bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid Internalization and Intracellular Metabolic Processing of Exogenous Ganglioside by Cerebellar Granule Cells Differentiated in Culture
Authors: Riboni Laura , Tettamanti Guido
Published: October 2006

Ganglioside GMl, tritiated at the level of the long chain base (sphingosine) ([Sph‐3H]GMl), sialic acid (N‐ace‐tylneuraminic acid) ([NeuAc‐3H]GMl), or terminal galactose ([Gal‐3H]GMl) was supplied to cerebellar granule cells differentiated in vitro, and its metabolic processing was followed with pulse time. Using [Sph‐3H]GM 1 and [NeuAc‐3H]GMl the formation of radioactive compounds of catabolic origin (GM2, GM3, lactosylceramide, glucosylceramide, and ceramide) started being detectable at 10–15 min of pulse, whereas compounds of biosynthetic origin (GD1a, GD1b, GT1b, O‐acetylated GT1b, sphingomyelin, and sialoglycoprotein) appeared after 15–30 min of pulse. Using [Gal‐3H]GM1 two radioactive substances were formed, GD1 a and GT1b, with the former (produced by direct sialosylation of GM1) appearing after 30 min of pulse and the latter (formed by bio‐synthetic recycling of released galactose) appearing after 2 h. The radioactivity linked to all metabolites increased with increasing pulse time until 4 h. The percentage of GM1 taken up and subjected to metabolic processing was found to increase from 1.8% after 10 min of pulse to 12.5% after 4 h. Cerebellar granule cells were able to release enzymes of lysosomal origin, β‐D‐N‐acetylhexosaminidase and β‐D‐galac‐tosidase, into the culture medium, with the release being markedly decreased by the absence in the medium of fetal calf serum, a condition that was used for studying exogenous GM1 uptake and metabolization. However, these enzymes exerted no activity at the pH of the culture medium, and no radioactive gangliosides, besides GM1, were detected in the culture medium during pulse. Addition of 50 μM chloroquine to granule cells during the pulse experiments with radioactive GM1 completely blocked the metabolic processing of exogenous GM1 with the exception of direct sialosylation, a result indicating the key role played by lysosomes in the process. The overall results show that cerebellar granule cells take up, internalize, and metabolize exogenous GM1 rapidly, with the lysosomes being involved immediately in degradation, and the biosynthetic apparatuses somewhat later in recycling metabolic intermediates. The hypothesis can be proposed that the ganglioside present in the membrane domain subjected to internalization is rapidly turned over.
Transient Expression of Opioid Receptors in Defined Regions of Developing Brain: Are Embryonic Receptors Selective?
Authors: Barg Jacob , Simantov Rabi
Published: October 2006

The developmental profile of opioid receptors was studied in rat and guinea pig striatum and hippocampus. The two brain regions show different receptor profiles during development, which are characteristic for each animal. Yet, both tissues and animal species share one common feature; the binding of the universal opioid ligand [3H]diprenorphine per milligram of protein is high at the early embryonic period, it decreases toward birth, and then gradually increases to the adult levels. This apparent transient expression of the receptors during the early developmental stage was manifested in the guinea pig as an actual decrease in the total receptor number. As an attempt to characterize the receptors involved in this process, the binding of the selective μ‐opioid ligand [3H]Tyr‐d‐Ala‐Gly‐MePhe‐NH(CH2)OH ([3H]DAGO) was studied in striatal membranes of young (P1) and adult (P60) rats. Competition between [3H]DAGO and the δ‐selective peptide Tyr‐d‐Pen‐GIy‐Phe‐d‐Pen (DPDPE) shows higher affinity of the S opioid to P1 membranes than to P60 membranes, though the number of δ receptors in P1 membranes is very small. This observation is in line with a previous study suggesting that opioid receptors in embryonic striatum and hippocampus are less selective to various opioids than those of adult brain. An additional difference between adult and embryonic tissue was observed on Scatchard analysis of [3H]DAGO binding; striatum P60 membranes exhibit one binding site with a KD of 0.8 ± 0.1 nM and Hill coefficient of 0.96, whereas striatum P1 membranes bind the peptide in an apparent cooperative fashion with an overall Hill coefficient of 1.30. The possibility that a cooperative binding pattern involves interactions of SH groups was tested with the sulfhydryl reducing agent dithiothreitol (DTT). Indeed, DTT‐treated membranes from adult rat striatum bind [3H]DAGO in a cooperative fashion (Hill coefficient, 1.32). The significance of a transient expression of apparently nonsetective opioid receptors in developing brain, and the possibility that receptors from embryonic and adult brain interact differently with one another or with other membrane constituents, such as G proteins and ion channels, is discussed.
Characterization of Rat ARPP‐21 mRNA: Sequence Analysis, Tissue Distribution, and Regulation
Authors: Ehrlich Michelle E. , Greengard Paul
Published: October 2006

ARPP‐21 (cyclic AMP‐regulated phosphoprotein; Mr= 21,000) is a cytosolic neuronal phosphoprotein that is highly enriched in the striatum and in other dopaminoceptive regions of the brain. The state of phosphorylation of ARPP‐21 is also regulated by vasoactive intestinal peptide in intact cells. We previously reported the sequence analysis of bovine ARPP‐21 cDNA and have now characterized rat ARPP‐21 cDNA to study further the molecular biology of this protein. The sequence of the coding region is 82 and 85% identical at the nucleotide and amino acid levels, respectively, between the two species. There are two major classes of clones, differing only in the lengths of their 3’untranslated ends, suggesting that the different ARPP‐21 mRNAs are derived from the use of alternate polyadenylation sites. Both major mRNA species, 2.6 and 0.7 kb, are present at the highest concentration in the striatum, followed by the cortex, consistent with previous immunocytochemical results. Southern blot analysis reveals a simple hybridization pattern, consistent with the presence of a single rat gene encoding ARPP‐21. The steady‐state levels of the ARPP‐21 mRNAs are developmentally regulated but, in the neonatal and mature animal, are not altered following 6‐hydroxydopamine lesions of the substantia nigra or by pharmacologic treatments that up‐regulate the D1‐ or D2‐dopamine receptors.
[3H]SCH 39166, A New D1‐Selective Radioligand: In Vitro and In Vivo Binding Analyses
Authors: McQuade Robert D. , Duffy Ruth A. , Anderson Cheryl C. , Crosby Gordon , Coffin Vicki L. , Chipkin Richard E. , Barnett Allen
Published: October 2006

