bluuebunny/crossref_metadata_2025 huggingface dataset:
Striatal Restricted Adenosine A2 Receptor (RDC8) Is Expressed by Enkephalin but Not by Substance P Neurons: An In Situ Hybridization Histochemistry Study
Authors: Schiffmann Serge N. , Jacobs Olivier , Vanderhaeghen Jean‐Jacques
Published: October 2006

RDC8 has been recently cloned and characterized as an adenosine A2 receptor. This receptor is expressed exclusively by medium‐sized neurons of the striatum as demonstrated by in situ hybridization. We have now studied the relationship of this receptor with three major components of the rat caudate‐putamen: enkephalin, substance P, and choline acetyltransferase. Our results demonstrate that the adenosine A2 receptor is expressed exclusively by the enkephalinergic striatal subpopulation but not by the substance P‐containing or cholinergic neurons.
Aminooxyacetic Acid Results in Excitotoxin Lesions by a Novel Indirect Mechanism
Authors: Beal M. Flint , Swartz Kenton J. , Hyman Bradley T. , Storey Elsdon , Finn Stephen F. , Koroshetz Walter
Published: October 2006

Aminooxyacetic acid (AOAA) is an inhibitor of several pyridoxal phosphate‐dependent enzymes in the brain. In the present experiments intrastriatal injections of AOAA produced dose‐dependent excitotoxic lesions. The lesions were dependent on a pyridoxal phosphate mechanism because pyridoxine blocked them. The lesions were blocked by the noncompetitive N‐methyl‐D‐aspartate (NMDA) antagonist MK‐801 and by coinjection of kynurenate, a result indicating an NMDA receptor‐mediated excitotoxic process. Electrophysiologic studies showed that AOAA does not directly activate ugand‐gated ion channels in cultured cortical or striatal neurons. Pentobarbital anesthesia attenuated the lesions. AOAA injections resulted in significant increases in lactate content and depletions of ATP levels. AOAA striatal lesions closely resemble Huntington's disease both neurochemically and histologically because they show striking sparing of NADPH‐diaphorase and large neurons within the lesioned area. AOAA produces excitotoxic lesions by a novel indirect mechanism, which appears to be due to impairment of intracellular energy metabolism, secondary to its ability to block the mitochondrial malate‐aspartate shunt. These results raise the possibility that a regional impairment of intracellular energy metabolism may secondarily result in excitotoxic neuronal death in chronic neurodegenerative illnesses, such as Huntington's disease.
Partial Purification of a Novel N‐Ethylmaleimide‐Activated Translational Inhibitor from Adult Rat Brain
Authors: Alcázar Alberto , Martín M. Elena , García Ana , Fando Juan L. , Salinas Matilde
Published: October 2006

A translational inhibitor that is activated by N‐ethylmaleimide treatment can be found in the postmicrosomal fraction prepared from the brain of adult rats, but it is almost undetectable in the same fraction prepared from suckling animals. The inhibitor is thermolabile and remains in the supernatant fraction after precipitation at pH 5. During the purification procedure, the inhibitor in its unactivated state binds to the anion exchanger (diethylaminoethyl‐cellulose) but not to the cation exchanger (phosphocellulose). Treatment with N‐ethylmaleimide increases inhibitor affinity for the cation exchanger, and this chromatographic step purifies the inhibitor by 143‐fold. Both the thermolabile nature and the behavior of the inhibitory activity during the different steps of the purification procedure suggest that this activity is most probably due to a protein. Although the addition of initiation factor 2 reverses the inhibition of protein synthesis in the presence of ATP and Mg2+, the inhibitor does not phosphorylate any of the initiation factor subunits “in vitro,” which indicates that it does not contain any intrinsic protein kinase activity. However, its presence in both a crude and a purified preparation of a kinase of the α subunit of a brain eukaryotic initiation factor increases the phosphorylation of the α subunit of the initiation factor. The mechanism of action of this inhibitor is discussed.
Noradrenaline‐Induced Prostaglandin Production by Sympathetic Postganglionic Neurons Is Mediated by α2‐Adrenergic Receptors
Authors: Gonzales Ralph , Sherbourne Caroline D. , Goldyne Marc E. , Levine Jon D.
Published: October 2006

In this study we have demonstrated that noradrenaline increases the levels of prostaglandin E2 and prostaglandin I2 (detected as the stable metabolite 6‐keto‐prostaglandin F1α) synthesized by homogenates of superior cervical ganglia from the adult rat. This noradrenaline‐induced prostaglandin production was further characterized: (a) Selective destruction of adrenergic sympathetic postganglionic neurons in the ganglia using 6‐hydroxydopamine abolished both basal and stimulated prostaglandin production, (b) Elimination of preganglionic cholinergic sympathetic nerve terminals in the ganglia had no effect, (c) Mepacrine (a phospholipase inhibitor) and indomethacin (a cyclooxygenase inhibitor) attenuated both basal and stimulated prostaglandin production. (d) Yohimbine, but not prazosin, suppressed the noradrenaline dose‐response curve for prostaglandin production. The results of these experiments show that, in vitro, noradrenaline stimulates de novo synthesis of prostaglandin E2 and prostaglandin I2 by sympathetic postganglionic neurons. This stimulation by noradrenaline appears to result from action at an α2‐adrenergic receptor.
Activation of High‐Affinity Uptake of Glutamate by Phorbol Esters in Primary Glial Cell Cultures
Authors: Casado Mariano , Zafra Francisco , Aragón Carmen , Giménez Cecilio
Published: October 2006

