bluuebunny/crossref_metadata_2025 huggingface dataset:
Retinoic acid regulates the development of oligodendrocyte precursor cells in vitro
Authors: Laeng P. , Décimo D. , Pettmann B. , Janet T. , Labourdette G.
Published: January 2005

Cultures of oligodendrocyte precursor cells can be grown from brain hemispheres of newborn rats. These cells, also called O‐2A progenitor cells, can differentiate in vitro into oligodendrocytes or type 2 astrocytes. Basic FGF and PDGF are known to stimulate their proliferation and delay their differentiation. Lack or excess of retinoic acid (RA) has been known for a long time to alter brain development suggesting that this compound is involved in normal brain development. Here we report that RA partially inhibits both the proliferation and the differentiation of oligodendrocyte precursor cells. It also down‐regulates the mitogenic effect of bFGF on these cells while keeping them in an immature stage. RA is more effective than bFGF in inhibiting myelin basic protein mRNA expression in these cells, and like bFGF, it preserves their bipotential character. RA nuclear receptors RAR‐α and their transcripts are expressed in oligodendrocyte precursor cells as seen by Western blot, Northern blot and in situ hybridization. The expression of RAR‐α transcripts is stimulated transiently by RA alone or associated to bFGF. The expression of RAR‐β transcripts is not constitutive and is induced by RA alone or associated to bFGF and to a lesser extent by bFGF alone. These results suggest that retinoids participate in the control of the development of glial cells of the oligodendrocyte lineage. © 1994 Wiley‐Liss, Inc.
Supraspinal delta opioid receptor mRNA levels are not altered in [D‐Ala2]Deltorphin II tolerant mice
Authors: Kest B. , Jenab S. , Brodsky M. , Elliott K. , Inturrisi C. E.
Published: January 2005

Delta opioid receptor (DOR) mRNA levels were studied in mice rendered tolerant to [D‐Ala2]deltorphin II by 4 days of repeated intracerebroventricular administration (10μg, [tid]). ED50 determinations on day 5 revealed a 10‐fold loss in [D‐Ala2]deltorphin II potency with the tail‐flick test. Utilization of a microdissection technique followed by quantitative solution hybridization of RNA extracts from mouse brain revealed mean levels of DOR mRNA ranging from 3.9 pg/μg RNA in the caudate‐putamen to 0.4 pg/μg RNA in the cerebellum. DOR mRNA levels were not different when RNA extracts from tolerant and nontolerant mice were compared. These data suggest that altered DOR mRNA levels are not one of the adaptive changes that occur with delta opioid ([D‐Ala2]deltorphin II) tolerance. © 1994 Wiley‐Liss, Inc.
Age‐related increases in glial fibrillary acidic protein do not show proportionate changes in transcription rates or DNA methylation in the cerebral cortex and hippocampus of male rats
Authors: Laping N. J. , Teter B. , Anderson C. P. , Osterburg H. H. , O'Callaghan J. P. , Johnson S. A. , Finch C. E.
Published: January 2005

Age‐related increases in the expression of glial fibrillary acidic protein (GFAP) in many brain regions are observed in short‐ and long‐lived mammals. Possible genomic mechanisms for the increase of GFAP mRNA and protein were studied in the hippocampus and cortex of male F344 rats and a longer‐lived hybrid F1(F344 × Brown Norway). No age‐related changes were found in the extent of cytosine methylation at 19 CpG sites in the 5′‐upstream GFAP promoter and in exon 1. With the nuclear runon assay, no change was found in the transcription rate of GFAP in the cerebral cortex or hippocampus. Thus, age‐related increases in GFAP are not associated with proportionate changes in transcription rates or DNA methylation. However, the transcription of glutamine synthetase was increased by about 60%. These findings contrast with age‐related loss of bulk tissue DNA methylation and decreased transcription rates of other genes reported in non‐neural tissues. © 1994 Wiley‐Liss, Inc.
NGF protects PC12 cells against ischemia by a mechanism that requires the N‐kinase
Authors: Boniece I. R. , Wagner John A.
Published: January 2005

Nerve growth factor (NGF), which has been shown to act as a morphological and neurochemical differentiating factor in PC12 cells, also protects PC12 cells from the toxicity of serum withdrawal and ischemia. By using a previously established in vitro model of ischemia, which incorporates the combination of anoxia with glucose deprivation (Boniece and Wagner: J Neurosci 13:4220–4228, 1993), we have been able to study the signal transduction pathways upon which NGF‐induced survival is dependent. Here we demonstrate that inhibitors of the N‐kinase and NGF‐induced neuritogenesis, 6‐thioguanine and 2‐aminopurine, prevent the protective effects of NGF, while they have little, if any, effect on the protection conferred by epidermal growth factor (EGF) or dbcAMP. This suggests that only NGF acts by a mechanism that depends strongly on the N‐kinase. Furthermore, the methyltransferase inhibitor 5′‐deoxy‐5′‐methylthioadenosine (MTA), which also inhibits NGF‐induced neuritogenesis, inhibits the protective effect of NGF but not the protective effects of EGF or dbcAMP. Thus, the neuroprotective effect of NGF requires some of the same signal transduction steps used by NGF to promote differentiation and neurite formation. Furthermore, we found that exposure of PC12 cells to retinoic acid, which promotes the differentiation and inhibits the growth of PC12 cells, also improves cell survival during ischemia. In addition, a combination of NGF and retinoic acid was more effective than either agent alone. It is likely that these two agents confer protection by independent pathways. © 1995 Wiley‐Liss, Inc.
Hypoosmotic volume regulation of astrocytes in elevated extracellular potassium
Authors: Olson James E. , Alexander C. , Feller D. A. , Clayman M. L. , Ramnath E. M.
Published: January 2005