SCH 39166 {(‐)‐trans‐6,7,7a,8,9,13b‐hexahydro‐3‐chloro‐2‐hydroxy‐N‐ methyl‐5H ‐ benzo ‐[d]naphtho[2,1b]azepine} has recently been described as a selective D1 antagonist and has entered clinical trials for the treatment of schizophrenia. The tritiated analogue of this compound, [3H]SCH 39166, has now been synthesized and characterized for its in vitro and in vivo binding profiles. [3H]SCH 39166 binds to D1 receptors in a saturable. high‐affinity fashion, with a KDof 0.79 nM. In competition studies, D1‐selective antagonists like SCH 23390 displaced the binding of [3H]SCH 39166 with nanomolar affinities, whereas antagonists of other receptors exhibited poor affinity. In vivo, [3H]SCH 39166 bound to receptors in rat striatum in a fashion suggestive of D1 selectivity. Further. when the time course for the binding of [3H]SCH 39166 was compared with the behavioral time course of the unlabeled compound, the two durations of action were virtually indistinguishable. Similar studies were performed for SCH 23390 and its tritiated analogue, but the in vivo binding of this radioligand exhibited a duration of action far greater than the behavioral activity of the unlabeled drug. In concert, these data demonstrate that [3H]SCH 39166 selectively labels D1 receptors in vitro and in vivo, and that this drug is superior for in vivo imaging of the D1 receptor.
Evidence for an Association of the 15‐kDa Proteolipid of Mediatophore with a 14‐kDa Polypeptide
Authors: Israël M. , Morel N. , Lesbats B.
Published: October 2006

The present report shows that mediatophore, a nerve terminal membrane protein that translocates acetyl‐choline on calcium action, forms a complex with a 14‐kDa polypeptide. The complex was identified based on the following results, (a) A polyclonal antimediatophore antiserum that immunoprecipitates activity precipitates both the 15‐and 14‐kDa polypeptides. (b) After HPLC purification of mediatophore, both antigens were found in the same peak, (c) After 3‐[(3‐cholamidopropyl)dimethylarnmonio]‐1‐pro‐panesulfonate solubilization of presynaptic membranes or of the purified mediatophore, an immunoaffinity column made with the anti‐14‐kDa antigen monoclonal antibody retained both the 14‐kDa and the 15‐kDa polypeptide. Similarly, im‐munoprecipitation experiments using protein A‐coated beads sedimented an immunocomplex in which both antigens were found, (d) The 14‐kDa antigen could be localized in the syn‐aptosomal membrane where mediatophore and its 15‐kDa component are found.
Redox Modulation of N‐Methyl‐D‐Aspartate‐Stimulated Neurotransmitter Release from Rat Brain Slices
Authors: Woodward John J. , Blair Robert
Published: October 2006

Rat brain cortical slices released tritiated norepi‐nephrine ([3H]NA) during a 2‐min stimulation with N‐methyl‐d‐aspartate (NMDA). Dithiothreitol (DTT; 0.1–5 mM), present for 6 min prior to stimulation, dose‐depen‐dently increased the release of [3H]NA from cortical slices stimulated with a maximally effective concentration of NMDA (500 μM). Similar results were observed for [3H]NA release from hippocampal slices and tritiated and endogenous dopamine release from striatal slices. DTT treatment also markedly shifted the dose‐response curve of NMDA to the left. Cortical slices released approximately the same amount of [JH]NA with 10 μM NMDA following DTT treatment (about 5%) as non‐DTT‐treated control slices did with 500 μM NMDA. The effects of DTT were fully reversed by subsequent treatment with 5,5′‐dithio‐bis(2‐nitrobenzoic acid) (DTNB; 0.5 mM). DTT treatment did not significantly alter the ability of magnesium (1.3 mM) or the polyamine antagonist arcaine to block the NMDA‐stimulated release of [3H]NA. In contrast, DTT treatment significantly attenuated the antagonist effects of the competitive glycine antagonist, 7‐chlorokynurenic acid, and the competitive NMDA antagonist, 2‐aminophosphonopentanoic acid. These results suggest that oxidation and reduction of disulfide bonds located within the NMDA receptor complex might regulate the activation of the NMDA receptor. This could have important consequences in vivo if endogenous oxidizing/reducing systems are found to have similar effects on NMDA‐stimulated responses.
Classical Conditioning‐Induced Changes in Low‐Molecular‐Weight GTP‐Binding Proteins in Rabbit Hippocampus
Authors: Nelson T. J. , Sanchez‐Andres J.‐V. , Schreurs B. G. , Alkon D. L.
Published: October 2006

Classical conditioning of Hermissenda, involving paired light‐rotation events, results in a 30–35% decrease in the levels of a 20‐kDa G protein (cp20). To test whether a similar protein exists in vertebrates, rabbits were trained to associate a tone with periorbital electrical stimulation and g proteins were analyzed by photoaffinity labeling with [œ‐32P]GTP‐azidoanilide. A 20‐kDa G protein similar to cp20 decreased by 36% in the hippocampus of rabbits subjected to paired tone and electrical stimulation, but not in unpaired controls. Learning‐specific decreases were also found in the amount of ras protein.
Activation of Tyrosine Hydroxylase by Nicotine in Rat Adrenal Gland
Authors: Fossom Linda H. , Sterling Carol , Tank A. William
Published: October 2006