The effects of 12‐O‐tetradecanoylphorbol 13‐acetate (TPA), a potent activator of protein kinase C, on high‐affinity Na+‐dependent glutamate transport were investigated in primary cultures of neurons and glial cells from rat brain cortex. Incubation of glial cells with TPA led to concentration‐ and time‐dependent increases in the glutamate transport that could be completely suppressed by the addition of the protein kinase C (PKC) inhibitor 1‐(5‐isoquinolinylsulfonyl)‐2‐methylpiperazine. The TPA effects could be mimicked by oleoylacetylglycerol and by the diacylglycerol kinase inhibitor R59022. The effects of TPA were potentiated by the Ca2+ ionophore A23187. Under the chosen experimental conditions TPA had no effect on glutamate transport in neurons. We conclude that PKC activates the sodium‐dependent high‐affinity glutamate transport in glial cells and that it has dissimilar effects on neurons and glial cells.
Mode of Action of Palytoxin on the Release of Acetylcholine from Rat Cerebrocortical Synaptosomes
Authors: Satoh Eiki , Nakazato Yoshikazu
Published: October 2006

Palytoxin (PTX; KT−4‐10−6 M) caused a dose‐dependent increase in the release of [3H] acetylcholine ([3H] ACh), cytosolic free Ca2+ concentration ([Ca2+]i), and uptake of 22Na+ and decrease in membrane potential in rat cerebrocortical synaptosomes. The dose‐response curves for the PTX‐induced increases in [3H] ACh release and in [Ca2+]iwere depressed by removing extracellular Ca2+ or by decreasing extracellular Na+ concentrations. The release of [3H] ACh induced by concentrations of PTX < 10−10M was more dependent on the simultaneous presence of both Ca2+ and Na+than the release induced by higher concentrations of PTX. The PTX‐induced increase both in [3H] ACh release and in {Ca2+]i was almost completely abolished by the combination of Ca2+ deprivation and Na+ concentration reduction. All responses to PTX were highly resistant to 10−6M tetrodotoxin. These results suggest that low concentrations of PTX cause depolarization as a result of an increase in Na+ permeability through tetrodotoxin‐insensitive channels. This, in turn, increases Ca2+ influx and leads to an increase in the release of ACh. It appears that at high concentrations PTX increases the release of [3H] ACh by directly increasing the influx of Ca2+ into synaptosomes and by releasing Ca2+ from intracellular storage sites via an Na+‐Ca2+ exchange mechanism.
Regulation of Prostaglandin E2 Receptor Binding Activity in Porcine Temporal Cortex by Protein Phosphorylation
Authors: Morii Hiroshi , Tanemura Mihoko , Watanabe Yasuyoshi
Published: October 2006

Regulation of prostaglandin (PG) E2 receptors was investigated in a 3‐[(3‐cholamidopropyl) dimethylammonio]‐ 1‐propanesulfonate‐solubilized fraction from the synaptic membrane of porcine temporal cortex. The fraction was preincubated with exogenous protein kinases, and then the binding of PGE2 was measured. PGE2 binding was increased approximately twofold by pretreatment with the catalytic subunit of cyclic AMP‐dependent protein kinase (A kinase) or calmodulin‐dependent protein kinase II but not by that with protein kinase C. The increase was dependent on the ATP concentration, with ED50 values being close to the ATmvalues of these protein kinases. Protein kinase inhibitors specific for A kinase and for calmodulin‐dependent protein kinase II abolished the effect in a dose‐dependent manner, with IC50 values being similar to those reported. Further study using the catalytic subunit of A kinase revealed that the maximal binding capacity apparently increased without affecting the affinity and the rate constants for association and dissociation. On the other hand, acid phosphatase treatment reduced the binding activity to the level of nonspecific binding. In addition, treatment by A kinase did not affect the binding of guanosine 5′‐(3‐thiotriphosphate) by the GTP‐binding proteins and the activation of adenylate cyclase mediated by stimulatory guanine nucleotide‐binding regulatory protein, and therefore the phosphorylation is believed to occur on the receptor protein. The results suggest that the PGE2 receptor can take active phosphorylated and inactive dephosphorylated forms, of which only the phosphorylated one can bind PGE2.
Agonist‐Dependent Internalization of γ‐Aminobutyric AcidA/Benzodiazepine Receptors in Chick Cortical Neurons
Authors: Tehrani Mohammad H. Jalilian , Barnes Eugene M.
Published: October 2006

An impermeant benzodiazepine receptor ligand was prepared by derivatization of the aminobenzodiazepine 1012‐S with 4‐sulfophenylisothiocyanate. The resulting N‐sulfophenyl)‐thiocarbamoyl derivative of 1012‐S (SPTC‐1012S) was purified by reverse‐phase HPLC, and the predicted structure was verified by mass spectrometry. The apparent affinity of SPTC‐1012S (IC50= 9.8 ± 2.9 nM) for displacement of [3H] flunitrazepam from intact chick cortical neurons was similar to that of 1012‐S (IC50= 4.0 ±0.3 nM). However, at concentrations from 0.1 to 10 μM, 1012‐S was consistently more efficacious than SPTC‐1012S, a finding indicating that 6‐8% of the benzodiazepine receptor pool was not accessible to the impermeant compound. This inaccessible pool was eliminated by permeabilization of the cells with saponin or Triton ±‐100, a result suggesting that ∼7% of neuronal benzodiazepine receptors are intracellular. Acute treatment (1–4 h at 37°C) of neurons with 100μMγ‐aminobutyric acid (GABA) or 100 nM clonazepam had little effect on the level of [3H] flunitrazepam binding but increased the proportion of intracellular receptors by 61 and 74%, respectively, compared with untreated controls. Similar treatment with 1 mM GABA increased the level of intracellular sites by 154–176%. The effect of GABA on receptor internalization was blocked by cotreatment with the GABAA receptor antagonist R 5135. The results suggest that SPTC‐1012S can be used as a probe to study the internalization of the GABAA/benzodiazepine receptor complex under normal conditions or following acute or chronic treatment with agonists.
Selective and Nonselective Effects of 1‐Methyl‐4‐ Phenylpyridinium on Oxygen Consumption in Rat Striatal and Hippocampal Slices
Authors: Martin Frederick R. , Sanchez‐Ramos Juan , Rosenthal Myron
Published: October 2006

