in electrically kindled animals. These findings provide evidence that increased glutamine synthetase activity is associated with developing, submaximal seizures and that decreased activity is characteristic of mature, chronic seizure states such as that associated with kindled amygdala seizures.
Prevention of chemically induced synaptosomal changes
Authors: Bondy S. C. , McKee M.
Published: January 2005

The ability of altered environmental conditions to modulate some properties of synaptosomes has been studied. Incubation conditions used included the presence of methyl mercury or an organochlorine insecticide: chlordecone. Other adverse chemical conditions during incubation were the absence of calcium salts from the incubation medium or the addition of agents bringing about enhanced oxidative conditions. Synaptosomal parameters studied were the cytosolic level of free, ionic calcium, [Ca2+]i, the extent of depolarization‐induced uptake of radioactive calcium, and the permeability of the limiting membrane. In addition, peroxidative activity was estimated by quantitation of thiobarbituric acid‐reactive material. All these facets of synaptosomal function were responsive to the presence of these potentially deleterious changes in the incubation medium. While the response of [Ca2+]i, was potentially in either direction, all adverse conditions increased synaptosomal permeability as evaluated by leakage of fura‐2 into the extracellular compartment. Pretreatment with ganglioside GM, in some situations or α‐tocopherol in others could either wholly or partially prevent the onset of such altered synaptosomal characteristics.
Phosphorylation of proteins of the postsynaptic density: Effect of development on protein tyrosine kinase and phosphorylation of the postsynaptic density glycoprotein, PSD‐GP180
Authors: Curd James W. , Bissoon N.
Published: January 2005

The effect of development on the tyrosine kinase activity of postsynaptic densities (PSDs) has been determined. PSDs were prepared from the forebrains of rats ranging in postnatal age from 13 to 90 days and the phosphorylation of both exogenous and endogenous substrates by tyrosine kinase measured. PSDs exhibited tyrosine kinase activity at all ages examined. Phosphorylation of the exogenous substrates polyglutamyltyrosine (4:1) and [val5] angiotensin II increased twofold between days 30 and 90 to levels̀ slightly lower than those present at 13 days. The phosphorylation of endogenous PSD proteins on tyrosine residues, assessed by alkali digestion of polyacrylamide gels of 32P‐labelled PSD proteins and by measuring the formation of [32P] phosphotyrosine by PSDs incubated in the presence of [γ‐32P] ATP, closely paralleled the changes in total tyrosine kinase activity. Tyrosine phosphorylation of the PSD‐specific glycoprotein, PSD‐GP180, also showed a transient increase between days 22 and 30, although its concentration within the PSD continued to increase slowly up to 90 days. The results indicate that the tyrosine kinase activity of PSDs is developmentally regulated and that tyrosine phosphorylation of PSD proteins is limited by enzyme rather than substrate availability.
Thy‐1‐like immunoreactivity in developing chicken skeletal muscle: Identification of a cross‐reactive slow‐fiber specific molecule that is not Thy‐1
Authors: Shahin S. , Jeffrey P. L. , Rostas J. A. P.
Published: January 2005

We investigated the Thy‐1‐like immunoreactivity during the development of chicken skeletal muscle using a group of monoclonal antibodies raised and characterised against purified chicken brain Thy‐1.The immunoreactivity attributable to bona fide Thy‐1 in muscle was present in nerves, connective tissue associated with the intrafusal capsule and blood vessels, and the extracellular matrix of the muscle fibres. During development there was no change in the staining of nerves, blood vessels or intrafusal capsules. However, the extracellular staining of muscle first appeared around hatching and gradually increased in intensity reaching maximal levels in the adult. The intensity of staining varied within and between the muscles examined. One of the antibodies (SB1 20.11)recognised an additional molecule that is not Thy‐1 and that was localised in the cytoplasm of slow muscle fibres. This immunoreactivity was first detectable at E10 in all myotubes that contained both alkali and acid stable mvosin ATPase activity (presumptive slow), but not in those myotubes with only alkalistable myosin ATPase activity (presumptive fast). Thereafter, the staining increased to a maximum in the newly hatched animal and then decreased until reactivity was undetectable in the adult (>25 weeks). All positive fibres initially stained with a uniform intensity but the time of commencement and the rate of loss of staining was variable. Those fibres that contained both acid stable and acid labile myosin ATPase activity lost the antigen much faster than the fibres containing only acid‐stable myosin ATPase activity, which also tended to increase in intensity for a longer period. These may represent, respectively, the slow tonic type III fibres and the slow twitch type I fibres classified by Barnard et al. (1982).
Influence of oxidative stress on the age‐linked alterations of the cerebral glutathione system
Authors: Benzi G. , Marzatico F. , Pastoris O. , Villa R. F.
Published: January 2005

