bluuebunny/crossref_metadata_2025 huggingface dataset:
Biochemistry of Somatodendritic Dopamine Release in Substantia Nigra: An In Vivo Comparison with Striatal Dopamine Release
Authors: Heeringa Marten J. , Abercrombie Elizabeth D.
Published: July 2010

Abstract: The somatodendritic release of dopamine in substantia nigra previously has been suggested to be nonvesicular in nature and thus to differ from the classical, exocytotic release of dopamine described for the dopaminergic nerve terminal in striatum. We have compared the effects of reserpine, a compound that disrupts vesicular sequestration of monoamines, on the storage and release of dopamine in substantia nigra and striatum of rats. Reserpine administration (5 mg/kg, i.p.) significantly decreased the tissue level of dopamine in substantia nigra pars reticulata, substantia nigra pars compacta, and striatum. In these brain areas, reserpine‐induced reductions in tissue dopamine level occurred within 2 h and persisted at 24 h postdrug. In vivo measurements using microdialysis revealed that reserpine administration rapidly decreased the extracellular dopamine concentration to nondetectable levels in substantia nigra as well as in striatum. In both structures, it was observed that reserpine treatment significantly attenuated the release of dopamine evoked by a high dose of amphetamine (10 mg/kg, i.p.) given 2 h later. In contrast, dopamine efflux in response to a low dose of amphetamine (2 mg/kg, i.p.) was not altered by reserpine pretreatment either in substantia nigra or in striatum. The present data suggest the existence, both at the somatodendritic and at the nerve terminal level, of a vesicular pool of dopamine that is the primary site of transmitter storage and that can be displaced by high but not low doses of amphetamine. The physiological release of dopamine in substantia nigra and in striatum is dependent on the integrity of this vesicular store.
The CCK‐B Antagonist CI‐988 Increases Dopamine Levels in Microdialysate from the Rat Nucleus Accumbens via a Tetrodotoxin‐ and Calcium‐Independent Mechanism
Authors: Corwin Rebecca L. , Jörn Andreas , Hardy Melva , Crawley Jacqueline N.
Published: July 2010

Abstract: CI‐988, a water‐soluble, selective cholecystokinin‐B antagonist, was perfused through a microdialysis probe into the anterior nucleus accumbens, posterior nucleus accumbens, or caudate nucleus of anesthetized rats. High concentrations of CI‐988 produced three‐ to fivefold increases in dopamine overflow, at all three sites, that were temporally correlated with the CI‐988 perfusion and returned to baseline levels upon cessation of CI‐988 perfusion. However, the cholecystokinin‐A antagonist CAM‐1481, and the relatively inactive enantiomer of CI‐988, CAM‐1241, also increased dopamine overflow in the nucleus accumbens. Furthermore, the ability of CI‐988 to increase dopamine overflow persisted in the absence of calcium in the perfusate and was not sensitive to tetrodotoxin treatment. The mechanism by which locally administered CI‐988 increases dopamine overflow appears not to be anatomically specific, not selective for one cholecystokinin receptor subtype, and may be nonvesicular.
Glucose Regulates Glutamate‐Evoked Arachidonic Acid Release from Cultured Striatal Neurons
Authors: Williams Robert J. , Murphy Nuala , Glowinski Jacques , Prémont Joël
Published: July 2010

Abstract: l‐Glutamate stimulates the liberation of arachidonic acid from mouse striatal neurons via the activation of N‐methyl‐d‐aspartic acid (NMDA) receptors and by the joint stimulation of α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionate (AMPA) and metabotropic receptors. In this study, we investigated whether starving cultured mouse striatal neurons of glucose would modify glutamatergic receptor‐mediated arachidonic acid release. Glucose deprivation for 30 min led to enhancement of the NMDA‐evoked release of arachidonic acid, compared with that observed in the presence of glucose. This enhanced response depended on both the concentration of glucose and the length of time of glucose deprivation. The enhanced NMDA response appeared to result from both a release of glutamate and the subsequent additional release of arachidonic acid due to the activation of AMPA and metabotropic receptors. Indeed, the increased NMDA response was completely reversed when extracellular glutamate was enzymatically removed. Moreover, glucose deprivation potentiated the combined AMPA/metabotropic receptor‐evoked release of arachidonic acid, even in the absence of extracellular glutamate. However, removing glucose did not improve the calcium rise induced by AMPA or NMDA. The ATP‐evoked release of arachidonic acid from striatal astrocytes was not altered by glucose starvation. In summary, glucose deprivation affected two properties of striatal neurons: (a) it induced an NMDA‐evoked release of glutamate from striatal neurons and (b) it selectively potentiated the AMPA/(1S,3R)‐1‐aminocyclopentane‐1,3‐dicarboxylic acid‐evoked release of [3H]arachidonic acid without altering the authentic NMDA‐mediated response.
Effect of Development of Habituation to Restraint Stress on Hypothalamic Noradrenaline Release and Adrenocorticotropin Secretion
Authors: Terrazzino S. , Perego C. , De Simoni M. G.
Published: July 2010