Cellular volume and potassium contents were determined in rat astrocytes from primary culture following suspension in isoosmotic (269 mOsm) and hypoosmotic (136 mOsm) phosphate‐buffered saline (PBS) containing various potassium concentrations. Within 1 min of suspension in hypoosmotic PBS, cells swelled to 135% of their volume in isoosmotic PBS. This initial swelling was not altered by varying the potassium concentration of the hypoosmotic PBS. After suspension in hypoosmotic PBS containing 3.2 mM potassium, a regulatory volume decrease (RVD) was observed. Higher concentrations of potassium in hypoosmotic PBS inhibited RVD following osmotic swelling. Cells swollen in hypoosmotic PBS containing 50 mM potassium continued to swell for 7 min, reaching a volume of 141 % of their initial isoosmotic volume. After 7 min, these cells demonstrated a subsequent decrease in volume. The swelling observed between 1–7 min after suspension in hypoosmotic PBS containing 50 mM potassium was not affected by 10 μM gadolinium, 1 mM quinine, 1mM DIDS (4,4′‐diisothiocyanato‐2,2′‐stilbenedisulfonic acid), 1 mM SITS (4‐acetamido‐4′‐isothiocyanato‐2,2′‐stilbenedisulfonic acid), 1 mM furosemide, or 100 μM bumetanide. Normal RVD was obtained in hypoosmotic PBS containing 50 mM potassium, if chloride was replaced with gluconate (but not nitrate) to reduce the extracellular K‐C1 product to that of hypoosmotic PBS containing 3.2 mM potassium. The volume decrease seen between 7–30 min after exposure to hypoosmotic PBS containing 50 mM potassium was blocked by 1 mM DIDS, 1 mM SITS, or 1 mM furosemide. Cellular potassium content was elevated by approximately 60% after 7 min exposure to isoosmotic or hypoosmotic PBS containing 50 mM potassium. In hypoosmotic PBS, this increase in cellular potassium was reduced with replacement of chloride by gluconate, but not by nitrate. The results indicate that astrocytes swollen in PBS containing elevated potassium concentrations continue to swell, in part, by accumulation of potassium plus chloride mediated by an approach to Donnan equilibrium. Cotransport carriers or stretch‐activated channels do not play a role in the enhanced swelling observed in hypoosmotic PBS containing 50 mM potassium. We suggest that a voltage‐sensitive chloride channel mediates this continuation of cell swelling. This mechanism may be important in the persistent swelling of astrocytes observed in pathologic conditions such as trauma and seizures where extracellular potassium is elevated, or when other factors are present which may cause astroglial depolarization. © 1995 Wiley‐Liss, Inc.
Characterization and localization of the BM88 antigen in the developing and adult rat brain
Authors: Patsavoudi E. , Merkouri E. , Thomaidou D. , Sandillon F. , Alonso G. , Matsas R.
Published: January 2005

Monoclonal antibody BM88 identifies a neuron‐specific antigen (BM88 antigen) present in the central and peripheral nervous system of the pig (Patsavoudi et al.: Neuroscience 30:463–478, 1989; J Neurochem 56:782–788, 1991). We have previously shown that the antigen is also expressed by cultured neurons derived from newborn rat brain. In the present study we have used the monoclonal antibody BM88 and a specific polyclonal antibody in order to identify the nature of the cross‐reactive antigen in rat brain and to investigate its expression and cellular localization in the developing and adult rat nervous system. Western blot analysis and immunocytochemistry revealed that the rat BM88 antigen displays very similar biochemical properties with its porcine homologue. It is a neuron‐specific integral membrane protein, apparently not glycosylated, consisting of two 23 kD polypeptide chains. Immunoperoxidase staining demonstrated that the BM88 antigen is widely distributed in the brain of 19‐day‐old rat embryos. At this stage, immunoreactivity was particularly prominent in differentiated cellular areas and developing fiber tracts of the embryonic rat brain, but was also present in the neuroepithelium. A similar wide distribution of the BM88 antigen was observed in the adult rat brain. Here, immunoreactivity was detected in the neuropil and neuronal perikarya. Immunocytochemical analysis of the expression of the BM88 antigen during postnatal development of the cerebellar cortex showed that this molecule is particularly concentrated in the Purkinje cells between postnatal days 10 to 15; their somata and developing dendrites were distinctly immunopositive during this period. An agedependent increase in the expression of the BM88 antigen both in brain and in the cerebellum was noted. Electron microscopy confirmed the presence of the BM88 reaction product within the perikarya, axons and dendrites of labeled neurons in the adult brain. The BM88 reaction product was preferentially associated with the limiting membrane of mitochondria, endoplasmic reticulum and small electron‐lucent vesicles, but was also present in the plasma membrane, especially at the level of synaptic densities. Our results show that the BM88 antigen participates in an activity common to all or most neurons, and demonstrate that the expression of this antigen is elevated upon neuronal differentiation and maturation. © 1995 Wiley‐Liss, Inc.
A potential interaction of p75 and trkA NGF receptors revealed by affinity crosslinking and immunoprecipitation
Authors: Huber L. J. , Chao M. V.
Published: January 2005

Nerve growth factor binds independently to two transmembrane receptors, the p75 neurotrophin receptor and the p140trk (trkA) tyrosine kinase receptor, which are both co‐expressed in the majority of neuronal cells that respond to NGF. Previous findings have suggested that appropriate co‐expression of the two receptors gives rise to high affinity NGF binding sites and increased neurotrophin responsiveness; however, evidence demonstrating a direct interaction between the two receptors in cell lines has been lacking. Here we have utilized affinity crosslinking agents with 125I‐NGF to detect an association of trkA and p75 receptors in embryonic spinal cord and brain tissues enriched in the two receptors. Although multimeric complexes of trkA and p75 were not detected by affinity crosslinking, immunoprecipitation of cross‐linked NGF‐receptor complexes with trk‐specific antibodies resulted in selective immunoprecipitation of crosslinked p75. Our results indicate that the trkA and p75 receptors can potentially interact, and that such an association may be responsible for the generation of high affinity NGF binding sites. © 1995 Wiley‐Liss, Inc.
Bovine opsin gene expression exhibits a late fetal to adult regulatory switch
Authors: DesJardin L. E. , Lockwood M. K. , Hauswirth William W.
Published: January 2005