The administration of nicotine activates tyrosine hydroxylase in the rat adrenal gland. This activation is apparently maximal 25 min after a single subcutaneous injection of nicotine at 2.3 mg/kg. Repeated injections of nicotine (seven injections once every 30 min) are associated with a persistent activation of adrenal tyrosine hydroxylase for at least 3 h. The nicotinic receptor antagonist hexamethonium docs not significantly inhibit the nicotine‐mediated activation of tyrosine hydroxylase in innervated adrenal glands. However, hexamethonium completely blocks the activation of adrenal tyrosine hydroxylase by nicotine in denervated adrenal glands. Furthermore, even though a single injection of nicotine activates tyrosine hydroxylase in both innervated and denervated adrenal glands, repeated injections of nicotine do not activate tyrosine hydroxylase in denervated adrenal glands. Our results suggest that the systemic administration of nicotine activates adrenal tyrosine hydroxylase by two mechanisms: (1) via direct interaction with adrenal chro‐maffin cell nicotinic receptors; and (2) via stimulation of the CNS leading to the release from the splanchnic nerve of substances that interact with adrenal chromaffin cell receptors other than the nicotinic receptor.
Incorporation of [3H]Arachidonic and [14C]Eicosapentaenoic Acids into Glycerophospholipids and Their Metabolism via Lipoxygenases in Isolated Brain Cells from Rainbow Trout Oncorhaynchus mykaiss
Authors: Tocher Douglas R. , Bell J. Gordon , Sargent John R.
Published: October 2006

The incorporation of [3H]arachidonate ([3H]AA) and [14C]eicosapentaenoate ([14C]EPA) into glycerophos‐pholipids was studied in isolated brain cells from rainbow trout, a teleost fish whose lipids are rich in (n‐3) polyunsaturated fatty acids (PUF As), EPA was incorporated into total lipid to a greater extent than AA, but the incorporation of both PUF As into total glycerophospholipids was almost identical. The incorporation of both AA and EPA was greatest into phosphatidylethanolamine (PE). However, when expressed per milligram of individual phosphoglycerides, both AA and EPA were preferentially incorporated into phospha‐tidylinositol (PI), the preference being significantly greater with AA. On the same basis, significantly more EPA than AA was incorporated into phosphatidylcholine (PC). When double‐labelled cells were challenged with calcium ionophore A23187, the 3H and 14C released from the cells closely paralleled each other, peaking at 10 min after addition of ionophore.

The 12‐monohydroxylated derivative was the predominant lipoxygenase product from both AA and EPA with a rank order of 12‐hydroxyeicosatetraenoic acid (12‐HETE) > leukotriene B4 (LTB4) > 5‐HETE > 15‐HETE for the AA products and 12‐hydroxyeicosapentaenoic acid (12‐HEPE) > 5‐HEPE > LTB5 > 15‐HEPE for EPA products. The 3H/14C (dpm/dpm) ratios in the glycerophospholipids, total released radioactivity, and the lipoxygenase products suggested that PC rather than PI was the likely source of eicosanoid precursors in trout brain cells.
Ranakinin: A Novel NK1 Tachykinin Receptor Agonist Isolated with Neurokinin B from the Brain of the Frog Rana ridibunda
Authors: O'Harte Finbarr , Burcher Elizabeth , Lovas Sandor , Smith D. David , Vaudry Hubert , Conlon J. Michael
Published: October 2006

An extract of the whole brain of the frog Rana ridibunda contained high concentrations of substance P‐like immunoreactivity, measured with an antiserum directed against the COOH‐terminal region of mammalian substance p and neurokinin b‐like immurtoreactivity, measured with an antiserum directed against the NH2‐terminus of neurokinin B. The primary structure of the substance p‐related peptide (ranakinin) was established as: Lys‐Pro‐Asn‐Pro‐Glu‐Arg‐Phe‐Tyr‐Gly‐Leu‐Met‐NH2. Mammalian substance P was not present in the extract. The primary structure of the neurokinin b‐related peptide was established as: Asp‐Met‐His‐Asp‐Phe‐Phe‐Val‐Gly‐Leu‐Met‐NH2. This amino acid sequence is the same as that of mammalian neurokinin B.

Ranakinin was equipotent with substance p and [Sar9,‐Met(O2)11]substance p in inhibiting the binding of 125I‐Bolton‐Hunter‐[Sar9, Met(O2)11]substance p, a selective radio‐ligand for the NK1 receptor, to binding sites in rat subman‐dibular gland membranes (IC50 1.6 ± 0.3 nM; n = 5). It is concluded that ranakinin is a preferred agonist for the mammalian NK1 tachykinin receptor subtype.
Characterization of a Novel Serotonin Receptor Subtype (5‐HTls) in Rat CNS: Interaction with a GTP Binding Protein
Authors: Zemlan Frank P. , Schwab Eileen F.
Published: October 2006