Abstract: The effect of repeated stress has been studied on noradrenaline release in the hypothalamic paraventricular nucleus and on adrenocorticotropin levels. Rats were stressed by 20‐min immobilization once a day for 5 days. On day 6 they were exposed to the same stress or to a different one (ether vapors for 2 min). Immobilization and ether stress increased noradrenaline release in naive rats (271 ± 43 and 197 ± 9%, respectively) and raised adrenocorticotropin levels, showing activation of the hypothalamus‐pituitary axis. Repeated daily restraint did not modify basal noradrenaline or adrenocorticotropin levels. The further immobilization session on day 6 did not change noradrenaline levels at any observation time (20–120 min). The adrenocorticotropin response was still present, although significantly reduced. In repeatedly restrained rats, exposure to ether vapors induced a maximal increase in noradrenaline level similar to that observed in naive rats, although prolonged. In these rats the adrenocorticotropin response did not differ from that in acutely stressed rats. These results suggest that habituation may develop to a stressful stimulus leading to suppression of the hypothalamic noradrenergic response and that this phenomenon is stress specific. Moreover, modifications of noradrenaline release in the paraventricular nucleus are not solely responsible for the adrenocorticotropin response during stress, suggesting that other pathways and/or neurotransmitters are involved too.
Effect of Acute Stress on Hippocampal Glutamate Levels and Spectrin Proteolysis in Young and Aged Rats
Authors: Lowy Martin T. , Wittenberg Lee , Yamamoto Bryan K.
Published: July 2010

Abstract: Aging in rats is associated with a loss of hippocampal neurons, which may contribute to age‐related cognitive deficits. Several lines of evidence suggest that stress and glucocorticoids may contribute to age‐related declines in hippocampal neuronal number. Excitatory amino acids (EAAs) have been implicated in the glucocorticoid endangerment and stress‐induced morphological changes of hippocampal neurons of young rats. Previously, we have reported that acute immobilization stress can increase extracellular concentrations of the endogenous excitatory amino acid, glutamate, in the hippocampus. The present study examined the effect of an acute bout of immobilization stress on glutamate levels in the hippocampus and medial prefrontal cortex of young (3–4‐month) and aged (22–24‐month) Fischer 344 rats. In addition, the effect of stress on spectrin proteolysis in these two brain regions was also examined. Spectrin is a cytoskeleton protein that contributes to neuronal integrity and proteolysis of this protein has been proposed as an important component of EAA‐induced neuronal death. There was no difference in basal glutamate levels between young and old rats in the hippocampus or medial prefrontal cortex. During the period of restraint stress a modest increase in glutamate levels in the hippocampus of young and aged rats was observed. After the termination of the stress procedure, hippocampal glutamate concentrations continued to rise in the aged rats, reaching a level approximately five times higher than the young rats, and remained elevated for at least 2 h after the termination of the stress. A similar pattern was also observed in the medial prefrontal cortex with an augmented post‐stress‐induced glutamate response observed in the aged rats. There was no increase in spectrin proteolysis in the hippocampus or medial prefrontal cortex of young or aged rats after stress or under basal nonstress conditions. The enhanced poststress glutamate response in the aged rats may contribute to the increased sensitivity of aged rats to neurotoxic insults.
Direct Detection of Ascorbyl Radical in Experimental Brain Injury: Microdialysis and an Electron Spin Resonance Spectroscopic Study
Authors: Kihara Tsuyoshi , Sakata Shigeru , Ikeda Masato
Published: July 2010

Abstract: To examine the role played by free radicals in brain injury, we performed experiments to detect radicals in the frontal cortex of rats, using electron spin resonance (ESR) and microdialysis. A dialysis probe was inserted into the frontal cortex, and spin adducts in perfusates were immediately detected by ESR. We obtained a relatively stable doublet signal, with parameters of g = 2.0057 and aH = 0.17 mT. This signal corresponded with that of the ascorbyl radical. Ascorbyl radical in the perfusate collected from the frontal cortex was augmented by microinjection of H2O2 and FeCl2 adjacent to the dialysis probe. When the rats were challenged with cold‐induced brain injury, ascorbyl radical and lactate dehydrogenase (LDH) level in the perfusate increased significantly. Pretreatment with superoxide dismutase and catalase attenuated the increase in ascorbyl radical and LDH level induced by the cold injury. Infusion of FeCl2 dissolved in perfusate caused a pronounced increase in ascorbyl radical and LDH level after the cold injury. We conclude that the direct detection of free radical formation further supports the hypothesis that free radicals play an important role in traumatic brain injury. Our findings also indicate that combined microdialysis with ESR spectroscopy is a useful in vivo method for monitoring free radical production in the brain.
Antioxidant Defense Systems in the Brains of Type II Diabetic Mice
Authors: Makar Tapas K. , Rimpel‐Lamhaouar Karin , Abraham Dicky G. , Gokhale Vinayak S. , Cooper Arthur J. L.
Published: July 2010

