bluuebunny/crossref_metadata_2025 huggingface dataset:
Nerve Growth Factor Promotes Neurite Regeneration in PC12 Cells by Translational Control
Authors: Twiss Jeffery L. , Shooter Eric M.
Published: July 2010

Abstract: When PC12 cells are primed with nerve growth factor (NGF) for periods of ≥1 week, they acquire the ability to regenerate neurites rapidly in response to NGF. It is not known how NGF promotes this regeneration, but it does not require ongoing RNA synthesis. Previous studies have suggested that NGF directs the accumulation of precursor molecules that are rapidly assembled to form the regenerated neurites. To address the nature of these precursor molecules, we have treated PC12 cells with macromolecular synthesis inhibitors during the priming and regeneration phases of neurite growth. Here we show that NGF promotes neurite regeneration by inducing the synthesis of new proteins. These proteins are encoded by short‐lived mRNAs that are generated during the NGF priming period. The isolation and identification of these mRNAs will allow a further understanding of how NGF promotes neurite regeneration.
l‐DOPA Inhibits Complex IV of the Electron Transport Chain in Catecholamine‐Rich Human Neuroblastoma NB69 Cells
Authors: Pardo Beatriz , Mena María Angeles , García de Yébenes Justo
Published: July 2010

Abstract: l‐3,4‐Dihydroxyphenylalanine (l‐DOPA) is toxic for human neuroblastoma cells NB69 and its toxicity is related to several mechanisms including quinone formation and enhanced production of free radicals related to the metabolism of dopamine via monoamine oxidase type B. We studied the effect of l‐DOPA on activities of enzyme complexes in the electron transport chain (ETC) in homogenate preparations from the human neuroblastoma cell line NB69. As a preliminary step we compared the activity of ETC in cellular homogenates with that of purified mitochondria from NB69 cells and rat brain. Specific activities for complex I, complex II–III, and complex IV in NB69 cells were, respectively, 65, 96, and 32% of those in brain mitochondria. Complex I activity was inhibited in a dose‐dependent way by 1‐methyl‐4‐phenylpyridinium ion with an EC50 of ∼150 µM. Treatment with 0.25 mMl‐DOPA for 5 days reduces complex IV activity to 74% of control values but does not change either complex I or citrate synthase. Ascorbic acid (1 mM), which protects NB69 cells from l‐DOPA‐induced neurotoxicity, increases complex IV activity to 133% of the control and does not change other ETC complexes. Ascorbic acid also reverses l‐DOPA‐induced reduction of complex IV activity in NB69 cells. This observation might indicate that the protection observed with ascorbic acid is related to complex IV activation. In vitro incubation with l‐DOPA (0.125–4 mM) for 2 min produced a dose‐dependent reduction of complex IV without change in complex I and II–III activities.
Thrombin Receptor Activation Stimulates Astrocyte Proliferation and Reversal of Stellation by Distinct Pathways: Involvement of Tyrosine Phosphorylation
Authors: Grabham Peter , Cunningham Dennis D.
Published: July 2010

Abstract: Treatment of cultured type‐1 astrocytes with thrombin leads to cell proliferation and reversal of stellation. The half‐maximal concentrations of thrombin required for each response are 500 and 2 pM, respectively. To test whether they might be mediated by different receptors, we examined the contribution of the G protein‐coupled thrombin receptor to these responses in purified rat astrocytes by using the agonist peptide SFLLRNP. In the absence of added growth factors, SFLLRNP fully mimicked the effects of thrombin at half‐maximal concentrations of 30 µM for an increase in cell number and DNA synthesis and 100 nM for the reversal of stellation. The role of protein tyrosine phosphorylation in these events was investigated using antiphosphotyrosine antibodies. Thrombin and SFLLRNP at concentrations at least 10‐fold greater than those required for half‐maximal reversal of stellation but below those required for mitogenesis induced an identical pattern of tyrosine phosphorylation on several proteins of 55–65, 106, 110–115, and 120–130 kDa. The response was rapid (<1 min) and transient with a peak response after ∼2 min. The specific tyrosine kinase inhibitor herbimycin A did not affect thrombin‐ or SFLLRNP‐mediated reversal of stellation at concentrations of up to 1 µM. In contrast, 1 µM herbimycin fully inhibited the ability of thrombin and SFLLRNP to increase cell number and stimulate DNA synthesis. Furthermore, this inhibition by 1 µM herbimycin A corresponded to inhibition of receptor‐induced tyrosine phosphorylation. Thus, cell proliferation but not reversal of stellation is dependent on thrombin receptor‐activated tyrosine kinase activity. These observations support the hypotheses that the thrombin receptor mediates the actions of thrombin in these cells and that activation of the thrombin receptor leads to multiple second messages that stimulate distinct cellular responses.
Metabotropic Glutamate Receptor (mGluR)‐Mediated Potentiation of Cyclic AMP Responses Does Not Require Phosphoinositide Hydrolysis: Mediation by a Group II‐Like mGluR
Authors: Winder Danny G. , Conn P. Jeffrey
Published: July 2010