Rates of bovine photoreceptor gene transcription, as measured by nuclear run‐on assays, exhibit gene‐specific patterns of regulation. Here we investigate initiation and elongation in nuclear run‐on assays with the use of sarkosyl to further understand the nature of these gene‐specific elements. Opsin transcription, alone among several genes tested, proved sarkosyl‐sensitive. This sensitivity is maximal in adult retinas, with inhibition first detected in mid‐third trimester fetal retinas. Therefore, opsin transcription appears to involve different regulatory elements in adult and fetal retinas, implying a fetal to adult switch in the control of opsin gene expression. Although this regulatory switch is initially activated at a time when the fetal outer nuclear layer of the retina first achieves adult‐like morphology, further maturation of opsin regulation takes place postpartum since levels of sarkosyl sensitivity are almost 5‐fold greater in adult retinas compared to the 7.5 month fetus. We also show that the sarkosyl‐induced reduction of opsin transcripton is not due to prevention of de novo RNA polymerase II initiation in the run‐on reaction, suggesting the detergent alters a positive‐acting, postinitiation component of the transcriptional apparatus. Since levels of opsin transcription with sarkosyl are similar to those of the other visual transduction genes with or without sarkosyl, this detergent‐sensitive transcriptional component appears to account for the singularly high, gene‐specific rate of opsin transcription in retinal photoreceptor cells. © 1995 Wiley‐Liss, Inc.
Analysis of proteoglycan expression in developing chicken brain: Characterization of a heparan sulfate proteoglycan that interacts with the neural cell adhesion molecule
Authors: Burg M. A. , Halfter W. , Cole Gregory J.
Published: January 2005

In the present study we have characterized the major proteoglycans of chick brain, focusing on their pattern of expression in development and on identifying the heparan sulfate proteoglycan (HSPG) that binds to the neural cell adhesion molecule (NCAM). The major chondroitin sulfate proteoglycans (CSPG) are a heterogeneous group of molecules with an average MW of 450 kDa. Protein core analysis reveals multiple protein cores between 100 and 350 k Da, The HSPGs are somewhat smaller, with an average MW of 350 kDa, and the major brain HSPG possesses a 250 kDa protein core. During development the relative percentage of HSPG decreases from approximately 50% of total sulfate‐labeled PG at E6 to 25% by E10. In order to begin to characterize the HSPG that interacts with NCAM, we initially used an antiserum produced against a HSPG which was previously shown to copurify with NCAM (Cole and Burg: Exp Cell Res 182:44‐60, 1989). This antiserum immunoprecipitated a HSPG core protein of 250 kDa, corresponding to the major HSPG of chick brain. We also show that the major brain HSPG binds to a synthetic peptide that encodes the heparan sulfate‐binding domain of NC AM, and that monoclonal antibodies to a recently identified chick retinal HSPG recognize this NCAM‐binding HSPG. This HSPG was immunopurified from E10 chick brain using the 6D2 monoclonal antibody, and has been shown to bind an affinity column containing the heparan sulfate‐binding peptide of NCAM. Consistent with its ability to bind NCAM, we show that the intact 6D2 HSPG inhibits cell adhesion to a HBD peptide substratum, and also binds chick brain cells when employed as a substratum. © 1995 Wiley‐Liss, Inc.
Spontaneously immortalized adult mouse Schwann cells secrete autocrine and paracrine growth‐promoting activities
Authors: Watabe K. , Fukuda T. , Tanaka J. , Honda H. , Toyohara K. , Sakai O.
Published: January 2005

We established spontaneously immortalized Schwann cell lines from long‐term cultures of adult mouse dorsal root ganglia and peripheral nerves. One of the cell lines, designated IMS32, responded to mitogenic stimuli by platelet‐derived growth factor (PDGF)‐BB, acidic and basic fibroblast growth factors (aFGF, bFGF), and transforming growth factors (TGF)‐b̃1 and ‐b̃2, as determined by bromodeoxyuridine (BrdU) incorporation and double immunofluorescence for S100 and BrdU. Furthermore, conditioned media (CM) obtained from IMS32 cells showed mitogenic activity for both IMS32 cells and long‐term cultured Schwann cells. Western blot analysis revealed TGF‐b̃‐like molecule in the CM, and the activity was absorbed with anti‐TGF‐b̃ neutralizing antibody. Reverse transcription followed by polymerase chain reaction (RT‐PCR) of IMS32 RNA revealed that these cells expressed TGF‐b̃1, b̃2, and ‐b̃3 transcripts. When rat pheochromocytoma PC 12 cells were incubated with the CM, they developed neurite growth. Coculture of PC12 and IMS32 cells also showed neurite growth of PC12 cells. RNA transcripts of nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), neurotrophin‐3 (NT3), ciliary neurotrophic factor (CNTF), and glial cell line‐derived neurotrophic factor (GDNF) were detected from IMS32 cells by RT‐PCR. In these, we sequenced the mouse GDNF cDNA coding region and observed 97% and 90% homologies to corresponding rat and human cDNA sequences, respectively. These results indicate that the immortalized Schwann cell line mitotically responds to various growth factors and secretes autocrine and paracrine growth‐promoting activities in vitro. © 1995 Wiley‐Liss, Inc.
Protein kinase C modulates exogenous acetylcholine current in Xenopus oocytes
Authors: Mileo A. M. , Palma E. , Polenzani L. , Limatola C. , Grassi F. , Eusebi F.
Published: January 2005