Three pharmacologically distinct high‐affinity [3H]serotonin ([3H]5‐HT) binding sites were identified in spinal cord synaptosomes. [3H]5‐HT competition studies using selective 5‐HT1A receptor ligands indicated that ∼25% of high‐affinity synaptosomal [3H]5‐HT binding was inhibited by 5‐HT1A‐selective compounds, an estimate consistent with [3H](±)‐8‐hydroxy‐2‐(di‐n‐propylamino)tetralin([3H]8‐OH‐DPAT) saturation experiments in which 5‐HT1A receptors were directly labeled. [3H]5‐HT competition studies using high‐affinity 5‐HT1B compounds performed in the presence of 100 nM 8‐OH‐DPAT (to block 5‐HT1A receptors) indicated that∼26% of all specific, high‐affinity [3H]5‐HT binding to spinal cord synaptosomes was to 5‐HT1B receptors. [3H]5‐HT competition studies performed in the presence of 100 nM 8‐OH‐DPAT and 10 nM RU 24969 (to block 5‐HT1A and 5‐HT1B receptors, respectively) indicated that the remaining 49% of [3H]5‐HT binding did not possess the phar‐macologic profile previous reported for 5‐HT1c, 5‐HT1D, 5‐ HT1E, 5‐HT2, or 5‐HT3 receptors. This residual 49% of [3H]5‐ HT binding to spinal cord synaptosomes observed in the presence of 100 nM 8‐OH‐DPAT and 10 nM RU 24969 (subsequently referred to as “5‐HTls”) displayed high affinity and saturability (KD= 4.7 nM) in association/dissociation and saturation experiments. Addition of 300 μM GTP or the nonhydrolyzable form of GTP, 5′‐guanylylimidodiphosphate, inhibited [3H]5‐HT binding to 5‐HT1S receptors in saturation experiments by 35 and 57%, respectively, whereas ATP was without effect. [3H]5‐HT competition studies identified several 5‐HT agonists and antagonists with a high or moderately high affinity for 5‐HT1s receptors. Although 5‐HT1s receptors were the predominant 5‐HT1 receptor subtype present in spinal cord, no significant density of 5‐HT1s receptors was found in brainstem or frontal cortex.
Dopaminergic Neurotoxicity In Vivo and Inhibition of Mitochondrial Respiration In Vitro by Possible Endogenous Pyridinium‐Like Substances
Authors: Sayre Lawrence M. , Wang Fengjiang , Arora Pramod K. , Riachi Naji J. , Harik Sami I. , Hoppel Charles L.
Published: October 2006

Elucidation of the mechanism(s) by which 1‐methyI‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and its active metabolite l‐methyl‐4‐phenylpyridinium (MPP+) cause parkinsonism in humans and other primates has prompted consideration of possible endogenous MPTP/MPP+‐like ncurotoxins in the etiology of idiopathic Parkinson's disease. Here we examined inhibition of mitochondrial respiration in vitro and neurotoxicity in rats in vivo produced by β‐carbolinium compounds that are presumed to form following Pictet‐Spengler cyclization of serotonin. We also evaluated N‐methyiisoquinolinium, a putative endogenous neurotoxin, in the same manner. The latter compound exhibited MPP+ like mitochondrial respiratory inhibition, whereas the β‐carbotinitim compounds, although more potent inhibitors of electron transport, exhibited weak accumulation‐ dependent enhancement of inhibition in intact mitochondria. It is interesting that the β‐carbolinium compounds inhibited succinate‐as well as glutamate‐supported respiration, and are best described as inhibitor‐uncouplers. The results of partitioning experiments suggest that both the low accumulation potential and the inhibition of succinate respiration may be a consequence of the β‐carboliniums being in equilibrium with neutral “anhydro” bases. Relative to MPP+, all compounds tested had weak dopaminergic uptake activity in vitro and weak dopaminergic toxicity in vivo, consistent with other findings of relatively low neurotoxic potential for presumed endogenous pyridiniums.
The Death Programme in Cultured Sympathetic Neurones Can Be Suppressed at the Posttranslational Level by Nerve Growth Factor, Cyclic AMP, and Depolarization
Authors: Edwards Susan N. , Buckmaster Anne E. , Tolkovsky Aviva M.
Published: October 2006

We have examined whether sympathetic neurones that have lost the potential to be rescued by protein and RNA synthesis inhibitors after a period of nerve growth factor (NGF) deprivation are irreversibly committed to die. We found that 15 h after withdrawal of NGF from 7‐day cultures of neonatal rat superior cervical ganglion neurones, 50% of the neurones lost the potential to be rescued by cycloheximide but that NGF rescued most of the neurones. By 22 h after NGF withdrawal, only 10% of the neurones were rescued by inhibition of macromolecular synthesis with cycloheximide, puromycin, or actinomycin D, but as many as 60‐80% of the neurones were rescued by NGF. This is after the time at which a DNA “ladder,” consistent with cell death by apoptosis, was first detected (18 h). As long as 27 h of NGF withdrawal was required before 50% of the neurones lost the potential to be rescued by NGF. The survival‐promoting agent 8‐(4‐chlorophenylthio)cyclic AMP (CPTcAMP) or depolarization with 50 mM K.C1 (HK) rescued neurones with kinetics similar to those of NGF, and rescue by all three agents did not require protein synthesis. Thus, NGF, CPTcAMP, and HK can rescue neurones deprived of NGF at much later times than either protein or RNA synthesis inhibitors by acting at the posttranslational level, a finding suggesting that initiation of the cell death programme in sympathetic neurones is not an irreversible step.
Expression of Various Microtubule‐Associated Protein 2 Forms in the Developing Mouse Brain and in Cultured Neurons and Astrocytes
Authors: Charrière‐Bertrand Cécile , Garner Craig , Tardy Marcienne , Nunez Jacques
Published: October 2006

Abstract: A cDNA probe specific to microtubule‐associated protein 2 (MAP2) was used to study the expression of the mRNAs encoding the high‐ and low‐molecular‐weight MAP2 variants in cultured neurons and astrocytes. The timing and relative abundance of these MAP2 transcripts and of their encoded proteins were also studied in the developing cerebral hemispheres and cerebellum of the mouse. A 9‐kb mRNA, known to encode high‐molecular‐weight MAP2, was expressed in cultured astrocytes, albeit at a lower level than in neurons. The 6‐kb transcript, recently shown to encode low‐molecular‐weight MAP2 (MAP2c), was expressed in neurons and was the predominant MAP2 transcript of the astrocytes. The level of the 9‐ and 6‐kb transcripts decreased at late stages of astroglial and neuronal cell culture. The 9‐kb mRNA was detected in the cerebellum and cerebral hemispheres at every developmental stage. Although the levels of this mRNA varied slightly in the cerebral hemispheres, its expression was biphasic in the cerebellum. This might be explained by the differences in timing of development of the various neuronal cell types formed in these two brain areas. The 6‐kb transcript was detected only at early developmental stages in the two brain areas. Correlating the temporal expression of the 9‐kb mRNA to that of high‐molecular‐weight MAP2 indicates that the accumulation of this protein is in part regulated at a cytoplasmic level.
Parallel Decrease of Glutamic Acid Decarboxylase and Preproenkephalin mRNA in the Rat Striatum Following Chronic Treatment with a Dopaminergic D1 Antagonist and D2 Agonist
Authors: Caboche Jocelyne , Vernier Philippe , Julien Jean‐François , Rogard Monique , Mallet Jacques , Besson Marie‐Jo
Published: October 2006