Abstract: Metabotropic glutamate receptors (mGluRs) in the CNS are coupled to a variety of second messenger systems, the best characterized of which is activation of phosphoinositide hydrolysis. Recently, we found that activation of mGluRs in rat brain slices by the selective mGluR agonist 1‐aminocyclopentane‐1S,3R‐dicarboxylic acid (1S,3R‐ACPD) potentiates cyclic AMP (cAMP) responses elicited by activation of other receptors coupled to Gs. It has been suggested that mGluR‐mediated potentiation of cAMP responses is secondary to activation of phosphoinositide hydrolysis. However, preliminary evidence suggests that this is not the case. Therefore, we designed a series of experiments to test more fully the hypothesis that mGluR‐mediated potentiation of cAMP responses is secondary to phosphoinositide hydrolysis. Inhibitors of both protein kinase C and intracellular calcium mobilization failed to antagonize 1S,3R‐ACPD‐stimulated potentiation of cAMP responses. Further, coapplication of phorbol esters and 1S,3R‐ACPD induced a cAMP response that was greater than additive. Finally, (RS)‐3,5‐dihydroxyphenylglycine, a selective agonist of mGluRs coupled to phosphoinositide hydrolysis, failed to potentiate cAMP responses, whereas (2S,1′R,2′R,3′R)‐2‐(2,3‐dicarboxycyclopropyl)glycine, an mGluR agonist that does not activate mGluRs coupled to phosphoinositide hydrolysis, elicited a robust potentiation of cAMP responses. In total, these data strongly suggest that mGluR‐mediated potentiation of cAMP responses is not secondary to activation of phosphoinositide hydrolysis and is likely mediated by a group II mGluR.
Expression of the Proenkephalin A Gene and [Met5]‐Enkephalin Secretion Induced by Arachidonic Acid in Bovine Adrenal Medullary Chromaffin Cells: Involvement of Second Messengers
Authors: Suh Hong W. , Hudson Pearlie M. , Hong Jau‐Shyong
Published: July 2010

Abstract: We have previously reported that arachidonic acid (AA) increases the long‐term secretion of [Met5]‐enkephalin (ME) and the expression of proenkephalin A (proENK) mRNA in bovine adrenal medullary chromaffin (BAMC) cells. To characterize the underlying signal transductional mechanisms for the AA‐induced responses, the interactions of AA with several second messenger systems were studied. Long‐term (24‐h) treatment with AA (100 µM) increased both the secretion of ME and the expression of proENK mRNA. Pretreatment of BAMC cells with nimodipine (1 µM), but not with ω‐conotoxin GVIA (1 µM), inhibited the secretion of ME and the expression of proENK mRNA induced by AA. Calmidazolium (1 µM), a calmodulin antagonist, also significantly inhibited AA‐induced responses. However, a protein kinase C (PKC) inhibitor, sphingosine (36 µM), was ineffective in blocking AA‐induced responses. In addition, the down‐regulation of PKC by phorbol 12‐myristate 13‐acetate (0.1 µM) for 48 h did not inhibit the AA‐induced responses. Forskolin (5 µM), an adenyl cyclase activator, alone increased the secretion of ME as well as proENK mRNA levels and, when coincubated with AA, showed an additive effect on the secretion of ME and the levels of proENK mRNA. The results suggest that the Ca2+/calmodulin pathway, but not the protein kinase A or PKC pathway, is partially involved in mediating the AA‐induced increases of the long‐term secretion of ME and the levels of proENK mRNA.
Regional Variations in the Pharmacology of NMDA Receptor Channel Blockers: Implications for Therapeutic Potential
Authors: Porter Richard H. P. , Greenamyre J. Timothy
Published: July 2010

Abstract: Quantitative receptor autoradiography was used to examine the regional binding characteristics of a diverse group of N‐methyl‐d‐aspartate (NMDA)‐receptor channel blockers that varied in potency 105‐fold. Full competition curves were generated in each of six brain regions for 11 different compounds. MK‐801 was the most potent compound studied, with an IC50 of ∼10 nM in the forebrain regions and 24 nM in the cerebellar granule cell layer (p < 0.05). The binding affinities of nine of the 11 compounds examined were significantly different in cerebellar granule cell layer than in forebrain regions. In addition, the apparent Hill slopes of five of the compounds were significantly different in cerebellum compared with forebrain. That the rank order of drug potencies in cerebellum diverges from that in forebrain suggests that cerebellar NMDA‐receptor ion channels differ pharmacologically from those in forebrain. There was a general trend that drugs known to be well tolerated in humans (remacemide hydrochloride and its metabolites, amantadine, budipine, and memantine) had lower affinities than compounds with severe neurobehavioral or psychotomimetic effects. Moreover, all of the compounds known to be well tolerated in humans had a significantly higher affinity in the cerebellum than in forebrain regions, in contrast to MK‐801, 1‐[1‐(2‐thienyl)cyclohexyl]‐piperidine hydrochloride (TCP), phencyclidine (PCP), and ketamine, which had lower affinities in cerebellum. Our results are consistent with the notion that low affinity (rapid kinetics) and, possibly, subunit specificity (as indicated by distinct regional pharmacologies) may be important determinants of the clinical tolerability of NMDA‐receptor channel blockers.
Determination of the Lumped Constant for the α‐Methyltryptophan Method of Estimating the Rate of Serotonin Synthesis
Authors: Vanier Michael , Tsuiki Ko , Grdiša Mira , Worsley Keith , Diksic Mirko
Published: July 2010