Insights into the etiology and pathophysiology of Parkinson's disease may derive from elucidation of the neurotoxic mechanisms of 1‐methyl‐4‐phenyl‐1, 2, 3, 6‐tetrahydropyridine (MPTP) and its active metabolite, I‐methyl‐4‐phenylpyridinium (MPP+). In previous studies, MPP+ provoked oxidation of cytochrome b and K+ leakage into the extracellular space of rat striatal slices. Magnitudes of these time‐dependent responses were far greater than expected had the MPP+ effects been limited to dopaminergic terminals. To determine whether cytochromes become oxidized from K+‐induced increases in ion transport activity or from electron transport inhibition at complex I, oxygen consumption was measured because this should be increased by the former and decreased by the latter mechanism. Low MPP+ concentrations (1 μM) decreased O2 consumption (∼40% in 3 h) in striatal slices. This decrease was diminished by mazindol and did not occur in hippocampal slices. High toxin concentrations (100 μM) inhibited oxygen consumption to a greater extent (∼60%) in striatal slices; this inhibition was still greater in hippocampal slices. These results support the hypothesis that acute effects of low (“selective”) MPP+ concentrations require the presence of dopaminergic terminals to trigger a sequence of destructive metabolic events but that the metabolic consequences of MPP+ spread to neighboring cells. In contrast, high MPP+ concentrations nonselectively inhibit metabolic and ion transport activity without requiring the presence of dopaminergic terminals. These results also suggest that physiological effects of “selective” MPP+ concentrations extend to nondopaminergic cells.
Mono‐ADP‐Ribosylation in Brain: Purification and Characterization of ADP‐Ribosyltransferases Affecting Actin from Rat Brain
Authors: Matsuyama Satoshi , Tsuyama Shingo
Published: October 2006

Four ADP‐ribosyltransferases that acted on non‐muscle actin were purified more than 3,000‐fold from rat brain extract. The molecular weights of these brain ADP‐ribosyltransferases were 66,000 as estimated by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and gel filtration on TSK gel G3000SW. The Km values for NAD were approximately 20 μM. These enzymes were not inhibited by thymidine or nicotinamide, but were inhibited by ADP and ADP‐ribose. Two soluble ADP‐ribosylation factors purified from rat brain had different effects on the four ADP‐ribosyltransferases during the ADP‐ribosylation of non‐muscle actin. These ADP‐ribosyltransferases modified not only actin but also the stimulatory guanine nucleotide‐binding protein of adenylate cyclase, Gs, and another guanine nucleotide‐binding protein in brain, Go. These findings suggest that the four brain ADP‐ribosyltransferases are concerned with nerve functions in the central nervous system.
Glucocorticoids Inhibit Glucose Transport and Glutamate Uptake in Hippocampal Astrocytes: Implications for Glucocorticoid Neurotoxicity
Authors: Virgin Charles E. , Ha Taryn P.‐T. , Packan Desta R. , Tombaugh Geoffrey C. , Yang Susan H. , Homer Heidi C. , Sapolsky Robert M.
Published: October 2006

Glucocorticoids (GCs), the adrenal steroid hormones secreted during stress, can damage the hippocampus and impair its capacity to survive coincident neurological insults. This GC endangerment of the hippocampus is energetic in nature, as it can be prevented when neurons are supplemented with additional energy substrates. This energetic endangerment might arise from the ability of GCs to inhibit glucose transport into both hippocampal neurons and astrocytes. The present study explores the GC inhibition in astrocytes. (1) GCs inhibited glucose transport approximately 15–30% in both primary and secondary hippocampal astrocyte cultures. (2) The parameters of inhibition agreed with the mechanisms of GC inhibition of glucose transport in peripheral tissues: A minimum of 4 h of GC exposure were required, and the effect was steroid specific (i.e., it was not triggered by estrogen, progesterone, or testosterone) and tissue specific (i.e., it was not triggered by GCs in cerebellar or cortical cultures). (3) Similar GC treatment caused a decrease in astrocyte survival during hypoglycemia and a decrease in the affinity of glutamate uptake. This latter observation suggests that GCs might impair the ability of astrocytes to aid neurons during times of neurologic crisis (i.e., by impairing their ability to remove damaging glutamate from the synapse).
Chronic Ethanol Treatment Alters Brain Levels of γ‐Aminobutyric AcidA Receptor Subunit mRNAs: Relationship to Genetic Differences in Ethanol Withdrawal Seizure Severity
Authors: Buck Kari J. , Hahner Lisa , Sikela James , Harris R. Adron
Published: October 2006

Chronic ethanol treatment is known to alter the function of the γ‐aminobutyric acidA (GABAA) benzodiazepine receptor complex. To determine if genetic differences in development of ethanol dependence are related to expression of GABAA receptor subunits, we measured whole brain levels of mRNA for the α1α3, α6, γ2s, γ2t, and γ3 receptor subunits in withdrawal seizure‐prone and ‐resistant (WSP and WSR, respectively) mice fed an ethanol‐containing liquid diet or a control diet Brain poly(A)+ RNA was converted to cDNA and amplified by the polymerase chain reaction using primers conserved among GABAA receptor subunits. Quantification was carried out by densitometric analysis of Southern blots generated using subunit‐specific probes. Chronic ethanol treatment decreased the content of α1, mRNA in WSP but not WSR mice and decreased the content of α6 mRNA in WSR but not WSP mice. The content of γ3 mRNA was increased by chronic ethanol in both lines. In untreated mice, the WSP line had lower levels of α3 and α6 mRNA than the WSR line. Thus, a decrease in the content of α1 mRNA is most clearly linked with development of withdrawal signs, although the amounts of α6 and α3 may also be important in the genetic differences between WSP and WSR mice. In contrast, levels of mRNA for γ2S and γ2L subunits do not appear to be altered in ethanol dependence.
Identification of B2 Bradykinin Binding Sites on Cultured Cortical Astrocytes
Authors: Cholewinski A. J. , Stevens G. , McDermott A. M. , Wilkin G. P.
Published: October 2006