Abstract: The levels of mRNA encoding glutamic acid decarboxylase (GAD) and preproenkephalin (PPE) were measured by Northern blot analysis, in the dorsal and the ventral part of the striatum, following long‐term treatments with drugs acting selectively on D1 or D2 dopaminergic receptors. Chronic injection of the selective D1 antagonist SCH 23390 elicited a significant decrease in level of both GAD and PPE mRNA (−30%) in the dorsal striatum, whereas no significant change was observed in the ventral striatum. Chronic administration of both SCH 23390 and RU 24926, a D2 agonist, decreased the GAD and PPE mRNA levels in the dorsal (−38 and −57%, respectively) as well as in the ventral (−70 and −60%, respectively) striatum. In the ventral striatum the marked reduction of GAD mRNA levels was paralleled by a significant decrease of Vmax values of GAD enzymatic activity (−41%). These results suggest that the decrease in content of both GAD and PPE mRNA, promoted by the chronic blockade of D1 receptors, is mainly due to the action of dopamine acting on unaffected D2 receptors. Indeed, this decrease is further amplified when the D2 agonist and the D1 antagonist are administered together. Our results substantiate further the molecular mechanisms by which dopamine acts on different populations of GABAergic and enkephalinergic neurons in the two striatal regions examined.
Iron and Aluminum Increase in the Substantia Nigra of Patients with Parkinson's Disease: An X‐Ray Microanalysis
Authors: Hirsch E. C. , Brandel J.‐P. , Galle P. , Javoy‐Agid F. , Agid Y.
Published: October 2006

Abstract: The levels of different elements were studied by x‐ray microanalysis in the substantia nigra and the central gray substance of patients with Parkinson's disease, progressive supranuclear palsy, and matched controls. In control brains, only iron, potassium, silicium, sodium, sulfur, and zinc were within the limit of detection of the technique. The abundance of each element was different, but their respective concentrations in the two brain regions were similar, except for sulfur levels which were higher on neuromelanin aggregates in the substantia nigra than in nigral regions lacking neuromelanin, and in the central gray substance. In Parkinson's disease, but not in progressive supranuclear palsy, nigral iron levels increased in regions devoid of neuromelanin and decreased on neuromelanin aggregates, but were unchanged in the central gray substance, when compared to control values. Concentrations of the other elements in the central gray substance and substantia nigra were not different from controls in brains from patients with Parkinson's disease and progressive supranuclear palsy. Analysis of Lewy bodies in the parkinsonian substantia nigra revealed high levels of iron and the presence of aluminum. Metal abundance was not affected in progressive supranuclear palsy, in spite of the nigral cell death. This suggests that the increased iron levels and the detection of aluminum observed in Parkinson's disease are not solely the consequence of the neuronal degeneration.
Effect of Varying the Ionic Concentration of a Microdialysis Perfusate on Basal Striatal Dopamine Levels in Awake Rats
Authors: Osborne P. G. , O'Connor W. T. , Ungerstedt U.
Published: October 2006

Abstract: In vivo microdialysis was used to study the effects of Ca2+, Mg2+, and K+ ion concentrations on basal extracellular (EC) levels of striatal DA and metabolites in awake rats on the second day (48 h) after implantation of a microdialysis probe. Basal EC striatal dopamine (DA) levels were markedly (90%) and reversibly reduced by removal and subsequent replacement of Ca2+ ions from the microdialysis perfusate. This implies that the EC DA in this preparation is primarily of synaptic origin. The addition and subsequent removal of 1.7 mM MgCl2 to the Mg2+‐free perfusate produced a reversible decrease (20%) in basal EC DA levels. This decrease may reflect a competitive interaction between Ca2+ and Mg2+ in the process of vesicular release. Basal EC DA levels were also reduced (27%) by decreasing the K+ concentration of the perfusate from 4 mM to 3 mM. However, after restoring the K+ concentration to 4 mM, EC DA levels were slow to return to pretreatment levels. Basal EC 3,4‐dihydroxyphenylacetic acid and homovanillic acid levels exhibited a parallel but diminished response to each manipulation of the ionic concentration of the perfusate. This study demonstrates that small variations in the concentrations of Ca2+, Mg2+, and K+ in the perfusate employed in microdialysis preparations will affect basal EC striatal DA and metabolite levels.
Developmental Regulation of β‐Thymosins in the Rat Central Nervous System
Authors: Lugo Delia Ines , Chen Shu‐Cheng , Hall Alan K. , Ziai Reza , Hempstead James L. , Morgan James I.
Published: October 2006