Abstract: The lumped constant (LC) for the α‐methyl‐l‐tryptophan method to convert the brain's uptake of labeled α‐methyl‐l‐tryptophan into the regional rate of serotonin synthesis was estimated. The method involved independently estimating the unidirectional uptake constant of the tracer (α‐[14C]methyl‐l‐tryptophan) to the tissue and the tracee (tryptophan) (with the addition of a radioactive compound) and calculating their ratio. The LC was estimated from logarithmically transformed data. Similar experiments were performed using rats treated with the drug probenecid, which blocks the efflux of 5‐hydroxyindoleacetic acid (a metabolite of serotonin) from the brain. The experiments using probenecid, corrected for the difference in the levels of plasma free tryptophan (increased in probenecid‐treated rats) relative to control experiments, gave an average LC for the rat brain of 0.46 ± 0.14 (mean ± SD). This value was not significantly different from the one obtained in controls (0.43 ± 0.13). In addition, the LC was also calculated using unidirectional uptake constants in the probenecid‐treated rats for α‐methyl‐l‐tryptophan and l‐tryptophan. This LC value was 0.39 ± 0.10. There was no significant difference between these three LC values. Thus, an average ± SD LC of 0.42 ± 0.07 for 28 brain structures investigated in this study was obtained. Statistically the LC obtained in different structures had a variability that could be accounted for by errors in measurements alone. In other words, dispersion in the LC values could be fully accounted for by chance alone. Data confirmed that the LC value did not change when the rate of serotonin synthesis was increased by probenecid treatment. We also showed that the rate of 5‐hydroxyindoleacetic acid accumulation in probenecid‐treated rats was 58 pmol g−1 min−1 (rat brain), which is about twice as much as reported by others for a normal rat. This difference could also be accounted for by the increase in the plasma level of free tryptophan in probenecid‐treated rats.
Stimulation of Cyclic GMP Accumulation by Sodium Nitroprusside Is Potentiated via a Gs Mechanism in Intact Pinealocytes
Authors: White Beatrix H. , Klein David C.
Published: July 2010

Abstract: Cyclic GMP accumulation in pinealocytes is elevated>100‐fold by norepinephrine (NE) through a mechanism involving conjoint activation of α1‐ and β1‐adrenergic receptors. Little or no stimulation occurs if either α1‐ or β1‐adrenergic receptors alone are activated. It appears that α1‐adrenergic effects are mediated by Ca2+ acting in part through nitric oxide (NO), and β1‐adrenergic effects are mediated by Gs. In the study presented here we investigated effects of adrenergic agonists or related postreceptor‐active agents on stimulation of pineal cyclic GMP accumulation by the NO generator sodium nitroprusside (NP). The cyclic GMP response to NP (1 mM) was potentiated by NE and isoproterenol (ISO) but not by phenylephrine, indicating that activation of β1‐adrenergic receptors potentiates the effects of NP. Similarly, vasoactive intestinal peptide (VIP), cholera toxin (CTX), and forskolin, all of which are known to mimic the effects of ISO in this system, also potentiated the effects of NP. In contrast, neither dibutyryl cyclic AMP nor agents that elevate intracellular Ca2+ levels caused marked potentiation of the effects of NP on pineal cyclic GMP. Depletion (90%) of Gsα by 21‐h treatment with CTX reduced β‐adrenergic potentiation of NP. These findings indicate that β‐adrenergic agonists and VIP potentiate the effects of NP through a mechanism involving Gs. The molecular basis of this action may be an increase in guanylyl cyclase responsiveness to NO.
Neurochemical Correlates of Dementia Severity in Alzheimer's Disease: Relative Importance of the Cholinergic Deficits
Authors: Bierer Linda M. , Haroutunian Vahram , Gabriel Steve , Knott Peter J. , Carlin Lorna S. , Purohit Dushyant P. , Perl Daniel P. , Schmeidler James , Kanof Philip , Davis Kenneth L.
Published: July 2010

Abstract: Cholinergic markers, neuropeptides, and amines and their metabolites were sampled from identical specimens across 10 neocortical regions in a large sample of Alzheimer's disease (AD) cases and controls. Levels of choline acetyltransferase, acetylcholinesterase, somatostatin, corticotropin‐releasing factor, serotonin, and 5‐hydroxyindoleacetic acid were significantly reduced in AD versus controls. After data reduction, the most descriptive neurochemical indices were used to examine the relationship of neurochemical measures and dementia severity within the AD sample, controlling for age effects. Dementia severity ratings were based on antemortem assessments (46.9% of AD sample) and postmortem chart review (53.1% of the AD sample). Choline acetyltransferase activity was highly correlated with clinical dementia ratings across the neocortex of the AD cases. Somatostatin and corticotropin‐releasing factor levels were correlated with dementia severity only when control cases were included in the analyses. None of the amines, their metabolites, or the neuropeptides quantified related significantly to dementia severity in the AD cohort. These data (a) confirm the strong association of cholinergic deficits with functional impairment in AD and show that this association is independent of age and (b) suggest that of all the neurochemical species quantified, the cholinergic indices may be unique in their association with dementia severity.
Increased Inositol 1,4,5‐Trisphosphate Accumulation Correlates with an Up‐Regulation of Bradykinin Receptors in Alzheimer's Disease
Authors: Huang H.‐M. , Lin T.‐A. , Sun G. Y. , Gibson G. E.
Published: July 2010

Abstract: An alteration in signal transduction systems in Alzheimer's disease (AD) would likely be of pathophysiological significance, because these processes control normal brain functions. Previously, a diminished β‐adrenergic‐mediated cyclic AMP response was found in cultured fibroblasts from AD patients. Because cross‐talk between the phosphoinositide and cyclic AMP pathways exists, the phosphoinositide cascade was studied under conditions that were similar to those for studying the cyclic AMP response. Cells from AD patients and age‐matched controls responded to bradykinin (BK) and released inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] in a time‐ and dose‐dependent manner. The level of Ins(1,4,5)P3 increased rapidly and transiently in response to BK, peaked at 5 s, but still remained 116–132% above the basal level by 30 s. Although the temporal patterns were similar in both groups, the Ins(1,4,5)P3 concentrations in AD fibroblasts were 73 and 89% above levels in the age‐matched controls at 5 and 10 s, respectively. Prostaglandin E1 also increased Ins(1,4,5)P3 formation, but this response was not different between the two groups. Although KD (affinity) values for the BK receptor were similar in both control and AD cells, the number of BK receptors (Bmax) was significantly elevated in AD fibroblasts (186.8 ± 0.8 fmol/mg of protein) as compared with control fibroblasts (57.2 ± 15.3 fmol/mg of protein). These results indicate that the elevated Ins(1,4,5)P3 production in response to BK in AD fibroblasts is positively correlated with an increase in the receptor numbers.
Cholesterol for Synthesis of Myelin Is Made Locally, Not Imported into Brain
Authors: Jurevics Helga , Morell Pierre
Published: July 2010