Bradykinin was found to bind to specific high‐affinity sites in cultured cortical astrocytes from rat brain, and this binding appeared to be specific for the B2 bradykinin receptor subtype. Nonlinear regression analysis of saturation experiments using a computer programme revealed a single KD of 16.6 ± 2.6 nM and a Bmax of 352.2 ±30.7 fmol/mg of protein. These results indicate that astrocytes possess bradykinin receptors and that these are predominantly of the B2 subtype.
Identification of the New Isoforms of Mouse Myelin Basic Protein: The Existence of Exon 5a
Authors: Aruga J. , Okano H. , Mikoshiba K.
Published: October 2006

Abstract: Myelin basic protein (MBP) is a major constituent in the myelin of the CNS. In mice, five forms of MBPs (14 kDa, two types of 17 kDa, 18.5 kDa, and 21.5 kDa) encoded by separate mRNAs have been identified based on cDNA cloning studies. These mRNAs are considered to be produced by alternative splicing from a single gene composed of seven exons. Here we report the existence of two novel MBP mRNAs encoding 19.7‐kDa and 21‐kDa MBPs identified by cDNA cloning using the polymerase chain reaction. Both of these MBPs contain a sequence of a previously unidentified exon of 66 nucleotides, which was mapped to be just 5’of exon 5 in the MBP gene. MBP mRNAs containing this novel exon (exon 5a) belong to a minor population in the whole brain and PNS and are somewhat enriched in the spinal cord. Exon 5a encodes a very hydrophobic segment rich in valine residues, which presumably forms a β‐pleated sheet.
Changes in the Activity of Protein Kinase C and the Differential Subcellular Redistribution of Its Isozymes in the Rat Striatum During and Following Transient Forebrain Ischemia
Authors: Wieloch T. , Cardell M. , Bingren H. , Zivin J. , Saitoh T.
Published: October 2006

Abstract: The changes in the levels of protein kinase C [PKC(α, βII, γ)] were studied in cytosolic and particulate fractions of striatal homogenates from rats subjected to 15 min of cerebral ischemia induced by bilateral occlusion of the common carotid arteries and following 1 h, 6 h, and 48 h of reperfusion. During ischemia the levels of PKC(βII) and ‐(γ) increased in the particulate fraction to 390% and 590% of control levels, respectively, concomitant with a decrease in the cytosolic fraction to 36% and 20% of control, respectively, suggesting that PKC is redistributed from the cytosol to cell membranes. During reperfusion the PKC(βII) levels in the particulate fraction remained elevated at 1 h postischemia and decreased to below control levels after 48 h reperfusion, whereas PKC(γ) rapidly decreased to subnormal levels. In the cytosol PKC(βII) and ‐(γ) decreased to 25% and 15% of control levels at 48 h, respectively. The distribution of PKC(α) did not change significantly during ischemia and early reperfusion. The PKC activity in the particulate fraction measured in vitro by histone IIIS phosphorylation in the presence of calcium, 4β‐phorbol 13‐myristate 12‐acetate, and phosphatidylserine (PS) significantly decreased by 52% during ischemia, and remained depressed over the 48‐h reperfusion period. In the cytosolic fraction PKC activity was unchanged at the end of ischemia, and decreased by 47% after 6 h of reperfusion. The appearance of a stable cytosolic 50‐kDa PKC‐immunoreactive peptide or an increase in the calcium‐and PS‐independent histone IIIS phosphorylation was not observed. Consequently, during ischemia PKC, preferentially PKC(γ) and PKC(βII), is translocated from the cytosol and inserted into cell membranes, concomitant with a decrease in PKC activity. In the reperfusion phase the depression of PKC activity persists and the enzyme is degraded. The observed translocation and downregulation of PKC during ischemia and reperfusion may be of significance for the development of ischemic neuronal damage.
A Comparison of [3H]MK‐801 and N‐[1‐(2‐Thienyl)cyclohexyl]‐3,4‐[3H]Piperidine Binding to the N‐Methyl‐D‐Aspartate Receptor Complex in Human Brain
Authors: Steele J. E. , Robinson T. N. , Cross A. J. , Bowen D. M. , Green A. R.
Published: October 2006

Abstract: The binding of (+)‐[3H]5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate ([3H]MK‐801) and N‐[1‐(2‐thienyl)cyclohexyl]‐3,4‐[3H]piperidine ([3H]TCP) to the N‐methyl‐D‐aspartate (NMDA) receptor complex of human brain has been investigated. Significant differences were noted between the binding of the two ligands in the same tissue samples. Binding of both ligands was stimulated by addition of glutamic acid or glycine. However, addition of both compounds resulted in an additional effect with [3H]MK‐801 but not [3H]TCP binding. Saturation analysis revealed approximately twice as many high‐affinity sites for [3H]MK‐801 (Bmax, 1,500 ± 300 fmol/mg of protein) than for [3H]TCP (Bmax, 660 ± 170 fmol/mg of protein). In addition, a low‐affinity site was detected for [3H]MK‐801 binding but not [3H]TCP binding. The pharmacology of the high‐affinity [3H]MK‐801 and [3H]TCP binding sites was similar with rank order of potency of inhibitors being MK801 > TCP > phencyclidine > N‐allylnormetazocine (SKF 10047). 2‐Amino‐5‐phosphonopentanoate inhibited binding of both ligands with comparable potency whereas both 7‐chlorokynurenic acid and ZnCl2 were more potent inhibitors of [3H]MK‐801 than of [3H]TCP binding. All compounds examined exhibited Hill coefficients of significantly less than unity. Saturation analysis performed in the striatum revealed that the number of binding sites was the same for both [3H]MK‐801 (Bmax, 1,403 ± 394 fmol/mg) and [3H]TCP (Bmax, 1,292 ± 305 fmol/mg). Addition of glutamate or glycine stimulated striatal binding but there was no further increase on addition of both together. It is concluded either that MK801 and TCP do not interact with NMDA receptors in an identical manner, or that NMDA receptors in human cortical membranes are heterogeneous.
γ‐Aminobutyric AcidA Receptor Heterogeneity Is Increased by Alternative Splicing of a Novel β‐Subunit Gene Transcript
Authors: Bateson Alan N. , Lasham Annette , Darlison Mark G.
Published: October 2006