The glutathione system (reduced and oxidized glutathione; redox index) was studied in the forebrain of male Wistar rats of 5, 15, and 25 months of age following the administration for 2 months in drinking water of chemicals that induce oxidative stress: paraquat and diethyldithiocarbamate (DDC) to increase superoxide radical formation, aminotriazole and hydrogen peroxide to increase hydroxyl radical generation, as well as diamide and ferrous chloride to decrease the glutathione cycle activity. Chronic oral administration of phosphatidylcholine for 2 months was evaluated in 25‐month‐old rats. Aging accentuated the changes produced by chemicals that induce oxidative stress; i.e., the changes in the glutathione redox index were most pronounced in the forebrains of the older paraquat‐, DDC‐, H2O2−, and diamidetreated rats. Markedly different adaptative changes occurred within the various drug groups. The reduced glutathione was increased (by paraquat, DDC and aminotrazole), decreased (by H2O2) or unchanged (by iron and diamide). Furthermore, in older rats, paraquat and DDC increased the glutathione redox index, whereas H2O2 and diamide decreased the glutathione redox index or were ineffective (i.e., aminotriazole, iron). The glutathione redox index alterted by chronic drug administration was modified by the concomitant administration of phosphatidycholine.
Protein synthesis in the neuropil of the rat dentate gyrus during synapse development
Authors: Phillips L. L. , Pollack A. E. , Steward O.
Published: January 2005

Previous studies have shown that there are dramatic accumulations of polyribosomes under developing synapses on dendrites of CNS neurons. The present study was designed to evaluate what types of proteins might be synthesized by the synapse‐associated polyribosomes. Hippocampal slics from rat pups sacrificed at 4,7,10,12,14, and 21 days after birth as well as slices from adult animals were incubated in a modified Eagle's medium containing 3H‐leucine. After a 30 min exposure to radiolabeled amino acids, the slices were microdissected, separating the dendritic enriched molecular layer from the cell bodies of the dentate gyrus and the hippocampus proper. The level of protein synthetic activity was assessed by comparing the incorporation in cell body and dendritic laminae. Polypeptides present in each dissected zone were separated electrophoretically on 1D SDS‐polyacrylamide gels according to their molecular weight and the newly synthesized proteins were analyzed through gel fluorography.

The overall level of 3H‐amino acid incorporation into protein (measured as cpm/μg protin) was higher than that of the adult at all postnatal ages. When the entire slice was analyzed, the maximum incorporation was at 12 days after birth. In the dissected subregions of the slice the peak protein synthetic activity in cell‐body‐enriched regions of dentate gyrus and hippocampus proper was at 4–7 days postnatal, declining between 7 and 21 days to values comparable to the adult. By contrast, protein synthesis in the molecular layer of the dentate gyrus did not peak until 12 days after birth, decreasing toward adult rates after 14 days. The overall pattern of Coomassie stained polypeptides present in the dentate molecular layer was comparable at all ages examined. Moreover, one‐dimensional gel analysis showed no qualitative differences in the proteins that were synthesized in the three dissected zones across ages. Gel fluorography identified four mid‐molecular weight ranges of polypeptides (43, 50, 52, and 56 kD) and a group of high molecular weight proteins (190‐220 kD) that were the most heavily labeled species in the dissected molecular layer. Quantitative gel analysis revealed that three of the midrange bands (43, 52, and 56 kD) accounted for a different percentage of the over all labeled protein across developmental age; prominent changes were found in the 43 kD band, where increased radiobeling was directly correlated with postnatal periods of rapid synaptogenesis.