Abstract: HPLC analysis of guanidinium hydrochloride extracts of neonatal and adult rat brain revealed a polypeptide that is present in high concentration in the immature nervous system, but whose levels decline dramatically in the adult. This polypeptide has been isolated and its complete amino acid sequence determined by gas‐phase Edman degradation following specific chemical and enzymatic cleavages. The molecule is identified as thymosin β10, a member of a multigene family that encodes a structurally conserved series of small acidic polypeptides of uncertain function. Thymosin β10 is present in the developing nervous system as early as embryonic day 9. Levels subsequently increase to peak values between embryonic day 15 and postpartum day 3, before falling to adult values (about a 20‐fold reduction) by postpartum day 14. The elevated levels of thymosin β10 in fetal and neonatal brain correlate with high levels of thymosin β10 mRNA, whereas the low values of the polypeptide in the adult and juvenile are mirrored by an approximate 15‐fold reduction in specific mRNA. In comparison, the levels of thymosin β4 polypeptide, a homologue of thymosin β10, only decline by about 20% during the same developmental period. However, the mRNA encoding thymosin β4 is elevated in fetal brain, and its levels decrease approximately four‐fold to a stable value around the time of birth. The reason for this discrepancy between thymosin β4 protein and mRNA levels is unknown. Thymosin β10 can also be detected by HPLC in fetal liver, where levels are approximately 5% of those in brain. In liver, thymosin β10 also declines following birth. It is concluded that β‐thymosin expression (as measured by steady‐state mRNA and polypeptide levels) is both up‐and down‐regulated during different phases of maturation of the mammalian nervous system.
Retinoic Acid Regulates Thymosin β Levels in Rat Neuroblastoma Cells
Authors: Hall Alan K. , Chen Shu‐Cheng , Hempstead James L. , Morgan James I.
Published: October 2006

Abstract: A small acidic polypeptide, termed thymosin β10, has been identified and is present in the nervous system of the rat by the ninth day of gestation. Thymosin β10 levels rise during the remaining days of life in utero, and then decline to nearly undetectable values between the second and fourth week post partum. The present study investigates the possible developmental signals and mechanisms that might regulate the expression of thymosin β10 during neuroembryogenesis. Many cell lines derived from tumors of the central nervous system express thymosin β10, as well as its homologue gene product, thymosin β4. Because some of these cell lines respond to exogenously applied agents by increasing their apparent state of differentiation, we have determined whether thymosin β10 levels are coordinately modulated. In several neuroblastomas, including the B103 and B104 lines, retinoic acid elicits a time‐ and dose‐dependent increase in the content of thymosin β10, but not that of thymosin β4. The increase in thymosin β10 polypeptide is associated with a marked increase in the specific mRNA encoding this molecule. The mRNA for thymosin β4 is unaffected by retinoic acid. This is in contrast with the situation in vivo, where the expression of both genes decreases after birth. Other agents that influence the morphology of B104 cells, such as phorbol esters and dibutyryl cyclic AMP, have no influence on β‐thymosin levels. A range of steroids, which like retinoids act upon nuclear receptors, was also inactive. The stimulatory action of retinoic acid is detectable within 4 h, and thymosin β10 peptide levels continue to rise for at least 4 days. The influence of the isoprenoid is fully reversible and exhibits structural specificity. We believe that this culture system is mimicking the early rising phase of thymosin β10 levels in brain and that endogenous retinoids may be candidate physiological regulators of this gene.
[3H]Harman Binding Experiments. II: Regional and Subcellular Distribution of Specific [3H]Harman Binding and Monoamine Oxidase Subtypes A and B Activity in Marmoset and Rat
Authors: May Torsten , Pawlik Monika , Rommelspacher Hans
Published: October 2006

Abstract: [3H]Harman (1‐[3H]methyl‐β‐carboline) was used in a novel radioligand binding assay to label selectively and with high affinity monoamine oxidase (MAO) type A. The concentration of the enzyme was determined in six CNS regions of the primate species marmoset (Callithrix jacchus) and of the rat: hypothalamus, hippocampus, cerebellum, cerebral cortex, striatum, and spinal cord. The specific [3H]harman binding in the CNS of the marmoset reveals the same pharmacological profile and other characteristics (affinity, saturability, and reversibility) as in the CNS of the rat. The regional distribution of the [3H]harman binding density (Bmax) in the CNS exhibits a distinct pattern in the marmoset and the rat and a 35 (hypothalamus) to 75% (hippocampus) lower Bmax in the marmoset than in the rat. The Bmax values of [3H]harman binding in the CNS of the marmoset and the rat combined as well as those from visceral organs of the rat (liver, heart, lung, thymus, spleen, and kidney) correlated positively and highly significantly with the respective Vmax values of specific MAO activity of the A type but not of the B type, determined with kynuramine as the substrate. In subcellular fractionation experiments with rat cerebral cortex, the highest [3H]harman binding density (Bmax) and MAO‐A activity (Vmax) were detected in mitochondrial fractions and severalfold lower values in the synaptosomal membrane fraction. In conclusion, we suggest that [3H]harman binding is a biochemical tool as a selective marker to quantify MAO‐A in the CNS of different mammalian species as well as in extraneuronal tissues.
Relationship Between [3H]Mazindol Binding to Dopamine Uptake Sites and [3H]Dopamine Uptake in Rat Striatum During Aging
Authors: Shimizu Ikuya , Prasad Chandan
Published: October 2006

Abstract: Certain drugs exhibit a remarkable correlation between their ability to inhibit synaptosomal uptake of dopamine and the binding of [3H]mazindol to striatal membranes. To investigate the role of mazindol binding sites in the dopamine uptake process and the fate of these sites (labeling dopaminergic neurons) during aging, we have examined the properties of mazindol binding and dopamine uptake in individual young and old rats. There was a 48% decrease (p= 0.0001) in the Bmax of mazindol binding and a 23% decrease (p= 0.0166) in the Vmax of dopamine uptake with no apparent change in their affinities with age. Regression analysis of the relationship between Bmax and Vmax exhibited a significant correlation in old (p= 0.0156) but not young rats (p= 0.1398). These data suggest that the number of mazindol binding sites decreases with age and that the number of sites on the dopamine transporter complex far exceeds the number required to elicit maximal dopamine uptake.
Adrenal Medullary Transplants Increase Spinal Cord Cerebrospinal Fluid Catecholamine Levels and Reduce Pain Sensitivity
Authors: Sagen Jacqueline , Kemmler James E. , Wang Hong
Published: October 2006