The modulation of acetylcholine‐activated current (IACh) by protein kinase C (PKC) was studied in Xenopus laevis oocytes microinjected with either mRNA extracted from C2C12 myotubes (C2C12 mRNA) or RNAs encoding murine αβγδ subunits of the nicotinic ACh receptor (nAChR). Voltage‐clamped oocytes were treated for 90 sec with 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA, 300 nM), a potent PKC activator. Transient increase in the amplitude and acceleration in the decay of IACh were invariably observed within minutes of TPA application, and were independent of extracellular Ca2+ concentration. Both parameters recovered to control within 20–30 min; then a slight depression of IACh developed. By this time, an initial PKC down regulation was observed. At the peak of TPA‐induced potentiation, dose‐response relations suggested an increased binding affinity of nAChR for the neurotransmitter. 4a‐phorbol 12,13‐didecanoate (300 nM), a biologically inactive analogue of TPA, did not affect IACh, while staurosporine (5–10 μM), a potent inhibitor of PKC activity, suppressed the action of TPA on IACh. In oocytes co‐injected with C2C12 mRNA and with rat brain mRNA, IACh was potentiated by 5‐hydroxytryptamine (10 μM), whose receptors are coupled to phosphoinositide hydrolysis. The nAChR‐channel activity in cell‐attached patches increased when TPA was applied to the oocytes. In 50% of the oocytes examined, a sustained depression of the single channel activity followed. We conclude that in Xenopus oocytes an endogenous PKC system regulates the function of embryonic‐type muscle nAChRs. © 1995 Wiley‐Liss, Inc.
Platelet‐derived growth factor is a survival factor for PSA‐NCAM + oligodendrocyte pre‐progenitor cells
Authors: Grinspan J. B. , Franceschini B.
Published: January 2005

Mature oligodendroglia, which synthesize and express lipids and proteins characteristic of myelin, are generated from precursor cells which are formed in germinal matrix, then migrate widely through the neuraxis. We now demonstrate that these precursor cells can be recognized at a very early stage by their surface expression of polysialylated neural cell adhesion molecules (PSA‐NCAM), and only later bind anti ganglioside antibodies that had previously been used to recognize “O‐2A” oligodendroglial precursor cells. PSA‐NCAM expression by these cells is likely to be of functional significance, since a recent study demonstrated that O‐2A cells become immobile when stripped of PSA‐NCAM. Platelet‐derived growth factor (PDGF) proved to be a survival factor for these PSA‐NCAM + cells, and in a defined medium, PDGF was sufficient to ensure maturation of immunopurified PSA‐NCAM + cells to oligodendroglia. © 1995 Wiley‐Liss, Inc.
Differential expression of exons 10 and 11 in normal tau and tau associated with paired helical filaments
Authors: Ksiezak‐Reding H. , Shafit‐Zagardo B. , Yen S.‐H.
Published: January 2005

Antibodies were raised to two synthetic peptides with amino acid sequences encoded by a variable region of exons 10 and 11 of the tau gene. The affinity‐purified antibodies, designated E‐10 and E‐11, were used to determine whether PHF‐tau and normal tau differ in variants containing three or four repeats in the microtubule‐binding domain, respectively. Normal adult human brain was shown by gel electrophoresis to contain six isoforms of tau. All of the isoforms reacted with E‐11, whereas only four of them with slower electrophoretic mobility were recognized by E‐10. Fetal brain tau was readily recognized by E‐11 but reacted poorly with E‐10. In PHF preparations, E‐11 bound to all three polypeptides of PHF‐tau of 68 kD, 64 kD, and 60 kD and reacted intensely with a material smearing from the top of the gel to about the 50‐kD region. In contrast, E‐10 only weakly recognized the two higher molecular weight PHF‐tau polypeptides of 68 kD and 64 kD, as well as smeared material, and the binding was not affected by phosphatase treatment. Using recombinant tau with four repeats as a reference, the immunoreactivity of E‐10 with PHF‐tau was estimated to be approximately 5% of that of E‐11. By comparison, the immunoreactivity of E‐10 with four isoforms of normal tau was comparable to that of E‐11. These results indicate that the ratio of three vs. four repeat variants in PHF‐tau is higher than in normal tau and suggest that Alzheimer disease may be associated with the disproportional expression of fetal (or juvenile) forms of tau. Alternatively, the weak reactivity of PHF‐tau with E‐10 antibody could be due to post‐translational modifications other than phosphorylation. © 1995 Wiley‐Liss, Inc.
Localization of constitutive and hyperthermia‐inducible heat shock mRNAS (hsc70 and hsp70) in the rabbit cerebellum and brainstem by non‐radioactive in situ hybridization
Authors: Foster J. A. , Rush S. J. , Brown I. R.
Published: January 2005

Neural expression of constitutive hsc70 mRNA and hyperthermia‐inducible hsp70 mRNA is examined using radioactive and non‐radioactive in situ hybridization procedures. A strong induction of hsp70 mRNA was noted in cell populations in cerebellar layers and in the brainstem which demonstrated expression of mRNA encoding proteolipid protein, an oligodendrocyte marker. The non‐radioactive in situ hybridization procedure using digoxigenin (DIG)‐UTP‐labeled riboprobes permitted improved signal localization, and stress‐inducible hsp70 mRNA was detected at the cytoplasmic cap areas of individual oligodendrocytes. Cell types which express constitutive members of the hsc/hsp70 multigene family were also identified. Neurons in the brainstem and in the deep white matter and molecular layer of the cerebellum showed expression of hsc70 mRNA while signal was not detected in adjacent glial cells. A neuron‐specific enolase riboprobe aided in the identification of neuronal cell types. The non‐radioactive DIG riboprobe revealed that hsc70 mRNA was highly localized to the cyto‐plasm of individual neurons. High constitutive levels of hsc70 in certain neurons may dampen hsp70 induction after hyperthermia in these cell populations. © 1995 Wiley‐Liss, Inc.
Exposure of tumor necrosis factor‐α to luminal membrane of bovine brain capillary endothelial cells cocultured with astrocytes induces a delayed increase of permeability and cytoplasmic stress fiber formation of actin
Authors: Deli M. A. , Descamps L. , Dehouck M.‐P. , Cecchelli R. , Joó F. , Ábrahám C. S. , Torpier G.
Published: January 2005