Abstract: We examined whether cholesterol needed for myelin formation is locally synthesized or whether it comes from the circulation. The experimental design was to inject [3H]water and to use incorporation of label into brain cholesterol as a measure of the rate of accumulation of newly synthesized cholesterol in brain. The contribution of the circulation to this labeled cholesterol pool was minimized by repressing liver synthesis of cholesterol with a high cholesterol diet. The rate of accumulation of total cholesterol was calculated from the increasing amounts of sterol in brain regions at successive time intervals during development. Thus, accumulating cholesterol not explained as being newly synthesized (radioactive) could be assumed to have come from the circulation. Long‐Evans rats, ranging in age from birth to 35 days, were injected intraperitoneally with [3H]water (0.3–1.0 mCi/g of body weight) and killed 2 h later. The brain was dissected into brainstem, cerebellum, and cerebral hemispheres, and total lipids were extracted. Cholesterol and its precursors were quantified by HPLC. The radioactivity associated with the sterol fractions and the specific activity of body water determined from serum were used to calculate the absolute amount of newly synthesized sterol. The rates of cholesterol synthesis were compared with the rates of accumulation of total cholesterol in each brain region. The rate of accumulation of total sterol (cholesterol and desmosterol) closely followed that of newly synthesized total sterol in all brain regions from the second through the fifth postnatal weeks. Thus, all sterol accumulation in brain during the period of rapid myelination can be explained by local synthesis; neither diet nor production of cholesterol by other organs plays a direct role in supplying cholesterol for myelination in brain.
Swim Stress Increases the Potency of Glycine at the N‐Methyl‐d‐Aspartate Receptor Complex
Authors: Nowak Gabriel , Redmond Anna , McNamara Mairead , Paul Ian A.
Published: July 2010

Abstract: We have previously demonstrated that chronic administration of antidepressants results in a reduction in the potency of glycine to displace 5,7‐[3H]dichlorokynurenic acid (5,7‐[3H]‐DCKA) from the strychnine‐insensitive glycine recognition site of the NMDA receptor complex. We now report that exposure of rats to the forced swim test results in a 56% increase in the potency of glycine to displace 5,7‐[3H]DCKA from frontal cortical homogenates. These data are consistent with the hypothesis that the forced swim test, a preclinical screen sensitive to acute administration of antidepressant drugs and NMDA receptor antagonists, also results in adaptation of the NMDA receptor complex. Moreover, these data lend further support to the hypothesis that glutamatergic pathways are involved in the neurobiological response to stress and, potentially, in the pathophysiology of depression.
[3H]Nemonapride and [3H]Spiperone Label Equivalent Numbers of D2 and D3 Dopamine Receptors in a Range of Tissues and Under Different Conditions
Authors: Vile John M. , D'Souza Ursula M. , Strange Philip G.
Published: July 2010

Abstract: [3H]Nemonapride and [3H]spiperone are very widely used to study dopaminergic systems in vitro and in vivo, but it has been reported that [3H]nemonapride and [3H]spiperone give markedly different Bmax values for preparations of D2 dopamine receptors from recombinant cell lines or animal tissues. We have used the two radioligands in parallel to study a range of dopamine receptors [D2(short), D2(long), and D3] in different buffers. Bmax values derived using either radioligand differ by an average of <20%, independent of receptor type or buffer conditions. All competition experiments show that the two ligands compete at a single site. It seems that [3H]spiperone and [3H]nemonapride do not differentiate between different forms or populations of D2‐like receptors.
Over‐Expression of the DM‐20 Myelin Proteolipid Causes Central Nervous System Demyelination in Transgenic Mice
Authors: Johnson Ravina Simons , Roder John C. , Riordan John R.
Published: July 2010

Abstract: We have created transgenic mice bearing varying copy numbers of a transgene coding for normal DM‐20, the alternatively spliced quantitatively minor isoform of myelin proteolipid protein. Demyelination of the CNS occurs as a consequence of 70 copies of this transgene. Overt symptoms begin at ∼3 months with a wobbling gait. Occasional seizures lasting a few seconds begin at 3–4 months. These symptoms progress in severity with age. Death occurs by 8–10 months. Myelination in 2‐month‐old animals, before the onset of any overt symptoms, appears morphologically normal at the electron microscopic level. However, the myelin in these 2‐month‐old animals has a reduced amount of the major myelin proteolipid protein and about three times as much DM‐20 as normal animals. In 7‐month‐old animals that appear to be undergoing demyelination in the CNS, both the major myelin proteolipid protein and DM‐20 are greatly reduced relative to the 2‐month‐old animal. Mice with 17 copies of the transgene also have a reduced amount of the major myelin proteolipid protein but appear to be otherwise normal and have normal life spans (>2 yr). Mice with low copy numbers of the transgene (2–4 copies) appear to be unaffected and have normal life spans.
Expression of the Choline Acetyltransferase Gene Depends on Protein Kinase A Activity
Authors: Inoue Hiroyasu , Li Yi‐Ping , Wagner John A. , Hersh Louis B.
Published: July 2010