We conclude that there is a high level of protein synthesis in dendritic laminae during developmental synaptogenesis. This burst of synthesis appears to result in the increased generation of a set of proteins which are normally synthesized within both granule cell body and dendrite enriched regions. The 43 kD member (s) of this set of polypeptides shows increased labeling during the most rapid phase of synaptogenesis.
Rapid, sensitive, and simple method for quantification of both neurotoxic and neurotrophic effects of NMDA on cultured cerebellar granule cells
Authors: Didier M. , Heaulme M. , Soubrié P. , Bockaert J. , Pin J.‐P.
Published: January 2005

A simple and sensitive method adapted from the staining of living cells with fluorescein diacetate was developed to rapidly estimate the number of living cells remaining in a culture dish 24 hr after a few min of NMDA treatment of cerebellar neurons. This method consists of the measurement, after cell lysis, of the total amount of fluorescein produced from fluorescein diacetate by the living granule cells present in each culture dish. We show that this method can also be used to quantify the survival effect of chronic exposure of granule cells to either K+ or NMDA In both cases, the fluorescence measured was found to be proportional to the number of fluorescein‐labelled cells counted under a fluorescence microscope, indicating that the present method can be used to quantify both toxic and trophic effects of NMDA on cerebellar granule cells. This study confirms that these two NMDA effects occur at the same NMDA concentration, and both are inhibited by MK 801 in the same concentration range. We showed, moreover, that granule neurons developed in the presence of NMDA are much less sensitive to NMDA toxicity than neurons developed in K+‐enriched medium.
Induction of oligodendrocyte differentiation by activators of adenylate cyclase
Authors: Raible D. W. , McMorris F. A.
Published: January 2005

Oligodendrocyte differentiation is accelerated by analogs of cyclic AMP (cAMP), such as N62‐′O‐dibutyryladenosine 3′,5′‐cyclic monophosphate (dbcAMP) or 8‐bromo cAMP, in developing rat glial cell cultures (Raible and McMorris, 1989). However, it is not known whether the immature developing oligodendrocytes have G protein‐coupled adenylate cyclase capable of generating cAMP, as would be required if development of the cells is normally regulated by endogenous cAMP. In the present study, we demonstrate that the diterpene forskolin, a potent activator of adenylate cyclase, or cholera toxin, which activates adenylate cyclase through G protein modification, can induce a precocious increase in oligodendrocyte number. When both forskolin and cholera toxin are added together, oligodendrocyte differentiation is induced to a level similar to that observed in response to cAMP analogs. These results establish for the first time that modulation of the endogenous cAMP regulatory system alters the schedule of oligodendrocyte differentiation.
Potassium‐stimulated release of taurine from cultured cerebellar granule neurons is associated with cell swelling
Authors: Schousboe A. , Morán J. , Pasantes‐Morales H.
Published: January 2005

Effects of increased concentrations of potassium and of hyposmolar conditions on release of taurine were investigated in cerebellar granule neurons cultured from mice. It was found that increases in the external potassium concentration as well as decreases in osmolarity dose‐dependently increased release of exogenously supplied [3H]‐taurine and endogenous taurine from the neurons. The release of endogenous taurine elicited by a reduction of the osmolarity of the incubation media to 70% or 50% was much more pronounced than that of other amino acids, particularly glutamine, the release of which was not affected at all. The potassium‐stimulated release of [3H]‐taurine was strictly chloride dependent and it was inhibited by an increase of the osmolarity of the media as well as by 4,4′‐diisothyocyanostilbene‐2,2′‐disulfonate (DIDS) (100 μM). Moreover, a similar increase in the potassium concentration led to an increase in intracellular volume (swelling), a process which was also chloride dependent. It is concluded that potassium‐stimulated taurine release from cerebellar granule neurons is associated with cell volume changes and that taurine is likely to play a role as an osmotically active substance in these neurons.
4‐Aminopyridine interrupts the modulation of acetylcholine release mediated by muscarinic and opiate receptors
Authors: Törőcsik A. , Vizi E. S.
Published: January 2005

The effect of 4‐aminopyridine, a potassium channel blocker on the muscarinic and opiate modulation of acetylcholine release, was investigated. Rat frontal cortical slices were loaded with [3H]choline, super‐fused continuously, and stimulated electrically. 4‐Aminopyridine enhanced the stimulation‐evoked release of tritium without affecting basal release. The electrically evoked release of radioactivity was reduced by the muscarinic agonist oxotremorine and the delta selective opiate receptor agonist Metenkephalin, and was enhanced—in the presence of the cholinesterase inhibitor physostigmine—by the muscarinic antagonist atropine. These effects were completely abolished by 4‐aminopyridine. Since 4‐aminopyridine blocks potassium permeability of the neuron, it is suggested that the changes in potassium permeability and the consequent alteration of membrane polarization are involved in the presynaptic modulation of acetylcholine release.
Specific antibodies for tyrosinated and detyrosinated tubulin recognize retina tubulin subpopulations that do not participate in the posttranslational tyrosination/detyrosination cycle
Authors: Arregui C. , Barra H. S.
Published: January 2005