Tumor necrosis factor‐α (TNF‐α), a proinflammatory cytokine, has long been known to be involved in the pathogenesis of central nervous system infections and of certain neurodegenerative diseases. However, the possible role of the blood‐brain barrier (BBB), the active interface between the blood circulation and brain tissue, remained unknown during these pathological conditions. In our in vitro reconstructed BBB model, 1‐hr exposure of recombinant human TNF‐α (in concentrations of 50, 250, and 500 U/ml, respectively) to the luminal membrane of bovine brain capillary endothelial cells (BBCEC) did not change significantly the transendothelia: flux of either sucrose (m.w. 342 Da), or inulin (m.w. 5 kDa) up to 4 hr (early phase), except for a slight decrease (P< 0.05) in sucrose permeation at 2–4 hr with the highest dose of TNF‐α On the other hand, at 16 hr after the 1‐hr challenge with TNF‐α (delayed phase) at all 3 concentrations, significant increase was induced in the permeability of BBCEC monolayers for both markers. These changes of permeability were accompanied by a selective reorganization of F‐actin filaments into stress fibers, while the intracellular distribution of vimentin remained similar to the control. These results suggest that BBCEC can respond directly to TNF‐α by a delayed increase of permeability and reorganization of actin filaments. ©1995 Wiley‐Liss, Inc.
Purine and pyrimidine nucleotides activate distinct signalling pathways in PC12 cells
Authors: De Souza L. R. , Moore H. , Raha S. , Reed J. K.
Published: January 2005

The role of extracellular nucleotides in intracellular signalling and neurosecretion was assessed in PC12 cells. Activation of phospholipase C and increased [Ca2+]i were mediated by purinoceptors with an agonist potency profile, ATP ∼ UTP > 2‐methylthioadenosine triphosphate (2‐MeSATP), typical of P2U. ATP also evoked a rapid acidification followed by a more gradual alkalinization (measured with 2′,7′‐biscarboxyethyl‐5(6)‐carboxyfluorescein (BCECF)), while UTP induced only a gradual alkalinization. The amiloride analogue 5‐(N‐ethyl‐N‐isopropyl) amiloride (EIPA) attenuated the alkalinization phase suggesting activation of the Na+/H+ exchanger by ATP and UTP. Using bisoxonol and [3H]tetraphenylphosphonium ([3H]TPP+) as potential‐sensitive probes, we showed that while ATP rapidly depolarized PC 12 cells in an Na+ ‐dependent manner, UTP evoked a much reduced and delayed response. The potency profile (ATP ∼ 2‐MeSATP ∼ adenosine 5′‐0‐(3thiotriphosphate) (ATPγS) ≫ UTP, α, beta;‐methyleneATP) suggested involvement of a receptor subtype distinct from P2U. Secretion of endogenous dopamine was also assessed. Those nucleotides that induced depolarization (ATP, 2‐MeSATP, ATPγS) were also the most potent secretagogues. UTP was ineffective. Our results suggest that ATP stimulates distinct purinoceptor subtypes and induces neurosecretion through the activation of multiple signalling path ways. © 1995 Wily‐Liss, Inc.
On identifying a second molecular antagonistic mechanism operative at the glycine receptor
Authors: Galvez‐Ruano E. , Lipkowitz K. B. , Aprison M. H.
Published: January 2005

We used molecular modeling techniques to examine six reported antagonists of glycine with varying K; values against strychnine. We found the data suggest two groups operating with different mechanisms. In group 1 (strychnine, brucine, Pitrazepin, and bicuculline methobromide) the antagonist contains two or three sites that can electrostatically bind to the three comparable groups of opposite charge in the recognition site where the natural neurotransmitter binds, thus opening the chloride channel. In addition, when in this position, the antagonist is able to also block the now opened chloride channel with a different portion of its structure. In many cases, this involves an interaction between a carbonyl group, on the antagonist and the guanidinium group of arginine which is part of the polypeptide segment of the outer mouth of the chloride channel (Grenningloh et al., Nature 330:2526, 1987). In group 2 (R5135 and 1,5‐diphenyl‐3,7‐diazaadamantan‐9‐ol) the antagonist contains charged sites but when one of these molecules attaches to the recognition site, the chloride channel is not opened. In addition, R5135 contains a carbonyl group which attaches to arginine as pointed out in the text, whereas 1,5‐diphenyl‐3,7‐diazaadamantan‐9‐ol contains a phenyl group that can block the channel. © 1995 Wiley‐Liss, Inc
Neuronal differentiation of P19 embryonal carcinoma cells in defined media
Authors: Yao M. , Bain G. , Gottlieb D. I.
Published: January 2005

The P19 embryonal carcinoma cell line is a useful model system for analyzing the factors that regulate neuronal differentiation. In order to analyze the extrinsic factors that are involved in differentiation, it is necessary to carry out experiments in fully defined media. Here we have investigated the neuronal differentiation of P19 cells in two defined media. Cells that are propagated and induced with retinoic acid in standard serum‐containing medium are capable of differentiating into neuron‐like cells in N2 medium. Dividing fibroblast‐like cells also appeared in these cultures. After about 10 days in culture in N2 medium, the great majority of neuron‐like cells died. On the other hand, culturing induced cells in N2 medium for 5 days and then switching to a defined medium consisting of Neurobasal medium plus B27 supplement allowed the neuron‐like cells to survive for prolonged periods of time. This defined medium thus provides a suitable system for analyzing extrinsic factors that affect the survival and differentiation of P19 neurons. P19 cells induced with retinoic acid and plated in N2 were exposed to bFGF and EGF, which are known to be mitogens for neuronal precursor cells. Both growth factors were mitogenic for a subpopulation of the induced cells. In separate experiments, cells cultured in N2 in the presence of RA were induced to differentiate into neuron‐like cells. © 1995 Wiley‐Liss, Inc.
GM1 ganglioside rescues substantia nigra pars compacta neurons and increases dopamine synthesis in residual nigrostriatal dopaminergic neurons in MPTP‐treated mice
Authors: Schneider J. S. , Kean A. , DiStefano L.
Published: January 2005