Abstract: Choline acetyltransferase activity is barely detectable in a mutant pheochromocytoma PC12 cell line, A123.7, which is deficient in protein kinase A activity. Northern blot and polymerase chain reaction analyses showed that this mutant cell line has dramatically reduced levels of choline acetyltransferase mRNA, which correlates with the low level of enzyme activity. Transient transfection analysis was used to assess the functionality, in these cells, of an enhancer element and a cholinergic‐specific repressor element derived from the human choline acetyltransferase gene. The results show that the enhancer element is inactive in the protein kinase A‐deficient cell line. Cotransfection experiments with plasmids expressing the catalytic subunit of protein kinase A support this conclusion. These data indicate that protein kinase A regulates expression of the choline acetyltransferase gene at the transcriptional level by controlling the activity of an enhancer element.
Coculture of Astroglial and Vascular Endothelial Cells as Apposing Layers Enhances the Transcellular Transport of Hypoxanthine
Authors: Ceballos Guillermo , Rubio Rafael
Published: July 2010

Abstract: In brain, astrocytes and endothelial cells are a major site of adenosine degradation. These two cell types, found in close apposition, constitute the wall of the brain's capillaries and serve as a site of hypoxanthine production and degradation. Both cell types possess the hypoxanthine salvage pathway and can incorporate hypoxanthine into nucleotides. This suggests that the endothelial‐astrocyte anatomical complex might play an important role in the brain's purine homeostasis. To test this hypothesis, cocultures of monolayers of vascular endothelial cells and astrocytes were grown over a porous membrane, in close apposition to one another, and studies on hypoxanthine transport and metabolism to uric acid were performed. The flux of hypoxanthine across the cell layers was simultaneously determined and compared with the flux of sucrose, as a probe of passive diffusion. Our results show that in endothelial, glial, and endothelial‐glial cell layers the hypoxanthine flux was greater than that of sucrose, and that the flux of hypoxanthine, but not of sucrose, was inhibited by adenine or by lowering the temperature. These results suggest that hypoxanthine moves across endothelial, glial, and endothelial‐glial cell layers by a transport process. Furthermore, we found that hypoxanthine transport is enhanced when glial and endothelial cells are cocultured compared with that in glial or endothelial monolayers. In addition the coculture also resulted in a depression of xanthine oxidase activity.
Effect of Noradrenergic Lesions on Subtypes of α2‐Adrenoceptors in Rat Brain
Authors: Ordway Gregory A.
Published: July 2010

Abstract: The binding of [3H]rauwolscine to α2A‐ (also referred to as α2D‐) and α2C‐adrenoceptors in homogenates of rat cerebral cortex was measured by exploiting the selectivity of oxymetazoline for α2A‐adrenoceptors. Inhibition of [3H]rauwolscine binding by oxymetazoline was modeled best assuming binding to two sites (p < 0.001). Competition curves for oxymetazoline were shifted rightward by the addition of GTP (250 µM) but were still fit best by a two‐site model (p < 0.001). A concentration of oxymetazoline was calculated that would optimally antagonize [3H]rauwolscine binding (with GTP present) to oxymetazoline‐sensitive α2A‐adrenoceptors, minimally inhibiting binding to α2C‐adrenoceptors. Subsequently, [3H]rauwolscine binding to α2A‐ and α2C‐adrenoceptors in cortex was examined 3 weeks after destruction of noradrenergic terminals. Binding to α2C‐adrenoceptors was increased significantly after treatment with 6‐hydroxydopamine (6‐OHDA) compared with vehicle‐treated controls, whereas binding to α2A‐adrenoceptors was unchanged. Pretreatment of rats with desipramine before 6‐hydroxydopamine, to protect noradrenergic neurons, resulted in no changes in binding to either α2A‐ or α2C‐adrenoceptors. Thus, α2C‐adrenoceptors are regulated by changes in synaptic availability of norepinephrine. α2A‐Adrenoceptors are either not regulated by synaptic norepinephrine or are located both post‐ and presynaptically so that up‐regulation of postsynaptic α2A‐adrenoceptors is offset by a loss of presynaptic α2A‐adrenoceptors.
Characterization of Acetylcholinesterase Secretion in Neuronal Cultures and Regulation by High K+ and Soluble Factors from Target Cells
Authors: Biagioni S. , Bevilacqua P. , Scarsella G. , Vignoli A. L. , Augusti‐Tocco G.
Published: July 2010

Abstract: We have observed that cultured neurons from chick spinal cord and the neuroblastoma hybrid line 108CC15 released lower amounts of acetylcholinesterase (AChE) when compared with the parental line, N18TG2. AChE activity extracted by hypotonic buffer, which can be regarded as the source of the released enzyme, was considerably higher in the parental than in the hybrid 108CC15 (respectively, ∼80% and ∼40% of cellular activity). On the other hand, evaluation of ectocellular, with respect to total, AChE activity showed that in N18TG2 cells only 7% of AChE was localized on the plasmalemma, whereas in the hybrid line the percentage of ectocellular activity was 3.7 times higher than in the parental line. We have also examined the effect of cytochalasin B and nocodazole. In the N18TG2 line, the former did not affect AChE release, which was significantly reduced by the latter. High K+ level in the culture medium, of both N18TG2 and hybrid 108CC15 cultures, induced an increase in AChE secretion; Ca2+ presence was required for high K+‐induced release. Muscle extracts increased AChE secretion in both the hybrid 108CC15 and the spinal cord neurons. The present data suggest that AChE secretion during neuronal development is modulated by depolarizing stimuli and by soluble factors produced by target cells and may be involved in the control of neuronal differentiation.
Fibroblast Growth Factors Stimulate Protein Tyrosine Phosphorylation and Mitogen‐Activated Protein Kinase Activity in Primary Cultures of Hippocampal Neurons
Authors: Creuzet C. , Loeb J. , Barbin G.
Published: July 2010