Abstract: DNA sequences encoding two variants of a novel γ‐aminobutyric acidA (GABAA) receptor β subunit were isolated from an embryonic chicken whole‐brain cDNA library and a chicken genomic library. The coding regions of these variants only differ from each other by the absence or presence of 12 bp in the region that encodes the presumed intracellular loop between transmembrane domains M3 and M4; the encoded subunits have been named β4 and β4′, respectively. The predicted mature polypeptides are 72–77% identical to the previously characterized mammalian and chicken β1,β2, and β3 subunits. Analysis of the β4‐subunit gene reveals that the different transcripts encoding the two variants arise by the use of one of two 5′‐donor splice sites that are separated by 12 bp. This is the first demonstration of alternative splicing of a GABAA receptor subunit gene transcript and represents a further mechanism for the generation of GABAA receptor heterogeneity.
Widespread Distribution of the 14‐3‐3 Protein in Vertebrate Brains and Bovine Tissues: Correlation with the Distributions of Calcium‐Dependent Protein Kinases
Authors: Ichimura Tohru , Sugano Hiroshi , Kuwano Ryozo , Sunaya Toshiyuki , Okuyama Tsuneo , Isobe Toshiaki
Published: October 2006

Abstract: A highly specific antiserum was prepared against bovine brain 14‐3‐3 protein, a protein kinase‐dependent activator of tyrosine and tryptophan hydroxylases. The immunoassay using this antiserum proved the presence of 14‐3‐3 protein in various bovine tissues and in brains of various vertebrate species. The quantitative analysis indicated that the tissue distribution of 14‐3‐3 protein is more closely related to the known distributions of the Ca2+‐dependent protein kinases, i.e., Ca2+/calmodulin‐ and Ca2+/phospholipid‐dependent protein kinases, rather than those of tyrosine and tryptophan hydroxylases. This result, together with the available data on this protein, suggests potential roles of the 14‐3‐3 protein in more diverse kinasemediated processes than the predicted role in monoamine synthesis.
Genetic Aspects of Dopamine Receptor Binding in the Mouse and Rat Brain: An Overview
Authors: Vadasz Csaba , Laszlovszky Istvan , De Simone Philip A. , Fleischer Arthur
Published: October 2006

Remarkable advances during the past few years have sparked new interest in the molecular identification of receptor types and in the spatiotemporal regulation of their expression. Because dopamine (DA) receptors have been implicated in major neuropsychiatric illnesses, such as schizophrenia and affective disorders, both of which have significant heritable components, new research is now aimed at testing hypotheses which deal with the genetic regulation of DA receptor expression and with linkage of such expression to behavioral disorders. The purpose of this article, which is not intended to be an exhaustive review, is to examine the findings of two decades of genetic studies on DA receptor expression in terms of radioligand binding and inbred mouse and rat strains, as well as to discuss some of the pitfalls in past research and new strategies in current work.
Microdetermination of 2‐Deoxyglucose and 2‐Deoxyglucose 6‐Phosphate to Determine Glucose Utilization Rates in Single Neurons and Small CNS Regions After Injecting Nontracer Amounts of 2‐Deoxyglucose
Authors: Akabayashi Akira , Kato Takahiko
Published: October 2006

Abstract: A nontracer amount (0.25 mmol/kg of body weight) of 2‐deoxyglucose (DG) was intravenously injected into rats, which were frozen 2 and 4 min later in liquid nitrogen. Freeze‐dried samples of CNS regions and cell bodies of spinal motor neurons were prepared, and the concentrations of glucose, glucose 6‐phosphate, DG, and DG 6‐phosphate (DG6P) in them were microassayed after 3,000–1,500,000‐fold amplification using an enzymatic amplification reaction, NADP cycling. Based on the time course of glucose, DG, and DG6P concentrations in arterial plasma and the anterior horn of the spinal cord, the Sokoloff‐type rate equations for DG and DG6P concentrations were mathematically solved, and the resultant DG and DG6P concentration functions were fitted to the data points using the nonlinear least‐squares fitting SALS package program. This fitting provided four rate constants for the functions and supported the theoretical basis for our calculations of glucose utilization rate (GUR) when DG was administered in nontracer amounts. The GUR was highest in the spinal motor neurons and lowest in the white matter of the cerebellum. Neuron‐rich structures, such as the cerebellar molecular and granular layers and the anterior horn of the spinal cord, had higher GUR values than the white matter of the cerebellum and spinal cord.
Induction of Nitric Oxide Synthase in Glial Cells
Authors: Simmons Martha L. , Murphy Sean
Published: October 2006

Abstract: Primary astrocyte cultures, C6 glioma cells, and N18 neuroblastoma cells were assayed for nitric oxide synthase (NOS) activity with a bioassay of cyclic GMP production in RFL‐6 fibroblasts. Treatment of astrocyte cultures for 16–18 h with lipopolysaccharide (LPS) induced NOS‐like activity that was l‐arginine and NADPH dependent, Ca2+ independent, and potentiated by superoxide dismutase. Induction was evident after 4 h, was dependent on the dose of LPS, and required protein synthesis. Treatment of astrocyte cultures with leucine methyl ester reduced microglial cell contamination from 7 to 1%, with a loss of 44% of NOS‐like activity. C6 cells treated with LPS also showed Ca2+‐independent and l‐arginine‐dependent NOS‐like activity. N18 cells demonstrated constitutive Ca2+‐dependent NOS‐like activity that was not enhanced by LPS induction. These data indicate that NOS‐like activity can be induced in microglia, astrocytes, and a related glioma cell line as it can in numerous other cell types, but not in neuron‐like N18 cells.
Cytokine Regulation of Nerve Growth Factor‐Mediated Cholinergic Neurotrophic Activity Synthesized by Astrocytes and Fibroblasts
Authors: Yoshida Kazunari , Kakihana Mitsuru , Chen Lan S. , Ong Michael , Baird Andrew , Gage Fred H.
Published: October 2006