GM1 ganglioside has been shown to stimulate recovery of the damaged dopamine system under a number of different circumstances. In addition to rescue of damaged dopamine neurons, the present study assessed the ability of GM1 to enhance the synthesis of dopamine in remaining nigrostriatal neurons following 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) exposure. There was a significantly greater accumulation of L‐dopa 30 min after aromatic amino acid decarboxylase inhibition with NSD‐1015 (100 mg/Kg) and an increase in the ratio of L‐dopa to dopamine in MPTP + GM1‐treated mice than in mice that received only MPTP. This effect of GM1 on dopamine synthesis was dependent upon the degree of initial damage to the nigrostriatal dopamine system. That is, the GM1 effect on dopamine synthesis could not be demonstrated in mice with greater than 95% striatal dopamine loss and 75% substantia nigra dopamine neuron loss. These results suggest that in addition to previously reported effects of GM1 on rescue and repair of dopaminergic neurons, GM1 may also have the ability to enhance dopamine synthesis in residual dopaminergic neurons. Direct effects on dopamine neurochemistry may contribute to functional improvements seen after GM1 treatment in various models of parkinsonism. © 1995 Wiley‐Liss, Inc.
Differentially expressed genes after peripheral nerve injury
Authors: Gillen C. , Gleichmann M. , Spreyer P. , Müller H. W.
Published: January 2005

In an attempt to identify genes associated with Wallerian degeneration and peripheral nerve regeneration we have performed differential hybridization screening of a cDNA library from crushed rat sciatic nerve (7 days postlesion) using radioactively labeled cDNA prepared from poly(A)+ RNA of normal vs. crushed nerve. Screening of 5,000 randomly selected colonies yielded 24 distinct clones that were regulated following nerve injury. Fifteen of the differentially expressed sequences could be classified as induced, whereas 9 sequences appeared to be repressed at 1 week postcrush. Sequencing and computer‐assisted sequence comparison revealed 3 classes of regulated cDNA clones representing (1) novel gene sequences (8 clones) including 3 transcripts containing a repetitive “brain identifier” (ID) element; (2) identified genes (7 clones) with previously undetected expression in the peripheral nervous system (PNS), such as apolipoprotein D, peripheral myelin protein 22kD (PMP22), SPARC (secreted protein, acidic and rich in cysteine), sulfated glycoprotein SGP‐1, apoferritin, decorin, and X16/SRp20; and (3) identified genes (9 clones) with known expression in the PNS including, e.g., the myelin protein P0, γ‐actin, vimentin, a‐tubulin, chargerin II, and cytochrome c‐oxidase subunit I. Northern blot and polymerase chain reaction analyses with RNA from crushed and transected nerve demonstrated that sequences with related function, like the group of myelin genes, cytoskeleton genes, genes involved in RNA processing and translation, in lipid transport or energy metabolism showed closely related temporal patterns of expression during nerve degeneration and regeneration. Finally, we compared the differentially expressed genes identified at 7 days after crush injury (this investigation) with the regulated sequences isolated previously by De Leon et al. (J Neurosci Res 29:437‐488, 1991) from a 3 day postcrush sciatic nerve cDNA library. © 1995 Wiley‐Liss, Inc.
Gene therapy in the central nervous system: Direct versus indirect gene delivery
Authors: Ridet J.‐L. , Privat A.
Published: January 2005

Over the last decade, the combination of molecular biology and cell transplantation techniques has given rise to a powerful method for gene therapy. The implantation of genetically modified cultured cells has been extensively used in the central nervous system (CNS) in various experimental models of neurologic disorders. More recently, viral and chemical methods have been developed to further efforts to shuttle transgenes into the relatively inaccessible brain. Adenoviral and liposomal synthetic vectors carry transgenes into neural tissue in situ and are beginning to show promise as new methods for CNS therapy. © 1995 Wiley‐Liss, Inc.
Dystroglycan expression in the wild type and mdx mouse neural retina: Synaptic colocalization with dystrophin, dystrophin‐related protein but not laminin
Authors: Montanaro F. , Carbonetto S. , Campbell K. P. , Lindenbaum M.
Published: January 2005

Alpha‐ and β‐dystroglycan (α‐ and β‐DG) are members of a dystrophin‐associaced glycoprotein complex (DGC) in skeletal muscle which binds to agrin and laminin, and has been postulated to be involved in myoneural synapse formation. The absence of functional dystrophin in Duchenne muscular dystrophy (DMD) and in one of its animal models, the mdx mouse, leads to a reduction of α‐ and β‐DG in muscle, and is often associated with mental retardation and abnormal retinal synaptic transmission in DMD. Using immunohistochemistry, we find that a‐ and β‐DG are expressed in the outer plexiform layer of both wild type and mdx retina, where both dystrophin and dystrophin‐related protein (DRP), but not laminin are present. In situ hybridization identifies two neuronal populations, photoreceptors and retinal ganglion cells, that express DG mRNA. α‐ and β‐DG are also expressed in the inner limiting membrane and around blood vessels where they colocalize with laminin and DRP. Western blot analysis revealed the expression of several dystrophin isoforms in wild type and mdx retina, possibly explaining the unaltered expression of α‐ and β‐dystroglycan in the mdx central nervous system (CNS). Our results support the hypothesis that a‐ and β‐DG can interact with dystrophin and DRP in the CNS and perform functions analogous to those of the DGC in muscle. © 1995 Wiley‐Liss, Inc.
Developmental expression of calmodulin mRNA and protein in regions of the postnatal rat brain
Authors: Berry F. , Brown I. R.
Published: January 2005