Abstract: The specific activities of superoxide dismutase, catalase, and glutathione S‐transferase (μ subtype) were significantly lower in the brains of mice with type II diabetes than in the brains of control mice. On the other hand, the specific activity of glutathione peroxidase was unaltered. The concentration of vitamin E, but not that of total glutathione and ascorbate, was increased in the brains of the type II diabetic mice. The relative amount of polyunsaturated fatty acids (as determined with soybean lipoxygenase) was increased in whole brains and crude synaptosomal membranes of the type II diabetic mice. Endogenous levels of thiobarbituric acid‐positive material were decreased in both whole brain homogenates and crude synaptosomal membranes of the db/db mice. Susceptibility of lipids within whole brain homogenates and crude synaptosomal membranes of mice with type II diabetes to peroxidation with iron/ascorbate was also markedly decreased compared with that of controls. Vitamin E is known to quench lipid peroxidation. Therefore, decreased lipid peroxidation in the type II mouse brain may be due to increased vitamin E content.
Effect of Protein Kinase C Modulators on 14,15‐Epoxyeicosatrienoic Acid Incorporation into Astroglial Phospholipids
Authors: Shivachar Amruthesh C. , Willoughby Karen A. , Ellis Earl F.
Published: July 2010

Abstract: Our previous studies have shown that 14,15‐epoxyeicosatrienoic acid (14,15‐EET) is a major product of arachidonic acid metabolism in astrocytes. The purpose of this study was to investigate cellular regulation of 14,15‐EET incorporation, distribution, and metabolism in primary cultures of rat brain cortical astrocytes. Incorporation of 14,15‐EET into astrocytes was lower (93,390 ± 11,121 dpm/5 × 106 cells) than incorporation of 8,9‐EET (226,500 ± 5,567 dpm/5 × 106 cells) and arachidonic acid (321,600 ± 1,200 dpm/5 × 106 cells). 14,15‐EET was distributed in the order neutral lipids and free fatty acids (solvent front) ≫ phosphatidylcholine (PC) > phosphatidylinositol (PI) > phosphatidylethanolamine. In contrast, 8,9‐EET and arachidonic acid were exclusively incorporated into PC. During incubation, astroglial epoxide hydrolase selectively metabolized 14,15‐EET, but not 8,9‐EET, to its vic‐diol. Although 4‐phenylchalcone oxide, a potent inhibitor of epoxide hydrolase, completely inhibited 14,15‐EET metabolism, a large amount of cell‐incorporated radioactivity remained as free 14,15‐EET. Long‐term exposure of astrocytes to 4β‐phorbol 12‐myristate 13‐acetate (4β‐PMA) resulted in a time‐dependent incorporation of 14,15‐EET into PI but not in control cells exposed to 4α‐phorbol 12,13‐didecanoate. PKC down‐regulation completely inhibited epoxide hydrolase metabolism of 14,15‐EET. Following recovery of down‐regulated PKC, 1 week after treatment with 4β‐PMA, astrocytes regained their normal pattern of low incorporation of 14,15‐EET. Protein kinase C (PKC) inhibition by staurosporine enhanced 14,15‐EET incorporation without affecting its metabolism to 14,15‐dihydroxyeicosatrienoic acid. Incorporation of 14,15‐EET by PKC‐down‐regulated cells was inhibited by thimerosal, a known inhibitor of fatty acyl‐CoA synthase. Our results suggest that the lower incorporation of 14,15‐EET into astroglial cells may be due to modulation of PKC‐mediated cellular mechanism(s).
Distribution of Calretinin, Calbindin D28k, and Parvalbumin in Subcellular Fractions of Rat Cerebellum: Effects of Calcium
Authors: Winsky Lois , Kuźnicki Jacek
Published: July 2010