We have used the monoclonal YL 1/2 (Tyr antibody) and polyclonal (Glu antibody) antibodies, specific for tyrosinated and detyrosinated tubulin, respectively, to determine the levels and cellular distribution of these tubulin species in chick retina during development. At embryonic day 4, detyrosinated tubulin was restricted to the ganglion cells of the fundic region. As development progresses, immunofluorescence also appears, first, in the outermost zone of the retina and then in the plexiform layers. The Tyr antibody staining was found in the different layers and it was fairly homogeneous in distribution. Analysis by dot immunobinding showed that the ratios of tyrosinated to detyrosinated tubulin obtained at different ages do not agree with those obtained previously by an enzymatic method based on the incorporation of [14C]tyrosine found that the lack of coincidence is due to the fact that a fraction of the tubulin species determined by the Tyr and Glu antibodies does not participate in the posttranslational tyrosination/detyrosination cycle. This is a novel concept that should be considered in the interpretations of immunofluorescence studies concerning the cellular distribution of tyrosinated and detyrosinated tubulin.
Carbohydrate epitopes involved in neural cell recognition are conserved between vertebrates and leech
Authors: Bajt M. L. , Schmitz B. , Schachner M. , Zipser B.
Published: January 2005

We are reporting on the evolutionary conservation of carbohydrate epitope families from vertebrate to leech. 1) The sulfated L2/HNK‐1 carbohydrate epitope Abo T, Balch CM (1981): J Immunol 127:1024–1029; Kruse J,Mailhammer R, Wernecke H, Faissner A, Timpl R, Schachner M (1984): Nature 311:153–155 is detected on glycoproteins of leech neurons using monoclonal antibodies (mAbs) L2 (336) and HNK‐1. 2) Three rat mAbs, L3, L4, and L5, bind to leech nerve and muscle. The L3, L4, and L5 epitopes are localized to a group of mannosidic leech glycoproteins originally identified through mAbs Lan3‐2 (Hogg N, Flaster M, Zipser B (1983): J Neurosci Res 9:445–457 and Laz6‐189 (McRorie JW III, Zipser B (1988): “Cell Culture Approaches to Invertebrate Neuroscience.” London: Academie Press, pp 33–52. MAb Lan3‐2, which binds to a mannosidic epitope of the 130 kD sensory protein, has recently been shown to perturb the penetration of sensory afferents into the synaptic area of the central neuropile (Zipser B, Morell R, Bajt ML (1989): Neuron 3:621–630). The L3, L4, and L5 mAbs have been described to recognize different mannosidic epitopes on glycoproteins, some of which have been identified as neural cell adhesion molecules, and on astrocyte‐specific proteoglycan from mouse brain (Kucherer A, Faissner A, Schachner M (1987): J Cell Biol 104:1597–1602; Fahrig T, Schmitz B, Weber D, (Kücherer‐Ehret A, Faissner A, Schachner M (1990): Eur J Neurosci 2:153–161. Streit A, Faissner A, Gehrig B, Schachner M (1990): J Neurochem In Press). The superposition of five different mannosidic epitopes on the axons of sensory afferents suggests complex, concerted participation of mannosidic epitopes in neuronal pathfinding and target recognition.
Glass micro‐fibers: A model system for study of early events in myelination
Authors: Bullock P. N. , Rome L. H.
Published: January 2005