Abstract: The neurotrophic activity of astrocytes and fibroblasts and its regulation by various cytokines were investigated. Astrocyte conditioned medium (ACM) enhanced the survival of neurons and the proliferation of astrocytes in embryonic cortical cultures grown in serum‐free defined medium. However, these results were not affected by acidic fibroblast growth factor, interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNFα), and transforming growth factor‐β1. In contrast, ACM induced choline acetyltranferase expression in septal cholinergic neurons via nerve growth factor (NGF)‐dependent and ‐independent mechanisms. However, neither acidic nor basic fibroblast growth factor is involved in this biological activity in ACM. The cytokines listed above mainly stimulate NGF‐mediated cholinergic neurotrophic activity in ACM. A combination of IL‐1β and TNFα significantly enhanced choline acetyltransferase activity in septal neurons co‐cultured with astrocytes, and this effect was found to be mediated by NGF produced by activated astrocytes. Effects of astrocytes on GABAergic neurons were also examined. ACM was found to increase glutamate decarboxylase activity in neuronal cultures from septum in the presence of Ara‐C. However, the cytokines did not enhance this activity in ACM. Moreover, a combination of IL‐1β and TNFα had no effect on glutamate decarboxylase activity in septal neurons co‐cultured with astrocytes. In a final set of experiments, cholinergic neurotrophic activity in skin‐derived fibroblast conditioned medium (FCM) was examined. FCM was found to possess biological activity similar to that of ACM on septal neurons grown in serum‐free defined medium with Ara‐C. The cytokines also enhanced NGF‐mediated cholinergic neurotrophic activity in FCM. Astrocytes and fibroblasts were found to possess NGF‐type and non‐NGF‐type cholinergic neurotrophic activity, and various cytokines were found to regulate the NGF‐type cholinergic neurotrophic activity in both types of cells. NGF produced by astrocytes and fibroblasts that are activated by cytokines is likely to be important for development and regeneration of NGF‐sensitive neurons in the central and peripheral nervous systems.
Ouabain‐Sensitive Choline Transport System in Capillaries Isolated from Bovine Brain
Authors: Galea Elena , Estrada Carmen
Published: October 2006

Abstract: In physiological conditions, there is a net transport of choline from brain to blood, despite the fact that the choline concentration is higher in plasma than in CSF. Because of the blood‐brain barrier characteristics, such passage against the concentration gradient takes place necessarily through endothelial cells. To get a better understanding of this phenomenon, [3H]choline uptake properties have been analyzed in capillaries isolated from bovine brain. [3H]‐Choline uptake was linear with time for up to 1 h. Nonlinear regression analysis of the uptake rates at different substrate concentrations gave the best fit to a system of two components, one of which was saturable (Km= 17.8 ± 4.8 μM; Vmax= 11.3 ± 3.4 pmol/min/mg of protein) and the other of which was nonsaturable at concentrations up to 200 μM. The [3H]choline transport was significantly reduced in the absence of sodium and after incubation with 10−4M ouabain for 30 min. Ouabain also inhibited choline uptake in purified cerebral endothelial cells, but not in the endothelium isolated from bovine aorta. Accordingly, cerebral endothelial cells were able to concentrate [3H]choline, with this effect being abolished by ouabain, whereas in aortic endothelial cells the [3H]choline intracellular concentration was never higher than that of the incubation medium. These results suggest that the blood‐brain barrier endothelium is specifically provided with an energy‐dependent choline transport system, which may explain the choline efflux from the brain and the maintenance of a low choline concentration in the cerebral extracellular space.
Effects of [Sar1] Angiotensin II on Proenkephalin Gene Expression and Secretion of [Met5]Enkephalin in Bovine Adrenal Medullary Chromaffin Cells
Authors: Suh Harold H. , Mar Eng‐Chun , Hudson Pearlie M. , McMillian Michael K. , Hong Jau‐Shyong
Published: October 2006

Abstract: We have studied the effect of [Sar1]angiotensin II [S1‐AII; a degradation‐resistant analogue of angiotensin II (All)] on the release of [Met5]enkephalin (ME) and proenkephalin A (proENK) gene expression. Short‐term (15‐min to 1‐h) stimulation of bovine adrenal medullary chromaffin (BAMC) cells with S1‐AII at concentrations from 0.1 to 100 nM had no significant effect on secretion of ME, whereas high concentrations of S1‐AII (3 to 100 μM) produced a concentration‐dependent increase in the concentration of ME in the incubation media. In contrast, long‐term (3‐ to 24‐h) stimulation with low concentrations (0.1 nM‐1μM) of S1‐AII increased the secretion of ME in a concentration‐dependent manner (EC50= 1 nM). The intracellular level of ME was not changed by long‐term treatment with S1‐AII (100 nM). In addition to increased ME secretion, long‐term (24‐h) stimulation with S1‐AII increased the expression of proENK mRNA in a concentration‐dependent manner (EC50= 4 nM). Losartan (2‐n‐butyl‐4 chloro‐5‐hydroxy‐methyl‐l‐[(2′‐(l H‐tetrazol‐5‐yl)biphenyl‐4‐yl)‐meth‐yl]imidazole potassium salt, a type 1 All receptor antagonist inhibited these effects, whereas PD123319 (50 μM, a type 2 All receptor antagonist) was inactive. Our results suggest that All in BAMC cells exerts a major effect on the long‐term regulation of expression of proENK mRNA and secretion of ME. These effects appear to be mediated by type 1‐like All receptors.
Inhibition by Calmodulin Antagonists of [3H]MK‐801 Binding in Brain Synaptic Membranes
Authors: Ogita Kiyokazu , Suzuki Takeo , Pingping Zuo , Yoneda Yukio
Published: October 2006