The expression of calmodulin (CaM) protein and mRNA was analyzed in specific regions of the rat brain during postnatal development. CaM levels in the adult brain were more abundant in the cerebral hemispheres and thalamus compared to brain stem and superior plus inferior colliculus. All brain regions contained higher CaM protein and mRNA levels than in non‐neural tissues such as the kidney. During postnatal development of the brain, maximal levels of CaM protein and CaM I mRNAs were attained at day 10 or 15. Protein levels declined thereafter in the adult in all regions except the thalamus. With respect to products of the rat CaM I gene, the 4.0 kb neural transcript demonstrated a pronounced increase during postnatal development, whereas the 1.8 kb message showed little change. © 1995 Wiley‐Liss, Inc.
Trophic and protective effects of growth/differentiation factor 5, a member of the transforming growth factor‐β superfamily, on midbrain dopaminergic neurons
Authors: Krieglstein K. , Suter‐Crazzolara C. , Hötten G. , Pohl J. , Unsicker K.
Published: January 2005

Growth/differentiation factor 5 (GDF5) is a novel member of the transforming growth factor‐β (TGF‐β) superfamily of multifunctional cytokines. We show here that GDF5 is expressed in the developing CNS including the mesencephalon and acts as a neurotrophic, survival promoting molecule for rat dopaminergic midbrain neurons, which degenerate in Parkinson's disease. Recombinant human GDF5 supports dopaminergic neurons, dissected at embryonic day (E) 14 and cultured for 8 days under serum‐free conditions, to almost the same extent as TGF‐β, and is as effective as glial cell line‐derived neurotrophic factor (GDNF), two established trophic factors for midbrain dopaminergic neurons. In contrast to TGF‐β and GDNF, GDF5 augments numbers of astroglial cells in the cultures, suggesting that it may act indirectly and through pathways different from those triggered by TGF‐β and GDNF. GDF5 also protects dopaminergic neurons against the toxicity of N‐methylpyridinium ion (MPP+), which selectively damages dopaminergic neurons through mechanisms currently debated in the etiology of Parkinson's disease (PD). GDF5 may therefore now be tested in animal models of PD and might become useful in the treatment of PD. © 1995 Wiley‐Liss, Inc.
Convergent influence of the central nucleus of the amygdala and the paraventricular hypothalamic nucleus upon brainstem autonomic neurons as revealed by c‐fos expression and anatomical tracing
Authors: Petrov T. , Krukoff T. L. , Jhamandas J. H.
Published: January 2005

Combinations of anatomical tracing with detection of Fos (the protein product of the immediate early gene c‐fos) consequent to the stimulation of the central nucleus of the amygdala were used to explore the possibility that the hypothalamic paraventricular nucleus participates in the activation of brainstem neurons in the nucleus of the solitary tract and ventrolateral medulla. After injections of the anterograde tracer Phaseolus vulgaris leucoagglutinin in the paraventricular nucleus, labeled fibers and varicosities were found to impinge on catecholaminergic and non‐catecholaminergic Fos‐positive neurons in the brainstem. After injections of a retrograde tracer in the nucleus of the solitary tract or ventrolateral medulla, we observed that some of the Fos‐positive neurons within the parvocellular paraventricular nucleus that project to the brainstem were catecholaminergic or oxytocinergic.

The results indicate that direct and indirect inputs from the amygdala may influence the activity of autonomic neurons in the brainstem. The paraventricular nucleus, via its direct projections onto catecholaminergic and non‐catecholaminergic neurons, may participate in activation of brainstem neurons. Activated catecholaminergic and oxytocinergic parvocellular neurons in the paraventricular nucleus may be involved in the transmission of autonomic signals from the amygdala toward the brainstem. ©1995 Wiley‐Liss, Inc.
CD9 of mouse brain is implicated in neurite outgrowth and cell migration in vitro and is associated with the α6/β1 integrin and the neural adhesion molecule L1
Authors: Schmidt C. , Künemund V. , Wintergerst E. S. , Schmitz B. , Schachner M.
Published: January 2005

We describe here a novel monoclonal antibody (mab INTRODUCTION H6) which recognizes CD9, an integral cell surface constituent previously described in cells of the hematopoietic lineage and involved in the aggregation of platelets. Mab H6 was raised against membranes of immature mouse astrocytes and reacted with a protein of 25–27 kD in detergent extracts of adult mouse brain membranes. Sequence analysis of the N‐terminal amino acids revealed an identity of 96% with CD9 from mouse kidney. CD9 was localized in the central and peripheral mouse nervous systems: in the spinal cord of 11‐day‐old mouse embryos, CD9 was strongly expressed in the floor and roof plates. In the adult mouse sciatic nerve, myelin sheaths were highly CD9immunoreactive. Mab H6 reacted with the cell surfaces of both glial cells and neurons in culture and inhibited migration of neuronal cell bodies, neurite fasciculation and outgrowth of astrocytic processes from cerebellar microexplants. Neurite outgrowth from isolated small cerebellar neurons was increased in the presence of mab H6 on substrate‐coated laminin, but not on substrate‐coated poly‐L‐lysine. Addition of mab H6 elicited an increase in intracellular Ca2+ concentration in these cells on substrate‐coated laminin. Immunoprecipitates of CD9 from cultured mouse neuroblastoma N2A cells contained the α6/β1 integrin. Moreover, preparations of CD9 immunoaffinity‐purified from adult mouse brain using a mab H6 column contained the neural adhesion molecule L1, but not other neural adhesion molecules. CD9 bound to L1, but not to NCAM or MAG. Both the α6/β1 integrin and L1 could be induced to coredistribute with CD9 on the surface of cultured neuroblastoma N2A cells. The combined observations suggest that CD9 can associate with L1 and α6/β1 integrin to influence neural cell interactions in vitro. © 1996 Wiley‐Liss, Inc.
Impaired synaptic functions with aging as characterized by decreased calcium influx and acetylcholine release
Authors: Tanaka Y. , Hasegawa A. , Ando S.
Published: January 2005