Abstract: Previous work in this laboratory has shown that adrenal medullary transplants into the spinal cord subarachnoid space can reduce pain sensitivity. This analgesia most likely results from the release of neuroactive substances, particularly catecholamines and opioid peptides, from the transplanted cells into the CSF of the spinal cord, since it can be attenuated or blocked by α‐adrenergic or opiate antagonists. The purpose of the present study was to more directly measure the release of catecholamines from adrenal medullary transplants in the spinal cord CSF using a spinal superfusion technique. CSF samples from rats with 6‐month‐old transplants were assayed for catecholamines using HPLC with electrochemical detection. Results indicated that norepinephrine levels were increased threefold, and epinephrine levels nearly 100‐fold, in animals with adrenal medullary transplants compared with control transplanted animals. There was no apparent increase in dopamine levels. Furthermore, the increased levels of total catecholamines were correlated with decreased pain sensitivity. Results of this study indicate that adrenal medullary transplants can survive for long periods in the rat spinal CSF and continue to release high levels of catecholamines. Together, the release of catecholamines and opioid peptides from adrenal medullary transplants may provide the ideal combination for the reduction of pain.
Regulation by Interleukin‐1 of Nerve Growth Factor Secretion and Nerve Growth Factor mRNA Expression in Rat Primary Astroglial Cultures
Authors: Čarman‐Kržan Marija , Vigé Xavier , Wise Bradley C.
Published: October 2006

Abstract: Primary cultures of neonatal rat cortical astrocytes contain low cellular levels (about 2 pg/mg of protein) of nerve growth factor (NGF), but secrete significant amounts of NGF into the culture medium (about 540 pg of NGF/mg of cell protein/38‐h incubation). Incubation of astrocytes with interleukin‐1 (IL‐1) increased the cellular content of NGF and the amount secreted by about threefold. In comparison, cerebellar astrocytes secreted significant amounts of NGF, and the secretion was also stimulated by IL‐1. The stimulatory action of IL‐1 on astrocytes prepared from cortex was dose‐and time‐dependent. Concentrations of IL‐1 causing half‐maximal and maximal stimulation of NGF secretion were 1 and 10 U/ml, respectively. Maximal NGF secretion induced by IL‐1 (10 U/ml) was seen following 38 h of incubation. The basal secretion of NGF was reduced by about 50% under Ca2+‐free conditions; however, the percent stimulation of NGF secretion by IL‐1 was the same in the absence or presence of Ca2+. The stimulatory action of IL‐1 was specific, because other glial growth factors and cytokines were almost ineffective in stimulating NGF secretion from cortical astroglial cells. IL‐1 treatment also increased cellular NGF mRNA content twofold. The results indicate that IL‐1 specifically triggers a cascade of events, independent of cell growth, which regulate NGF mRNA content and NGF secretion by astrocytes.
Neurotensin Regulation of Endogenous Acetylcholine Release from Rat Striatal Slices Is Independent of Dopaminergic Tone
Authors: Lapchak Paul A. , Araujo D. M. , Quirion R. , Beaudet A.
Published: October 2006

Abstract: The effects of neurotensin (NT) alone or in combination with the dopamine antagonist sulpiride were tested on the release of endogenous acetylcholine (ACh) from striatal slices. NT enhanced potassium (25 mM)‐evoked ACh release from striatal slices in a dose‐dependent manner. This effect was tetrodotoxin‐insensitive, suggesting an action directly on cholinergic elements. The dopamine antagonist sulpiride (5 × 10−5M) significantly increased (63%) potassium‐evoked ACh release from striatal slices; potassium‐evoked ACh release was further increased (90%) in the presence of NT (10−5M) and sulpiride (5 × 10−5M). The second set of experiments tested the effects of 6‐hydroxydopamine (6‐OHDA) lesions of the substantia nigra on NT‐induced increases of potassium‐evoked ACh release. These lesions did not alter the NT regulation of potassium‐evoked ACh release from striatal slices, but did significantly increase spontaneous (33%) and potassium‐evoked (40%) ACh release from striatal slices. Striatal choline acetyltransferase activity was not affected by 6‐OHDA lesions. In addition, following 6‐OHDA lesions, sulpiride was ineffective in altering ACh release from striatal slices. Furthermore, evoked ACh release in the presence of the combination of NT and sulpiride was not different from that in the presence of NT alone. These results suggest that in the rat striatum, NT regulates cholinergic interneuron activity by interacting with NT receptors associated with cholinergic elements. Moreover, the NT modulation of cholinergic activity is independent of either an interaction of NT with D2 dopamine receptors or the sustained release of dopamine.
GTP and Ca2+ Modulate the Inositol 1,4,5‐Trisphosphate‐Dependent Ca2+ Release in Streptolysin O‐Permeabilized Bovine Adrenal Chromaffin Cells
Authors: Föhr K. J. , Ahnert‐Hilger G. , Stecher B. , Scott J. , Gratzl M.
Published: October 2006

Abstract: The inositol 1,4,5‐trisphosphate (IP3)‐induced Ca2+ release was studied using streptolysin O‐permeabilized bovine adrenal chromaffin cells. The IP3‐induced Ca2+ release was followed by Ca2+ reuptake into intracellular compartments. The IP3‐induced Ca2+ release diminished after sequential applications of the same amount of IP3. Addition of 20 μM GTP fully restored the sensitivity to IP3. Guanosine 5′‐O‐(3‐thio)triphosphate (GTPγS) could not replace GTP but prevented the action of GTP. The effects of GTP and GTPγS were reversible. Neither GTP nor GTPγS induced release of Ca2+ in the absence of IP3. The amount of Ca2+ whose release was induced by IP3 depended on the free Ca2+ concentration of the medium. At 0.3 μM free Ca2+, a half‐maximal Ca2+ release was elicited with ∼0.1 μM IP3. At 1 μM free Ca2+, no Ca2+ release was observed with 0.1 μM IP3; at this Ca2+ concentration, higher concentrations of IP3 (0.25 μM) were required to evoke Ca2+ release. At 8 μM free Ca2+, even 0.25 μM IP3 failed to induce release of Ca2+ from the store. The IP3‐induced Ca2+ release at constant low (0.2 μM) free Ca2+ concentrations correlated directly with the amount of stored Ca2+. Depending on the filling state of the intracellular compartment, 1 mol of IP3 induced release of between 5 and 30 mol of Ca2+.
Morphological, Biochemical, and Functional Characterization of Bulk Isolated Glial Progenitor Cells
Authors: Lubetzki C. , Goujet‐Zalc C. , Gansmüller A. , Monge M. , Brillat A. , Zalc B.
Published: October 2006