Abstract: Fibroblast growth factors (FGFs) are not only mitogens, but they also promote the differentiation of various cell types. For instance, basic FGF (bFGF) provides a critical trophic support for hippocampal neurons in culture. To elicit their biological effects, FGFs interact with high‐affinity receptors that are transmembrane proteins with a cytoplasmic portion containing a tyrosine kinase activity. The tyrosine phosphorylation pattern was examined in primary cultures of hippocampal neurons derived from rat embryos. In these cultures grown for 3 days in the absence of serum, the addition of bFGF causes a rapid increase of tyrosine phosphorylation for various proteins with an optimal level after 5 min of bFGF exposure. Concomitantly, bFGF activates mitogen‐activated protein kinase (MAP kinase) activity measured with a selective MAP kinase peptide. The activity increased rapidly after the addition of bFGF and remained elevated even when cultures were treated for 1 h with bFGF. Both acidic and basic FGF were able to enhance protein tyrosine phosphorylation and MAP kinase activity, whereas nerve growth factor and epidermal growth factor did not elicit any of these responses. These data indicate that some of the transduction signals (i.e., tyrosine phosphorylation and activation of MAP kinase) that have been described for the proliferative effect of FGFs are also involved when FGFs act as trophic factors for postmitotic neurons in culture.
Release of Glutamate and Aspartate from CA1 Synaptosomes: Selective Modulation of Aspartate Release by Ionotropic Glutamate Receptor Ligands
Authors: Zhou Ming , Peterson Cathleen L. , Lu Ya‐Bin , Nadler J. Victor
Published: July 2010

Abstract: Synaptosomes prepared from area CA1 of the rat hippocampus were used to determine (a) whether Schaffer collateral‐commissural‐ipsilateral associational terminals release both aspartate and glutamate in a Ca2+‐dependent manner when reuptake of released glutamate is minimal and (b) whether autoreceptor mechanisms described in CA1 or hippocampal slices could reflect direct actions of glutamate receptor ligands on the synaptic terminal. When challenged for 1 min with either 25 mM K+ or 300 µM 4‐aminopyridine, CA1 synaptosomes released both glutamate and aspartate in a Ca2+‐dependent manner. The glutamate/aspartate ratio was ∼5:1 in each case. K+‐evoked glutamate release was unaffected by ligands active at NMDA or (RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA) receptors. Unlike glutamate release, the release of aspartate was enhanced by NMDA, and this effect was blocked by d‐2‐amino‐5‐phosphonovalerate (d‐AP5). Kainate selectively depressed and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) selectively increased the K+‐evoked release of aspartate. AMPA enhanced aspartate release, like the antagonist CNQX. When applied in the presence of diazoxide, which blocks the desensitization of AMPA receptors, AMPA and kainate both depressed aspartate release. These findings support the view that Schaffer collateral‐commissural‐ipsilateral associational terminals release aspartate as well as glutamate and that these two release processes are regulated by different autoreceptor mechanisms.
Cystic Fibrosis Transmembrane Conductance Regulator Is Found Within Brain Ventricular Epithelium and Choroid Plexus
Authors: Hincke M. T. , Nairn A. C. , Staines W. A.
Published: July 2010

Abstract: The cystic fibrosis gene product, cystic fibrosis transmembrane conductance regulator (CFTR), functions as a CI− channel that is regulated by cyclic AMP‐dependent phosphorylation. We have investigated the expression of CFTR protein in the rodent brain by both western blotting of samples prepared by microdissection and immunohistochemistry. CFTR was found to be expressed in choroid plexus and ependyma. In tissue sections, CFTR‐like immunoreactivity was concentrated in fine puncta localized about 1–2 µm from the CSF‐contacting side of ependyma and choroid plexus. CFTR in choroid plexus may play a role in the regulation of the composition of CSF by cyclic AMP‐elevating agents, but the role of this chloride transporter in ependymal function remains to be determined.
Long‐Term Induction of Tyrosine Hydroxylase Expression: Compensatory Response to Partial Degeneration of the Dopaminergic Nigrostriatal System in the Rat Brain
Authors: Blanchard Véronique , Chritin Muriel , Vyas Sheela , Savasta Marc , Feuerstein Claude , Agid Yves , Javoy‐Agid France , Raisman‐Vozari Rita
Published: July 2010

Abstract: The present study was undertaken to examine the adaptive changes occurring 1 and 6 months after moderate or severe unilateral 6‐hydroxydopamine‐induced lesions confined to the lateral part of the rat substantia nigra pars compacta (SNC). The expression of tyrosine hydroxylase (TH) enzyme was analyzed in the remaining dopaminergic nigral cell bodies and in the corresponding striatal nerve endings. In the cell bodies of the lesioned SNC, TH mRNA content was increased (+20 to +30%) 6 months after the lesion without changes in cellular TH protein amounts. The depletion of TH protein in the nerve terminal area was less severe than the percentage of cell loss observed in the SNC at 1‐ and 6‐month postlesion intervals. Moreover, the decrease in TH protein in the ipsilateral striatum was less pronounced 6 months after lesion than 1 month after. That no corresponding change in TH protein content was observed in the cell bodies at a time when TH increased in nerve terminals suggests that the newly synthesized protein is probably rapidly transported to the striatal fibers. These results suggest the existence of a sequence of changes in TH expression between cell bodies and fibers, occurring spontaneously after partial denervation of the nigrostriatal pathway.
Free Fatty Acids, Neutral Glycerides, and Phosphoglycerides in Transient Focal Cerebral Ischemia
Authors: Zhang Jian‐Ping , Sun Grace Y.
Published: July 2010