A system was developed to analyze early events in the process of myelination. Primary cultures of rat oligodendrocytes were maintained in the presence of glass micro‐fibers which served as artificial axons. A culture chamber was constructed which allowed the close apposition of fibers and cells in a three‐dimensional arrangement designed to resemble an in vivo environment. Cells cultured in the presence of glass micro‐fibers coated with a glial cell matrix extract were induced to organize into clusters around the fibers. Examination of oligodendrocyte‐fiber sandwiches by SEM revealed the presence of a number of cell contacts with the fibers. TEM images showed that, in most cases, fibers were surrounded by the cells and not multiply wrapped. Only occasionally was a loose wrapping of cell membrane observed around the fibers. Cells cultured in the presence of matrix‐coated glass micro‐fibers showed an increased production of sulfolipids that was at least partially dependent on the presence of the matrix coating. Coating of these “artificial axons” may aid in the identification of signal molecules produced by neurons which enable them to be myelinated.
Type 1 astrocytes and oligodendrocyte‐type 2 astrocyte glial progenitors migrate toward distinct molecules
Authors: Armstrong R. C. , Harvath L. , Dubois‐Dalcq M. E.
Published: January 2005

During central nervous system (CNS) development, glial precursors proliferate in subventricular zones and then migrate throughout the CNS to adopt their final destinations and differentiate into various types of mature glial cells. Although several growth factors promoting the proliferation and/or differentiation of glial precursors have been identified, very little is known about the nature of signals that guide glial cell migration in the CNS. Therefore, we have investigated whether polypeptide growth factors and/or extracellular matrix molecules may mediate the migration of two major glial cell types, type 1 astrocytes and oligodendrocyte‐type 2 astrocyte (O‐2A) progenitor cells. We show that, in a microchemotaxis chamber assay, type 1 astrocytes move toward laminin and complement‐derived C5a. Astrocyte migration toward laminin is inhibited by a laminin‐specific pentapeptide, YIGSR‐NH2. In contrast, O‐2A progenitors migrate toward platelet‐derived growth factor (PDGF), which also functions as a mitogen for these cells. Using a new method to simultaneously assay migration and DNA synthesis, we also demonstrate that O‐2A progenitors can migrate toward PDGF even when DNA replication is inhibited with an antimitotic agent. Thus, migration of different types of glial cells can be induced in vitro by specific signaling molecules, which are present in the developing brain and may stimulate migration of glial cells prior to CNS myelination.
Isolation and localization of a slow troponin (TnT) gene on chromosome 19 by subtraction hybridization of a cDNA muscle library using myotonic dystrophy muscle cDNA
Authors: Samson F. , Lee J. E. , Hung W. ‐Y. , Potter T. G. , Herbstreith M. , Roses A. D. , Gilbert J. R.
Published: January 2005

Subtraction hybridization techniques were used to isolate 91 cDNA clones which are overexpressed in normal control skeletal muscle relative to muscle from patients with myotonic muscular dystrophy. The gene responsible for myotonic dystrophy (DM) has been localized to the 19q13.2–13.3 region of chromosome 19. To test as a candidate gene for DM, clones which represent differences in transcription are analyzed for localization to chromosome 19. One clone, designated MSL 366, was found to be on the long arm of chromosome 19 distal to the CKMM gene at 19q13.2. Sequence analysis confirmed that MSL 366 is the cDNA for human slow skeletal muscle troponin T. A genomic clone has been isolated and linkage studies with DM are in progress.
Glial fibrillary acidic protein and βA4 protein deposits in temporal lobe of aging brain and senile dementia of the Alzheimer type: Relation with the cognitive state and with quantitative studies of senile plaques and neurofibrillary tangles
Authors: Harpin M. L. , Delaère P. , Javoy‐Agid F. , Bock E. , Jacque C. , Delpech B. , Villarroya H. , Duyckaerts C. , Hauw J. J. , Baumann N.
Published: January 2005

The aim of this study was to compare brain glial fibrillary acidic protein (GFAP) levels to the modifications of cognitive functions (Blessed test score [BTS]), the density of the main neuropathological lesions (senile plaques [SP] and neurofibrillary tangles [NFT]), and the density of the two main subtypes of βA4 deposits (classic plaques and diffuse deposits) in a series of patients with normal aging and senile dementia of the Alzheimer type of various degrees of severity. GFAP levels (enzymelinked immunosorbent assay [ELISA] technique) and the densities of changes were measured in the temporal lobe of 12 women over 75 years of age. Under these conditions, the ELISA assay could determine GFAP in brain homogenates (aqueous‐Triton buffer soluble extract) in a range from 2.5 ng to 600 ng per assay. Least affected patients (with a BTS of 19 and over) all ranged below 60 μg/mg protein. Most affected patients (with a BTS under 6) ranged above 150 μg/mg protein. However, interindividual variations were wide. A significant correlation between the BTS and the amount of GFAP could be found only when using the non parametric test of Spearman. There was a significant positive correlation between the amount of GFAP and the density of (1) SP, (2) NFT both revealed by Bodian's silver stain, and (3) classic βA4 plaques shown by immunocytochemistry. On the contrary, no correlation was observed with diffuse βA4 deposits. One case with very large amounts of diffuse βA4 deposits without SP or NFT showed no associated GFAP reactivity. This suggests that GFAP production is a critical event in the formation of classic SP.
Calcium‐dependent regulation of the enkephalin phenotype by neuronal activity during early ontogeny
Authors: Agoston D. V. , Eiden L. E. , Brenneman D. E.
Published: January 2005