Abstract: We describe a simple, rapid, and efficient method, based on separation on a Percoll centrifugation gradient, to purify glial progenitor cells from newborn rat brains. Cytofluorimetry analysis of the isolated cell population showed that 75 ± 8 and 86 ± 7% of the cells were A2B5‐ and R24‐positive, respectively. Transmission electron microscopy examination of the purified cell population confirmed their homogeneity and illustrated their typical morphology, as previously described in situ. Assay of UDP‐galactose‐ceramide galactosyltransferase, 3′‐phosphoadenosine 5′‐phosphosulfate galactosylceramide sulfotransferase, and 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase activities showed that the levels of these enzymes were 446, 76, and 11 times lower, respectively, than the levels measured in mature oligodendrocytes. Low levels of mRNA coding for 2′,3′‐cyclic nucleotide 3′‐phosphohydrolase and myelin proteolipid protein, but not for myelin basic protein, were present in the glial progenitor cells. At the time of isolation, 40% of the cells in the population were dividing, and the cells could easily be expanded in culture. After 3 weeks of culture in the presence of 1% fetal calf serum, 75% of the cells had differentiated into galactosylceramide‐positive oligodendrocytes. When the culture took place in the presence of 10% fetal calf serum, only 2% of the cells expressed galactosylceramide, and 60% were glial fibrillary acidic protein‐positive astrocytes; half of them were also A2B5 positive.
Generation of Two Forms of the γ‐Aminobutyric AcidA Receptor γ‐2‐Subunit in Mice by Alternative Splicing
Authors: Kofuji Paulo , Wang Jia Bei , Moss Stephen J. , Huganir Richard L. , Burt David R.
Published: October 2006

Abstract: γ‐Aminobutyric acidA (GABAA) receptors are multisubunit ligand‐gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (α, β, γ, and δ). The γ2‐subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine γ2‐subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids, LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the γ2‐subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight‐amino‐acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.
Evidence for Nonrandom Distribution of GD1 a Ganglioside in Rabbit Brain Microsomal Membranes
Authors: Palestini P. , Masserini M. , Fiorilli A. , Calappi E. , Tettamanti G.
Published: October 2006

GD1a is the major ganglioside of rabbit brain microsomal membranes and occurs mainly with two molecular species, containing the C18:1 (62.3%) and C20:1 (37.7%) long‐ chain bases. The membranes were exposed to Vibrio cholerae (VC) sialidase under conditions where the enzyme hydrolyzed only GD1a(∼9%), producing GM1 ganglioside, whereas the other gangliosides remained virtually unaffected. The long‐ chain‐base analysis showed that newly‐formed GM1 contained ∼68% of the C20:1 molecular species. This indicates that VC sialidase did not randomly affect the two molecular species of GD1a but hydrolyzed preferentially the C20:1 one. In similar experiments, GD1a was inserted into the external layer of phosphatidylcholine vesicles and incubated with VC sialidase under conditions producing ∼ 10% hydrolysis. Long‐ chain‐base analysis showed that the proportion of C20:1 species in GM1 was 25.1% using vesicles composed of dipal‐mitoylphosphatidylcholine and 42.3% with egg phosphatidylcholine, whereas it was 39.2% in the starting GD1a. Therefore, in artificial membranes, VC sialidase acted preferentially on the C18:1 or C20:1 molecular species, depending on the length and unsaturation of the phospholipid fatty acids. Because VC sialidase is known to affect molecular dispersions more easily than packed aggregations of the gangliosidic substrate, the data suggest that in rabbit brain microsomal membranes the GD1a ganglioside molecular species carrying C20:1 long‐chain base are more molecularly dispersed than those containing C18:1 long‐chain base.
Differential Turnover Rates of D1 Dopamine Receptors in the Retina and in Distinct Areas of the Rat Brain
Authors: Giorgi O. , Pibiri M. G. , Biggio G.
Published: October 2006

The present study was designed to examine the steady‐state density and the turnover rates of D1 dopamine (DA) receptors in the striatum, nucleus accumbens, substantia nigra, and retina of the rat. The turnover rates were measured by monitoring the repopulation kinetics of D1 DA receptors labelled with [3H]SCH 23390 after the irreversible inactivation induced by a single dose of N‐ethoxycarbonyl‐2‐ethoxy‐ 1, 2‐dihydroquinoline (10 mg/kg s.c.). The repopulation of D1 DA receptors could be described adequately in all the neural tissues investigated by a theoretical model that assumes a constant rate of receptor production (i.e., zero order) and a rate of degradation that is dependent on the receptor density at any time (i.e., first order). The quantitative analysis of the experimental data using this theoretical model revealed significant regional differences in the rates of receptor production and degradation. Thus, the receptor production rates determined in the nucleus accumbens and striatum (8.03 and 9.96 fmol/mg of protein/h, respectively) were four‐ to sixfold larger than those measured in the substantia nigra (1.80 fmol/mg of protein/h) and retina (1.50 fmol/mg of protein/h). On the other hand, the receptor degradation rates in the striatum, nucleus accumbens, and retina (0.0093 h−1, 0.0110 h−1, and 0.0123 h−1, respectively) were 2.6–3.5‐fold larger than the receptor degradation rate in the substantia nigra (0.0035 h−1).