Abstract: In brain synaptic membranes not extensively washed, (+)‐5‐[3H]methyl‐10,1 l‐dihydro‐5H‐dibenzo[a,d]‐cyclohepten‐5,10‐imine ([3H]MK‐801) binding was markedly inhibited in a concentration‐dependent manner (at concentrations above 1 μM) by several compounds having antagonistic activity at the Ca2+‐binding protein calmodulin. Scatchard analysis revealed that N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7) inhibited the binding through a significant decrease in the density of binding sites without affecting the affinity at 10 μM. In membranes extensively washed and treated with a low concentration of Triton X‐100, L‐glutamic acid (Glu) drastically accelerated the initial association rate of [3H]MK‐801 binding with glycine (Gly), almost doubling the initial association rate found in the presence of Glu alone. The addition of W‐7 invariably reduced the initial association rate observed in the presence of either Glu alone or both Glu and Gly, without significantly altering the dissociation rate of bound [3H]‐MK‐801, irrespective of the presence of the two stimulatory amino acids. The maximal potencies of Glu, Gly, and spermidine in potentiating the binding were all attenuated by W‐7. These results suggest that calmodulin antagonists may interfere with opening processes of an ion channel associated with an N‐methyl‐D‐aspartate‐sensitive subclass of excitatory amino acid receptors in rat brain.
Distribution of Protein Phosphatase Inhibitor‐1 in Brain and Peripheral Tissues of Various Species: Comparison with DARPP‐32
Authors: Hemmings Hugh C. , Girault Jean‐Antoine , Nairn Angus C. , Bertuzzi Gloria , Greengard Paul
Published: October 2006

Abstract: The distribution of inhibitor‐1, a cyclic AMP‐regulated inhibitor of protein phosphatase‐1, was analyzed in various brain regions and peripheral tissues of various species by immunolabeling of sodium dodecyl sulfate‐polyacrylamide gel transfers using specific antibodies. The distribution of inhibitor‐1 was directly compared to that of DARPP‐32, a structurally related cyclic AMP‐regulated inhibitor of protein phosphatase‐1. In rat CNS, a single immunoreactive protein of Mr 30,000, identified as inhibitor‐1, was widely distributed. In contrast, DARPP‐32 was highly concentrated in the basal ganglia. Inhibitor‐1 was detected in brain tissue from frog (Mr 27,000), turtle (Mr 29,000/33,000), canary (Mr 26,000), pigeon (Mr 28,000), mouse (Mr 30,500), rabbit (Mr 26,500), cow (Mr 27,000), and monkey (Mr 27,500), but not from goldfish. Inhibitor‐1 was detected at various levels in most peripheral tissues of the species studied; however, it was not detectable in certain tissues of particular species (e.g., rat and cow liver). DARPP‐32 was detected in brain tissue of all the species tested except frog and goldfish, but was not detectable in most peripheral tissues. Both inhibitor‐1 and DARPP‐32 were concentrated in the cytosol and synaptosomal cytosol of rat striatum. The developmental expressions of inhibitor‐1 and DARPP‐32 in rat striatum differed: the level of inhibitor‐1 peaked in the first postnatal week and then declined by the third postnatal week, whereas the level of DARPP‐32 increased to a peak level by the third postnatal week and remained elevated thereafter. Because inhibitor‐1 and DARPP‐32 have distinct but partially overlapping regional distributions and developmental expression in rat CNS and have distinct tissue distributions in a number of species, it appears that their functions are not fully interchangeable.
Hippocampal 8‐[3H]Hydroxy‐2‐(Di‐n‐Propylamino)Tetralin Binding Site Densities, Serotonin Receptor (5‐HT1A) Messenger Ribonucleic Acid Abundance, and Serotonin Levels Parallel the Activity of the Hypothalamopituitary‐Adrenal Axis in Rat
Authors: Burnet Philip W. J. , Mefford Ivan N. , Smith Craig C. , Gold Philip W. , Sternberg Esther M.
Published: October 2006

Abstract: We have previously demonstrated that susceptibility of the Lewis rat to inflammatory disease, compared with the relatively resistant Fischer F344/N rat, is related to a hyporesponsive hypothalamopituitary‐adrenal axis to inflammatory and other stress mediators. Because serotonin (5‐HT) and the 5‐HT1A receptor are important stimulators of this axis, we have investigated the levels of 8‐[3H]‐hydroxy‐2,3‐(di‐n‐propylamino)tetralin binding sites, 5‐HT1A mRNA, 5‐HT, and 5‐hydroxyindoleacetic acid in various brain regions of Lewis, outbred Harlan Sprague Dawley, and Fischer F344/N rats. Lewis rats expressed significantly fewer hippocampal and frontal cortical 8‐[3H]‐hydroxy‐2,3‐(di‐n‐propylamino)tetralin binding sites and less 5‐HT1A mRNA than Harlan Sprague Dawley and Fischer F344/N rats. Adrenalectomy increased the number of 8‐[3H]hydroxy‐2,3‐(di‐n‐propylamino)tetralin binding sites and 5‐HT1A mRNA expression in the hippocampus of all three strains. Levels of hippocampal 5‐HT in Fischer F344/N rats were significantly greater than levels detected in the same regions from Lewis and Harlan Sprague Dawley rats. Hypothalamic 5‐HT and 5‐hydroxyindoleacetic acid levels in Harlan Sprague Dawley rats were higher than the same area from the other two strains. Adrenalectomy increased the levels of 5‐hydroxyindoleacetic acid in the hypo‐thalamus of all three strains. We conclude that hippocampal 5‐HT1A receptor densities and 5‐HT levels in the rat parallel the activity and responsiveness of the hypothalamopituitary‐adrenal axis.
A Novel Correlation Between the Levels of β‐Amyloid Protein Precursor and τ Transcripts in the Aged Human Brain
Authors: Oyama Fumitaka , Shimada Hiroyuki , Oyama Rieko , Titani Koiti , Ihara Yasuo
Published: October 2006