Abstract: Cerebral ischemia is known to cause an increase in levels of free fatty acids (FFAs) and diacylglycerols (DGs), although the mechanism(s) leading to these changes is not well understood. In this study, we examined FFA and DG levels along with those of other lipids in rats during and after transient focal cerebral ischemia induced by temporary occlusion of the right middle cerebral artery (MCA) and both common carotid arteries. During the duration of ischemia (15–60 min), there was a time‐dependent increase (two‐ to 10‐fold) in FFA levels in the right MCA cortex, whereas levels of DG and other lipids were not altered appreciably. FFA levels in right MCA cortex returned to near control values after reperfusion. However, following a 60‐min ischemic insult, there was a second phase of FFA level increase that was evident after 16 h. The FFAs accumulated during the ischemia period were different from those after reperfusion, suggesting differences in mechanisms for their release. During the second phase of FFA release, there were increases in levels of DGs and triacylglycerols (TGs) with unusually high proportions of 20:4(n‐6) and 22:6(n‐3). The increases in FFA, DG, and TG levels were marked by a decrease in content of phosphoglycerides (PGs). It is interesting that the increases in levels of FFAs and neutral glycerides accounted only for 10% of the total PGs depleted. The lipid changes during this reperfusion period correlated well with the development of cortical infarct. Because FFAs are potent inhibitors of mitochondrial respiratory function, the time‐dependent FFA accumulation during the ischemia period may be an important determinant for the extent of ischemia‐induced injury after reperfusion.
Iron‐Mediated Oxidation of 3,4‐Dihydroxyphenylalanine to an Excitotoxin
Authors: Newcomer Timothy A. , Rosenberg Paul A. , Aizenman Elias
Published: July 2010

Abstract: 2,4,5‐Trihydroxyphenylalanine (TOPA) oxidizes in solution to form a quinone derivative that is a non‐NMDA glutamatergic agonist and neurotoxin. DOPA can autoxidize in physiological solutions to form small amounts of both TOPA and TOPA quinone. We report here that this conversion can be dramatically enhanced by iron (II) alone, but more so by iron (II) in the presence of hydrogen peroxide. This conversion is of sufficient magnitude that the resulting product can elicit non‐NMDA, glutamate receptor‐mediated electrical responses in cultured cortical neurons isolated from rat. This finding suggests that TOPA quinone may play a role in pathological processes involving abnormal iron metabolism in catecholaminergic neurons.
Excitotoxicity at Both NMDA and Non‐NMDA Glutamate Receptors Is Antagonized by Aurintricarboxylic Acid: Evidence for Differing Mechanisms of Action
Authors: Zeevalk Gail D. , Schoepp Darryl , Nicklas William J.
Published: July 2010

Abstract: In this study, the endonuclease inhibitor aurintricarboxylic acid (ATA) was examined for its ability to attenuate both acute and delayed excitotoxicity mediated through NMDA and non‐NMDA glutamate receptors. Ex vivo embryonic chick retina, a model system frequently used for studies of excitotoxicity, was exposed to either 100 µM NMDA or kainate (KA) ± various concentrations of ATA for 60 min, then allowed to recover for 24 h. Lactate dehydrogenase release into the medium and histology were assessed as measures of delayed toxicity. ATA attenuated lactate dehydrogenase release due to NMDA or KA in a dose‐dependent manner. Histology revealed that ATA decreased the number of pyknotic profiles in response to either glutamate agonist. The mechanism of ATA protection was addressed. ATA was found to block NMDA‐ but not KA‐mediated 22Na+ influx and cyclic GMP formation. In membrane binding studies, ATA was relatively selective for displacement at the NMDA receptor. The IC50 values for displacement of [3H]CGS 19755, α‐[3H]amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid ([3H]AMPA), or [3H]KA were 29.9 ± 1.3, 313 ± 46, and >1,000 µM± SEM, respectively. ATA also fully attenuated NMDA‐induced and partially attenuated KA‐induced acute excitotoxicity as monitored histologically by tissue swelling and by the increase in GABA in the medium. Temporal studies of ATA efficacy indicated that ATA needed to be present during NMDA exposure to afford protection but, versus KA, was equally effective if administered immediately after KA exposure. Questions regarding the cellular penetration of ATA were raised because incubation with 100 µM ATA for 60 min had no effect on lactate formation or [3H]leucine incorporation into trichloroacetic acid‐precipitable material, even though, in cell‐free systems, ATA is a potent inhibitor of phosphofructokinase activity and protein synthesis. These studies demonstrate that ATA can protect against excitotoxicity mediated through NMDA or non‐NMDA glutamate receptors. The mechanism of protection versus NMDA is through interruption of NMDA receptor interactions. ATA has no direct effect at the KA receptor; thus, its mechanism of protection versus KA is distinct from that versus NMDA and is, at present, unknown.
Involvement of τ Protein Kinase I in Paired Helical Filament‐Like Phosphorylation of the Juvenile τ in Rat Brain
Authors: Takahashi Miho , Tomizawa Kayoko , Ishiguro Koichi , Takamatsu Masako , Fujita Shinobu C. , Imahori Kazutomo
Published: July 2010

Abstract: τ protein kinase I (TPKI) phosphorylates τ and forms paired helical filament epitopes in vitro. We studied temporal expression and histochemical distribution of τ phosphoserine epitopes at sites known to be phosphorylated by TPKI. Antibodies directed against phosphorylated Ser199 (anti‐PS 199) or phosphorylated Ser396 (C5 or anti‐PS 396) were used. TPKI is abundantly expressed in the young rat brain and the highly phosphorylated juvenile form of τ occurs in the same period. The activity peak of TPKI coincided with the high level of phosphorylation of Ser199 and Ser396 in juvenile τ at around postnatal day 8. By immunohistochemistry on the hippocampus and neocortex of 3–11‐day‐old rats, phosphorylated Ser396 was found in young axonal tracts and neuropil, where TPKI immunoreactivity was also detected. TPKI and phospho‐Ser199 immunoreactivities were also detected in the perikarya of pyramidal neurons. TPKI immunoreactivity had declined to a low level and phosphorylated serine immunoreactivities were undetectable in the sections of adult brain. These findings implicate TPKI in paired helical filament‐like phosphorylation of juvenile form of τ in the developing brain.
Widespread Neurotrophin Receptor Expression in the Immune System and Other Nonneuronal Rat Tissues
Authors: Lomen‐Hoerth Catherine , Shooter Eric M.
Published: July 2010