Age‐related alterations of presynaptic functions were studied in terms of acetylcholine (ACh) synthesis and release using synaptosomes isolated from mouse brain cortices. The following three findings were obtained: (1) Choline acetyltransferase activity and ACh production rate remained constant throughout all ages tested. This observation, obtained with synaptosomes, was not consistent with data reported for brain slices (Gibson GE, Peterson C: J Neurochem 37:978–984, 1981). Various conditions, such as low glucose or membrane depolarization, modulated ACh synthesis to similar extents in young and aged synaptosomes. (2) Depolarization‐induced release of ACh from synaptosomes significanty decreased in the senescent stage. The fraction of ACh released from aged synaptosomes was less than that released from young synaptosomes, although the ACh contents in the synaptosomes did not change with age. (3) Calcium influx induced by depolarization was lower in the synaptosomal preparations from aged mice than in those from young mice. A strong positive correlation was observed between the amounts of ACh released and the increased calcium levels when the data for all preparations, both from young and aged mice, were plotted. This indicates that diminished calcium influx may cause the reduced ACh release by aged synapses. The present study provides evidence for an age‐related decrease in presynaptic functions, that is, a reduction in calcium influx via voltage‐dependent calcium channels followed by a decreased ACh release from synapses despite an abundance of ACh within the synapses. © 1996 Wiley‐Liss, Inc.
Tics in a patient with Parkinson's disease
Authors: Shale Heidi , Fahn Stanley , Mayeux Richard
Published: January 2005

A patient with Gilles de la Tourette syndrome later developed Parkinson's disease in middle age. This was accompanied by a marked reduction in the frequency of tics but levodopa toxicity exacerbated the tics. The dopamine hypothesis of tic disorders is supported by this observation.
Electrophoresis and immunoblot of cerebrospinal fluid proteins in spasmodic torticollis
Authors: Cullis Paul A. , Townsend Laurace , Lewitt Peter , Pomara Nunzio , Reitz Darlene
Published: January 2005

Protein patterns of cerebrospinal fluid (CSF) from patients with spasmodic torticollis (ST) were investigated to determine whether abnormalities previously reported could be detected and further identified. CSF was collected from 12 patients with ST and 6 normal controls. The CSF proteins were analyzed using sodium dodecyl sulfate (SDS) polyacrylmide gel electrophoresis and silver staining. In 11 of the 12 patients with ST, a CSF protein pattern was observed which differed from that in the controls. The identity of the abnormal proteins was ascertained by blotting and immunostaining with specific antisera to IgG and ceruloplasmin (Cp). CSF from 2 of 12 patients had distinct bands staining for IgG and 7 had abnormal immunostaining for Cp.
Gait disorder of subcortical arteriosclerotic encephalopathy: Binswanger's disease
Authors: Thompson P. D. , Marsden C. D.
Published: January 2005

The abnormality of gait that may occur in patients with subcortical arteriosclerotic encephalopathy (Binswanger's disease) is described in 12 patients in whom difficulty walking was the presenting symptom. Their gaits had elements of both Parkinsonism and ataxia, which were most market in 4 patients unable to stand unaided. The difficulty in using their legs to walk was out of proportion to that of other movements of the lower limbs when lying or seated. In contrast, upper limb mobility and facial expression were relatively preserved. Attention is drawn to the similarity of this gait pattern to that of some patients with hydrocephalus, frontal lobe lesions, and “senile” disorders of gait. A possible mechanism for this Parkinsonian‐ataxia in these various conditions may be their propensity to damage the afferent and efferent interconnections of the leg areas of the motor and supplementary motor areas of the cerebral cortex with, the cerebellum and basal ganglia.
Antiparkinsonian activity of (+)‐PHNO in the MPTP‐treated common marmoset
Authors: Nomoto M. , Stahl S. , Jenner P. , Marsden C. D.
Published: January 2005

Administration of MPTP (1–4 mg/kg ip daily for 5–7 days) to common marmosets induced persistent parkinsonian motor deficts. The subcutaneous adminstration of (+)‐PHNO [(+)‐4‐propyl‐9‐hydroxynaphtoxazine; 1–4 μ/kg] caused a dose‐dependent reversal of the akinesia and incoordination of movement. Similarly, oral administration of (+)‐PHNO (5–20 μ/kg) caused an equivalent reversal of the motor abnormalities. No dyskinetic phenomena were induced by (+)‐PHNO on oral or subcutaneous administration. Oral or subcutaneous administration of (+)‐PHNO to normal control marmosets also increased the usual repetoire of motor behaviour, but this was not as marked as in MPTP‐treated animals. (+)‐PHNO is a potent dopamine agonist drug of potential use in the treatment of Parkinson's disease.
Antiparkinsonian activity of single oral dose of PHNO
Authors: Grandas F. , Quinn N. , Critchley P. , Rohan A. , Marsden C. D. , Stahl S. M.
Published: January 2005

PHNO, a new D‐2 agonist, was investigated in five patients with Parkinson's disease. In an acute, open, oral, dose‐ranging study comparing benefit from single doses, 4 mg of PHNO was found to be equivalent to one tablet of Sinemet 25/250 mg. Adverse reactions were those anticipated for a dopaminomimetic agent. Because of its novel structure and apparent transcutaneous penetration, further studies on PHNO are desirable.