Abstract: The distribution of calretinin, calbindin D28k, and parvalbumin was examined in subcellular fractions prepared from rat cerebellum and analyzed by immunoblot. Calretinin was also quantified by radioimmunoassay. As expected, all three soluble, EF‐hand calcium‐binding proteins were predominantly localized in the cytosolic fraction. Calretinin and calbindin D28k were also detected in membrane fractions. Calretinin was more abundant in synaptic membrane than in microsomal fractions. The cerebellar microsomal fraction contained the greatest concentration of membrane‐associated calbindin D28k. The association of calretinin and calbindin D28k with membrane fractions was decreased in samples prepared or incubated in low calcium. Quantification of calretinin in subcellular fractions of rat cerebellum revealed a greater amount of calretinin in cytosolic fractions prepared or incubated in low calcium and reduced amounts of calretinin in all membrane fractions incubated in low calcium with the exception of the mitochondrial fraction. These results imply that calretinin and calbindin D28k might have physiological target molecules that are associated with, or are components of, brain membranes.
Rapid Dephosphorylation of τ in Heat‐Shocked Fetal Rat Cerebral Explants: Prevention and Hyperphosphorylation by Inhibitors of Protein Phosphatases PP1 and PP2A
Authors: Papasozomenos Sozos Ch. , Su Yuan
Published: July 2010

Abstract: We heat shocked 21‐ and 35‐day‐old fetal rat cerebral explants at 45°C for 18 min and performed immunocytochemistry and immunoblot analysis of sodium dodecyl sulfate extracts using the monoclonal anti‐τ antibodies Tau‐1, Tau‐5, Tau‐46, and PHF‐1 and the peroxidase‐antiperoxidase technique or 125I‐labeled protein A. Tau‐1 and PHF‐1 recognize nonphosphorylated and phosphorylated epitopes, respectively, and both Tau‐5 and Tau‐46 recognize phosphate‐independent epitopes. τ immunoreactivity was confined to neurons and increased in heat‐shocked perikarya but not axons. At 0 h after heat shocking, there was dephosphorylation of τ exemplified by (1) faster migration of τ isoforms with resultant loss or attenuation of the 60‐ and 52‐kDa τ isoforms recognized by all four anti‐τ antibodies and concomitant accentuation of the fastest moving 50‐kDa τ isoform recognized by Tau‐1, Tau‐5, and Tau‐46; and (2) significant increase in the nonphosphorylated Tau‐1 epitope with resultant decreases in the ratio of total (phosphorylated plus nonphosphorylated) τ to nonphosphorylated τ and the difference of total τ minus nonphosphorylated τ. τ was phosphorylated back to the control level by 12 h and remained so at 24 and 48 h after heat shocking. Treatment of explants with cycloheximide, a protein synthesis inhibitor, did not prevent the heat shocking‐induced dephosphorylation of τ. Treatment of explants with the inhibitors of protein phosphatases PP1 and PP2A, okadaic acid or calyculin A, produced hyperphosphorylated τ polypeptides, prevented the heat shocking‐induced dephosphorylation of τ, and intensified the immunoreactivity of the neurofilament subunit H with the only antiphosphoneurofilament antibody that reacts with intraneuronal neurofibrillary tangles. In 35‐day‐old explants, in addition to the three 50‐, 52‐, and 60‐kDa τ isoforms seen in 21‐day‐old explants, a 66‐kDa τ polypeptide was also present.
Lack of Effects of Inhibition of Nitric Oxide Synthesis on Local Glucose Utilization in the Rat Brain
Authors: Takahashi Shinichi , Cook Michelle , Jehle Jane , Kennedy Charles , Sokoloff Louis
Published: July 2010

Abstract: The effects of chronic treatment with NG‐nitro‐l‐arginine methyl ester, a potent inhibitor of nitric oxide synthase activity, on local cerebral glucose utilization were examined in conscious rats. Intraperitoneal injections of 50 mg/kg of the nitroarginine twice daily for 4 days have been found to result in almost complete inhibition of nitric oxide synthase activity in brain. Local cerebral glucose utilization was determined by means of the quantitative autoradiographic [14C]deoxyglucose method in an experimental group (n = 7) of rats that were treated with the nitroarginine according to this schedule and in a normal control group (n = 7) treated similarly with saline. The rats were conscious but partially restrained during the determinations of local cerebral glucose utilization. The nitroarginine treatment raised mean arterial blood pressure statistically significantly to 147 ± 3 mm Hg (mean ± SEM) from a level of 120 ± 5 mm Hg in the saline controls (p < 0.001 by grouped t test), but there were no statistically significant effects on glucose utilization in any of 39 brain structures examined. It is concluded that nitric oxide normally exerts no significant influence on energy metabolism in the rat brain.
Neurokinin Receptors Could Be Differentiated by Their Capacity to Respond to the Transglutaminase‐Synthesized γ‐(Glutamyl5)Spermine Derivative of Substance P
Authors: Esposito C. , Mancuso F. , Calignano A. , Di Pierro P. , Pucci P. , Porta R.
Published: July 2010