Abstract: RNase protection analysis using p75, trk A, and trk B RNA probes was used to examine mRNA expression in rat tissues, with particular emphasis on the immune system. Every tissue examined, with the exception of postnatal day 0 spleen, expressed p75 mRNA. Trk A mRNA was observed in tissues previously reported to be negative for the trk A receptor, such as kidney, thymus, lymph node, muscle, and lung. Neuronal tissues expressed only the long form of trk A, whereas nonneuronal tissues expressed both trk A forms. Trk B mRNA was expressed by the same tissues as trk A, plus heart and spleen. Neuronal tissues expressed full‐length and truncated trk B, whereas nonneuronal tissues only expressed truncated trk B. During development of the thymus p75 mRNA levels increased and trk A mRNA levels decreased. Similarly, for the spleen, p75 mRNA levels increased and those of trk B decreased during development. The expression of p75, trk A and trk B was localized primarily to the stroma of the thymus and spleen, but there was some expression by the splenocytes and thymocytes. The widespread expression of neurotrophin receptors in areas not known to be targets for neurotrophins suggests broader functions for neurotrophins outside of the nervous system.
High Levels of Ascorbic Acid, Not Glutathione, in the CNS of Anoxia‐Tolerant Reptiles Contrasted with Levels in Anoxia‐Intolerant Species
Authors: Rice Margaret E. , Lee Edward J. K. , Choy Youyin
Published: July 2010

Abstract: Ascorbic acid and glutathione (GSH) are antioxidants and free radical scavengers that provide the first line of defense against oxidative damage in the CNS. Using HPLC with electrochemical detection, we determined tissue contents of these antioxidants in brain and spinal cord in species with varying abilities to tolerate anoxia, including anoxia‐tolerant pond and box turtles, moderately tolerant garter snakes, anoxia‐intolerant clawed frogs (Xenopus laevis), and intolerant Long‐Evans hooded rats. These data were compared with ascorbate and GSH levels in selected regions of guinea pig CNS, human cortex, and values from the literature. Ascorbate levels in turtles were typically 100% higher than those in rat. Cortex, olfactory bulb, and dorsal ventricular ridge had the highest content in turtle, 5–6 µmol g−1 of tissue wet weight, which was twice that in rat cortex (2.82 ± 0.05 µmol g−1) and threefold greater than in guinea pig cortex (1.71 ± 0.03 µmol g−1). Regionally distinct levels (2–4 µmol g−1) were found in turtle cerebellum, optic lobe, brainstem, and spinal cord, with a decreasing anterior‐to‐posterior gradient. Ascorbate was lowest in white matter (optic nerve) in each species. Snake cortex and brainstem had significantly higher ascorbate levels than in rat or guinea pig, although other regions had comparable or lower levels. Frog ascorbate was generally in an intermediate range between that in rat and guinea pig. In contrast to ascorbate, GSH levels in anoxia‐tolerant turtles, 2–3 µmol g−1 of tissue wet weight, were similar to those in mammalian or amphibian brain, with no consistent pattern associated with anoxia tolerance. GSH levels in pond turtle CNS were significantly higher (by 10–20%) than in rat for several regions but were generally lower than in guinea pig or frog. GSH in box turtle and snake CNS were the same or lower than in rat or guinea pig. The distribution GSH in the CNS also had a decreasing anterior‐to‐posterior gradient but with less variability than ascorbate; levels were similar in optic nerve, brainstem, and spinal cord. The paradoxically high levels of ascorbate in turtle brain, which has a lower rate of oxidative metabolism than mammalian, suggest that ascorbate is an essential cerebral antioxidant. High levels may have evolved to protect cells from oxidative damage when aerobic metabolism resumes after a hypoxic dive.
Effect of Peroxynitrite on the Mitochondrial Respiratory Chain: Differential Susceptibility of Neurones and Astrocytes in Primary Culture
Authors: Bolaños Juan P. , Heales Simon J. R. , Land John M. , Clark John B.
Published: July 2010

Abstract: The effect of the neurotoxic nitric oxide derivative, the peroxynitrite anion (ONOO−), on the activity of the mitochondrial respiratory chain complexes in cultured neurones and astrocytes was studied. A single exposure of the neurones to ONOO− (initial concentrations of 0.01–2.0 mM) caused, after a subsequent 24‐h incubation, a dose‐dependent decrease in succinate‐cytochrome c reductase (60% at 0.5 mM) and in cytochrome c oxidase (52% at 0.5 mM) activities. NADH‐ubiquinone‐1 reductase was unaffected. In astrocytes, the activity of the mitochondrial complexes was not affected up to 2 mM ONOO−. Citrate synthase was unaffected in both cell types under all conditions studied. However, lactate dehydrogenase activity released to the culture medium was increased by ONOO− in a dose‐dependent manner (40% at 0.5 mM ONOO−) from the neurones but not from the astrocytes. Neuronal glutathione concentration decreased by 39% at 0.1 mM ONOO−, but astrocytic glutathione was not affected up to 2 mM ONOO−. In isolated brain mitochondria, only succinate‐cytochrome c reductase activity was affected (22% decrease at 1 mM ONOO−). We conclude that the acute exposure of ONOO− selectively damages neurones, whereas astrocytes remain unaffected. Intracellular glutathione appears to be an important factor for ameliorating ONOO−‐mediated mitochondrial damage. This study supports the hypothesis that the neurotoxicity of nitric oxide is mediated through mitochondrial dysfunction.