Abstract: β‐Amyloid protein precursor (APP) and τ are implicated in the pathogenesis of Alzheimer's disease. We quantified the levels of APP and τ transcripts in the three cortical regions of 38 aged human brains obtained from consecutive autopsied patients. The level of APP mRNA was directly proportional to that of τ mRNA to a remarkable extent, suggesting coordinate expression of the APP and τ genes, whereas much weaker correlations were noted among mRNAs encoding other neuronal proteins. From the previous data on the differential expression of APP and τ mRNAs, the levels of APP‐751 and ‐695 mRNAs were calculated and found to be proportional to those of four‐repeat and three‐repeat τ mRNAs, respectively, whereas that of APP‐770 mRNA was rather constant. These results suggest that the mRNA concentrations of APP isoforms are linked to those of τ isoforms in the aged human brain.
Age‐Dependent Changes in the Ultrastructure and in the Molecular Composition of Rat Brain Microtubules
Authors: Leterrier J. F. , Eyer J.
Published: October 2006

Abstract: An age‐dependent increase of a cathepsin D‐like protease activity that preferentially degrades high molecular weight microtubule‐associated proteins (MAPs) has been previously described. Microtubules (MT) purified from rat brain of different ages in the presence of several protease inhibitors retained undegraded MAPs through cycles of polymerization, and revealed several age‐dependent changes in the relative amounts of MAPs and MT‐associated kinases. MAP2 immunoreactivity was found significantly lower in MT preparations from aged animals in contrast with a relative increase of t molecules. In addition, the phosphorylation of MAP2 by its associated cyclic AMP‐de‐pendent protein kinase was also altered, consecutively to the partial loss of the enzyme during polymerization cycles and an age‐dependent decrease in the ability of the cyclic nucleotide to stimulate MAP2‐bound kinase activity. The evidence of an unusually high packing density of sedimented MT from old rat brains further suggested the modification with aging of the physical structure of the arm‐like projections of MAPs, in addition to a lower amount in high molecular weight MAPs. These results support the hypothesis of a selective alteration with aging of the mechanical and regulatory properties of brain MT, consecutive to a change in the composition and/or the structure of MAPs.
Simultaneous Effects of p‐Chloroamphetamine, d‐Fenfluramine, and Reserpine on Free and Stored 5‐Hydroxytryptamine in Brain and Blood
Authors: Martín Francisco , Artigas Francesc
Published: October 2006

Abstract: The effects of acute treatment with p‐chloroamphetamine, d‐fenfluramine, and reserpine on intracellular (brain tissue and whole blood) and extracellular (CSF and platelet‐free plasma) compartments of 5‐hydroxytryptamine (5‐HT) in the brain and blood of the same rats have been examined. These treatments affected 5‐HT in brain tissue and whole blood similarly (r= 0.823). Reserpine significantly reduced both intracellular pools at 2 and 24 h. p‐Chloroamphetamine and d‐fenfluramine were more effective on brain tissue 5‐HT. The concentration of 5‐HT in CSF was significantly increased by all treatments. p‐Chloroamphetamine induced a dramatic 70‐fold increase of CSF 5‐HT, paralleling a 42% decrease in brain tissue. d‐Fenfluramine significantly increased CSF 5‐HT to 212% of controls and reduced whole brain 5‐HT (‐23%). The effects of p‐chloroamphetamine and d‐fenfluramine on 5‐HIAA in brain, CSF, and plasma were nonsignificant. Individual values of 5‐hydroxyindoleacetic acid (5‐HIAA) in CSF and brain were highly correlated (r= 0.855), indicating that CSF 5‐HIAA reflects well the concentration of 5‐HIAA in brain tissue. Yet the intra‐ and extracellular concentrations of 5‐HIAA were unrelated to the 5‐HT changes. This indicates that CSF 5‐HIAA does not reflect the active (extracellular) compartment of 5‐HT in brain.
Striatal Protection Induced by Lesioning the Substantia Nigra of Rats Subjected to Focal Ischemia
Authors: Buisson A. , Callebert J. , Mathieu E. , Plotkine M. , Boulu R. G.
Published: October 2006

Abstract: Unilateral 6‐hydroxydopamine lesion of the substantia nigra reduced the volume of striatal necrosis and suppressed the increase in extracellular glutamate concentration in the striatum induced by middle cerebral artery occlusion in rats. These results indicate that the dopaminergic nigrostriatal pathway is highly involved in the vulnerability of the striatum to ischemia and suggest that glutamate‐dopamine interactions may play a key role in the striatal ischemic insult.
Chronic Administration of Lithium or Other Antidepressants Increases Levels of DARPP‐32 in Rat Frontal Cortex
Authors: Guitart Xavier , Nestler Eric J.
Published: October 2006

Abstract: We studied the chronic actions of lithium on rat brain by investigating its effects on cyclic AMP‐dependent protein phos‐phorylation by use of a back‐phosphorylation procedure. We identified one heavily regulated phosphoprotein in frontal cortex as the 32‐kDa dopamine‐ and cyclic AMP‐regulated phosphoprotein (DARPP‐32). Immunoblot experiments demonstrated that chronic lithium regulation of DARPP‐32 back‐phosphorylation is associated with equivalent increases in levels of DARPP‐32 immunoreactivity. Lithium regulation of DARPP‐32 immunoreactivity required chronic drug administration and was not observed in several other brain regions examined. Moreover, chronic administration of the antidepressant imipramine or tranylcypromine produced a similar increase in levels of DARPP‐32 in frontal cortex, whereas other types of psychotropic drugs, including haloperidol. morphine, and cocaine, did not influence DARPP‐32 levels. Increased levels of DARPP‐32 could reflect a common functional effect on frontal cortex of long‐term exposure to lithium and some other antidepressant medications, an effect possibly related to the clinical actions of these drugs.