Abstract: Four different γ‐(glutamyl5)amine derivatives of substance P (SP) were synthesized in vitro in the presence of purified guinea pig liver transglutaminase and Ca2+. The 1,3‐diaminopropane, spermidine, spermine (Spm), and monodansylcadaverine adducts of the neuropeptide were purified by HPLC on a reversed‐phase column and characterized by fast atom bombardment mass spectrometry. The γ‐(glutamyl5)Spm derivative of SP (Spm‐SP) was found to be able, like the parent neuropeptide, to provoke rabbit aorta relaxation, to decrease rat arterial blood pressure, and to inhibit collagen‐induced platelet aggregation. Unlike SP, only a weak inflammatory response was observed when Spm‐SP was injected in the rat hind limb. All these effects were found to be prevented by Nω‐nitro‐l‐arginine methyl ester, a well‐known nitric oxide synthesis inhibitor. In contrast, Spm‐SP was completely ineffective in contracting guinea pig ileal segments, thus confirming our preliminary observations indicating that Spm‐SP does not evoke SP‐like spasmogenic effects on isolated smooth muscle preparations. The specificity of the effects due to the selective introduction of a Spm moiety at the glutamine5 level was demonstrated by the SP agonist pharmacological profile of the other γ‐(glutamyl5)amine derivatives tested. These results suggest that neurokinin receptors could be differentiated by their capacity to respond to Spm‐SP.
Processing of Procarboxypeptidase E into Carboxypeptidase E Occurs in Secretory Vesicles
Authors: Song Lixin , Fricker Lloyd
Published: July 2010

Abstract: Carboxypeptidase E (CPE) functions in the posttranslational processing of bioactive peptides. Like other peptide processing enzymes, CPE is initially produced as a precursor (“proCPE”) that undergoes posttranslational processing at a site containing five adjacent Arg residues near the N‐terminus and at other sites near the C‐terminus of proCPE. The time course of the N‐terminal processing step suggests that this conversion occurs in either the Golgi apparatus or the secretory vesicles. To delineate further the site of proCPE processing, pulse/chase analysis was performed under conditions that block transit out of the Golgi apparatus (brefeldin A, carbonyl cyanide m‐chlorophenylhydrazone, or 20°C) or that block acidification of vesicles (chloroquine, monensin, or ammonium chloride). The results of these analysis suggest that efficient proCPE processing requires an acidic post‐Golgi compartment. To test whether known processing enzymes can perform this cleavage, purified proCPE was incubated with furin, prohormone convertase 1, or a dynorphin converting enzyme, and the products were analyzed on denaturing polyacrylamide gels. Furin cleaves proCPE within the N‐terminal region, although the reaction is not very efficient, requiring relatively large amounts of furin or long incubation times. The other two peptide processing enzymes did not cleave proCPE, whereas a relatively small amount of secretory granule extract was able to convert proCPE into CPE. Taken together, these findings suggest that the conversion of proCPE into CPE occurs primarily in secretory vesicles.
Ceramide Prevents Neuronal Programmed Cell Death Induced by Nerve Growth Factor Deprivation
Authors: Ito Akira , Horigome Kazuhiko
Published: July 2010

Abstract: We examined the ability of ceramide and sphingomyelinase (SMase) to prevent neuronal programmed cell death (PCD). We found that a cell‐permeable ceramide analogue prevented neuronal PCD when applied to established sympathetic neuron primary cultures at the time of nerve growth factor (NGF) deprivation. Other amphiphilic lipids such as oleic acid failed to prevent cell death. Exogenous SMase also showed the same effect, probably by raising the intracellular ceramide level by sphingomyelin (SM) breakdown. Phosphocholine, another hydrolytic product of SM by SMase, did not prevent cell death. Other phospholipases, such as phospholipase C and phospholipase A2, could not prevent cell death. Given the recent findings that the SM cycle is activated to increase the intracellular ceramide level on NGF binding to the low‐affinity NGF receptor (LNGFR) and that NGF binding to LNGFR suppresses apoptosis in neural cell lines, our results suggest the possibility of the SM cycle as a signaling mechanism transducing the PCD‐preventing activity of NGF.
Developmental Changes in the Differential Expression of Two Serotonin 5‐HT3 Receptor Splice Variants in the Rat
Authors: Miquel M.‐C. , Emerit M. B. , Gingrich J. A. , Nosjean A. , Hamon M. , El Mestikawy S.
Published: July 2010