Genetic components of the neuronal phenotype are regulated by epigenetic factors—trophic molecules and neuronal activity—during neurodifferentiation. Developing neurons in dissociated cultures of embryonic mouse spinal cord show spontaneous electrical activity after one week in culture. We now report that the blockade of this spontaneous electrical activity for two days with tetrodotoxin (TTX) causes virtually complete down‐regulation of preproenkephalin A gene transcripts in embryonic spinal cord cultures. This TTX‐induced down‐regulation is fully reversed upon reinitiation of neuronal activity (removal of TTX from cultures). This reversible, tetrodotoxin‐induced down‐regulation kephalin mRNA is confined to a restricted period of early neurodevelopment (days 7 to 21 in culture). Since depolarization triggers calcium entry through voltage‐activated calcium channels, we have investigated the involvement of calcium in the mechanism of this activity‐ and age‐dependent regulation of preproenkephalin A expression. The selective activation of the L‐type of voltage‐sensitive calcium channels by a dihydropyridine derivative [( + ) 202‐791] prevented this TTX‐induced down‐regulation without reducing methionine enkephalin secretion. This effect was observed only when the drug was applied to electrically active cultures, prior to the addition of TTX. Simultaneous application of ( + ) 202‐791 and TTX, or pretreatmont with TTX, failed to prevent TTX‐induced down‐regulation. Thus, activity‐dependent phenotypic plasticity of met‐enkephalinergic neurons in spinal cord is:(1) maximum at an early age of neuronal development (<10 days in culture) and becomes less apparent in old cultures (>30 days); (2) reversible throughout; and (3) mediated by calcium entry through L‐type channels.
Transplantation of a temperature‐sensitive, nerve growth factor‐secreting, neuroblastoma cell line into adult rats with fimbria–fornix lesions rescues cholinergic septal neurons
Authors: Whittemore Scott R. , Holets V. R. , Keane R. W. , Levy D. J. , McKay R. D. G.
Published: January 2005

The HT4 cell line was derived from infection of a mouse neuroblastoma cell line with a retrovirus that encoded the temperature‐sensitive (ts) mutant of SV40 large T antigen. At nonpermissive temperature, HT4 cells differentiated with neuronal morphology, expressed neuronal antigens, synthesized nerve growth factor (NGF) mRNA, and secreted biologically active NGF in vitro. We sought to establish whether transplanted HT4 ceils expressed class I major histocompatibility complex (MHC) antigens, a partial requirement for recognition by cytotoxic T lymphocytes (CTL), and thus be susceptible to xenograft rejection. Differentiated HT4 cells expressed marginally detectable levels of class I MHC antigens, but demonstrated higher levels of class I MHC expression after treatment with interferon‐γ. However, HT4 cells were resistant to direct lysis by perforin the pore‐forming protein of CTLs, and thus may have potential use in xenograft experiments. To address whether HT4 cells secrete NGF in vivo, HT4 cells were transplanted into adults rats with unilateral fimbria‐fornix transections. A ts cell line derived from P4 cerebellum, BTl, that does not differentjate with neuronal phenotype or synthesize NGF in vitro, was transplanted as a control. Six weeks posttransplant. HT4 cells had integrated into host CNS without forming tumors. In BT1 transplants, the number of medial septal acetylcholinesterase (AChE)‐positive cells was reduced to 26‐39% of the contralateral control side, depending on the rostrocaudal level. In HT4 transplants, the number of cholinergic septal neurons was 58‐78% of the contralateral side. This percentage was significantly (P < 0.005) greater than that seen with BT1 transplants, indicating that transplanted HT4 cells secrete NGF in vivo and rescue cholinergic septal neurons following fimbria‐fornix transection.