Abstract: PCR was used to isolate identical partial cDNA clones encoding a serotonin 5‐HT3 receptor subunit from rat nodose and superior cervical ganglia. The amino acid sequence predicted from these clones, extending from the putative transmembrane domain I to the stop codon, demonstrated a 93% homology with the 5‐HT3 receptor A (R‐A) subunit cloned from NCB 20 hybridoma mouse neuroblastoma/Chinese hamster embryonic brain cells. Comparison of the sequences of the rat gene and cDNA encoding this subunit revealed a five amino acid deletion, GSLLP, located within the putative second intracellular loop of the receptor subunit. This deletion was shown to occur at an intron/exon junction. Therefore, alternative splicing was probably responsible for the presence of short (5‐HT3 R‐AS) and long (5‐HT3 R‐AL) forms of 5‐HT3 R‐A mRNA in these ganglia. PCR experiments, with specific primers located upstream and downstream of the GSLLP deletion, were used to detect reverse transcribed 5‐HT3 R‐A mRNAs. A short fragment (92 bp), corresponding to the deleted form, and a long fragment (107 bp), corresponding to the nondeleted form, were amplified from various regions of the CNS and peripheral ganglia of the rat, as well as from NG108‐15 hybridoma cells. In the adult rat, the ratio of the two forms varied very little from one tissue to another, the long form corresponding to only ∼10% of the total 5‐HT3 R‐A mRNA. Study of their respective distributions during ontogeny demonstrated a differential expression of the short and long forms in some tissues during late embryonic development, at embryonic day 17 (E17) or E20. In particular, the long form amounted to about one‐third of the total 5‐HT3 R‐A mRNA in the cerebral cortex and hippocampus at E17, and this proportion reached 50 and 75% in the superior cervical ganglion and nodose ganglion, respectively, at E20. These data indicate that alternative splicing of the 5‐HT3 R‐A mRNA is regulated in the CNS and PNS during development in the rat.
The Human Choline Acetyltransferase Gene Encodes Two Proteins
Authors: Grosman Daren D. , Lorenzi Matthew V. , Trinidad Alma C. , Strauss William L.
Published: July 2010

Abstract: Both, 2,500‐ and 6,000‐nucleotide (nt) mRNAs are generated by alternative splicing of the primary transcript from the human gene for choline acetyltransferase (ChAT), the 68‐kDa enzyme that synthesizes acetylcholine. In vitro translation of cRNA derived from a clone of the 2,500‐nt mRNA produced a protein with ChAT activity demonstrating that this transcript encodes the human ChAT enzyme. An antibody directed against a unique amino acid sequence predicted from the 6,000‐nt ChAT gene transcript identified a 27‐kDa protein on immunoblots of human nucleus basalis proteins. This protein was further shown to cross‐react with antibodies prepared against the 68‐kDa human ChAT enzyme. Gel‐filtration chromatography of human nucleus basalis proteins demonstrated that the 27‐kDa protein does not have ChAT activity, which eluted as a single peak coincident with that of the 68‐kDa enzyme. The 27‐kDa protein was, however, shown to colocalize with the ChAT enzyme in cholinergic neurons of the human spinal cord using immunohistochemical techniques.
Expression of Cytochrome P450 Side‐Chain Cleavage Enzyme and 3β‐Hydroxysteroid Dehydrogenase in the Rat Central Nervous System: A Study by Polymerase Chain Reaction and In Situ Hybridization
Authors: Sanne Jean‐Luc , Krueger Karl E.
Published: July 2010

Abstract: In examining steroid synthesis in the CNS, expression of the mRNAs encoding for cytochrome P450 side‐chain cleavage enzyme (P450SCC) and 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 isomerase (3β‐HSD) has been studied in the rat brain. P450SCC transforms cholesterol into pregnenolone and 3β‐HSD transforms pregnenolone into progesterone. PCR was used to amplify cDNA sequences from total RNA extracts. Classical steroidogenic tissues, like adrenal and testis, as well as the non‐steroidogenic tissue lung have been used as controls. The expression of P450SCC and 3β‐HSD have been demonstrated by PCR in cortex, cerebellum, and spinal cord. In addition, primary cultures of rat cerebellar glial cells and rat cerebellar granule cells were found to express P450SCC and 3β‐HSD at comparable levels. Furthermore, three of the four known isoenzymes of 3β‐HSD were identified, as determined using selective PCR primers coupled with discriminative restriction enzymes and sequencing analysis of the amplified brain products. Using RNA probes, in situ hybridization indicated that P450SCC and 3β‐HSD are expressed throughout the brain at a low level and mainly in white matter. Enrichment of glial cell cultures in oligodendrocytes, however, does not increase the relative abundance of P450SCC and 3β‐HSD mRNA detected by PCR. This discrepancy suggests that the developmental state of cultured cells and their intercellular environment may be critical for regulating the expression of these enzymes. These findings support the proposal that the brain apparently has the capacity to synthesize progesterone from cholesterol, through pregnenolone, but that the expression of these enzymes appears to be quite low. Furthermore, the identification of these messages in cerebellar granule cell cultures implies that certain neurons, in addition to glial cells, may express these steroidogenic enzymes.
Expression of the Human β2‐Adrenoceptor in NCB20 Cells Results in Agonist Activation of Adenylyl Cyclase and Agonist‐Mediated Selective Down‐Regulation of Gsα
Authors: Mullaney Ian , Shah Bukhtiar H. , Wise Alan , Milligan Graeme
Published: July 2010

Abstract: Murine neuroblastoma × embryonic Chinese hamster brain NCB20 cells were transfected with a construct containing the human β2‐adrenoceptor under the control of a β‐actin promoter. Two clones were selected for detailed analysis: D1, which expressed some 12.7 pmol/mg of membrane protein, and L9, which expressed 1.2 pmol/mg of membrane protein of the receptor. Incubation with the β‐adrenoceptor agonist isoprenaline resulted in stimulation of adenylyl cyclase activity in both of the clones, whereas no such activation was observed in wild‐type NCB20 cells. The EC50 for isoprenaline stimulation of adenylyl cyclase activity in membranes of clone D1 (0.8 nM) was significantly lower, however, than in membranes of clone L9 (10.4 nM). Although the maximal adenylyl cyclase stimulation by isoprenaline was similar in both clones, D1 had a higher basal activity. Immunoblotting studies with specific antipeptide antisera directed against various G protein α subunits showed that treatment of the cells with isoprenaline resulted in a 35% reduction in the membrane‐associated levels of Gsα in membranes of clone L9 cells and a 50% reduction in Gsα levels in membranes prepared from clone D1. Isoprenaline treatment had no effect on the levels of Gsα in wild‐type NCB20 cells, and such treatment had no effect on the levels of other G protein α subunits such as Gq/G11 and Gi2 in any of the cell lines investigated. Time course analysis revealed that half‐maximal loss of Gsα in clone D1 was achieved within 1–2 h of addition of agonist. Dose‐response curves to isoprenaline in clone D1 indicated that half‐maximal down‐regulation of Gsα was produced by ∼1 nM agonist. Measurement of Gs mRNA levels in both clones, however, using both reverse transcriptase‐polymerase chain reaction and northern blotting revealed no significant change following treatment with isoprenaline.
Identification of Inducible Mixed Function Oxidase System in Rat Glioma C6 Cell Line
Authors: Geng Jun , Strobel Henry W.
Published: July 2010

Abstract: The mixed function oxidase system consists of NADPH‐cytochrome P450 reductase (P450 reductase) and various isoforms of cytochrome P450 (P450), which can catalyze the oxidation of a broad range of endogenous and exogenous compounds. In this study, we examined the rat glioma C6 cell line for the presence of P450 reductase and three isozymes of cytochrome P450, 1A1, 2B1, and 2B2, by reverse transcription followed by PCR (RT‐PCR). Rat glioma C6 cells were treated with hepatic P450 inducers phenobarbital (PB) or benzo(a)anthracene (BA). Cytochromes P450 1A1, P450 2B1, and P450 2B2, and P450 reductase, were detected in all the different treatment groups. Restriction digestion was used to confirm the PCR fragments and the expected digestion products were obtained. The induction of P450 1A1 and 2B was quantified using competitive PCR. Ten‐ and five‐fold inductions of P450 1A and 2B mRNA after BA or PB treatments, respectively, were detected by competitive PCR. Microsomes prepared from rat glioma C6 cells showed cytochrome P450 spectra with absorption at 450 nm. Ethoxyresorufin O‐deethylase activity (11.5 ± 1.7 pmol/min/mg of microsomal protein) and pentoxyresorufin O‐dealkylation activity (8.9 ± 1.4 pmol/min/mg of microsomal protein) confirmed the induction of P450 1A and 2B at the protein level in response to BA or PB treatments, respectively. These experiments provide further evidence that the rat glioma C6 cell line contains an active mixed function oxidase system that can be induced by hepatic P450 inducers.
Involvement of Protein Kinase C in Ca2+‐Signaling Pathways to Activation of AP‐1 DNA‐Binding Activity Evoked via NMDA‐ and Voltage‐Gated Ca2+ Channels
Authors: Ohtani Ken‐ichi , Sakurai Hiroaki , Oh Esther , Iwata Emi , Tsuchiya Tomofusa , Tsuda Masaaki
Published: July 2010

Abstract: Stimulation of cultured cerebellar granule cells with N‐methyl‐d‐aspartate (NMDA) or kainic acid (KA) leads to activation of activator protein‐1 (AP‐1) DNA‐binding activity, which can be monitored by an increase in 12‐O‐tetradecanoylphorbol 13‐acetate (TPA)‐responsive element (TRE)‐binding activity, in concert with c‐fos induction. For this increase in TRE‐binding activity, Ca2+ influx across the plasma membrane is essential. Treatment of cells with an intracellular Ca2+ chelator, BAPTA‐AM, abolished this increase. Close correspondence between the dose‐response curves of 45Ca2+ uptake and TRE‐binding activity by NMDA or KA suggested that Ca2+ influx not only triggered sequential activation of Ca2+‐signaling processes leading to the increase in TRE‐binding activity, but also controlled its increased level. Stimulation of non‐NMDA receptors by KA mainly caused Ca2+ influx through voltage‐gated Ca2+ channels, whereas stimulation of NMDA receptors caused Ca2+ influx through NMDA‐gated ion channels. The protein kinase C (PKC) inhibitors staurosporine and calphostin C inhibited the increase in TRE‐binding activity caused by NMDA and KA at the same concentration at which they inhibited that caused by TPA. Furthermore, down‐regulation of PKC inhibited the increase in TRE‐binding activity by NMDA and KA. Thus, a common pathway that includes PKC could, at least in part, be involved in the Ca2+‐signaling pathways for the increase in TRE‐binding activity coupled with the activation of NMDA‐ and non‐NMDA receptors.
Neuroprotection by the N‐Methyl‐d‐Aspartate Receptor Antagonist CGP 40116: In Vivo and In Vitro Studies
Authors: Maier Carolina M. , Sun Guo Hua , Kunis David M. , Giffard Rona G. , Steinberg Gary K.
Published: July 2010

Abstract: The goal of this study was to evaluate the effects of a novel competitive N‐methyl‐d‐aspartate (NMDA) receptor antagonist, d‐(E)‐2‐amino‐4‐methyl‐5‐phosphono‐3‐pentoic acid (CGP 40116), on neuronal damage in vivo and in vitro. We studied 20 rabbits that underwent a 2‐h occlusion of the left internal carotid, anterior cerebral, and middle cerebral arteries followed by 4 h of reperfusion. Ten minutes after occlusion the animals were treated with either normal saline (n = 7) or CGP 40116 at two different doses (20 mg/kg, n = 6; 40 mg/kg, n = 7) administered over a 5‐min period. Somatosensory evoked potentials were used to confirm adequate ischemia and neuronal injury was assessed by histopathology and magnetic resonance imaging. CGP 40116 decreased cortical ischemic neuronal damage by 74 and 77% (control, 37.8%± 13.1%; CGP 20 mg/kg, 9.9 ± 3.6%; CGP 40 mg/kg, 8.7 ± 3.7%; p < 0.01) and reduced cortical ischemic edema by 52 and 35% (control, 42.3 ± 10.4%; CGP 20 mg/kg, 20.1 ± 6.7%; CGP 40 mg/kg, 27.5 ± 13.3%; p < 0.05) but did not protect against striatal injury. We performed a second study using primary cell cultures from mouse neocortex to determine the effects of CGP 40116 on neuronal death induced by a 10‐min exposure to 500 µM NMDA or by 45 min of oxygen‐glucose deprivation (OGD). Our results demonstrate that CGP 40116 was effective at attenuating neuronal death in a concentration‐dependent manner (ED50 of 3.2 µM against NMDA toxicity and 23.1 µM against OGD) as measured by lactate dehydrogenase levels 24 h after the insult. The neuroprotective effects of CGP 40116 in vivo and in vitro suggest it may be of potential clinical therapeutic value.
Mice Transgenic for Copper/Zinc Superoxide Dismutase Exhibit Increased Markers of Biogenic Amine Function
Authors: Schwartz Paul J. , Berger Urs V. , Coyle Joseph T.
Published: July 2010

Abstract: Mice that are transgenic for and overexpress human copper/zinc superoxide dismutase were used to investigate the role of this enzyme in the pathophysiology of Down's syndrome (DS; trisomy 21). Previous studies have indicated that overexpression of copper/zinc superoxide dismutase leads to deficits in peripheral markers of neurochemical function, which are consistent with the hypothesis that this enzyme plays a role in the pathophysiology of DS. We have measured concentrations of amino acids and biogenic amines (catecholamines, serotonin, and their metabolites), uptake of biogenic amines into crude synaptosomes, and activities of synthetic enzymes in both control mice and mice transgenic for human copper/zinc superoxide dismutase that overexpress it by two‐ to fivefold above control values. We find that these transgenic mice exhibit higher concentrations of the biogenic amines in specific brain regions, with little or no change in amino acid concentration. Furthermore, tyrosine hydroxylase activity is increased in the striatum of the transgenics, whereas glutamic acid decarboxylase and choline acetyltransferase activities are unchanged in all but one brain region. These findings indicate that over‐expression of copper/zinc superoxide dismutase, by itself, is not sufficient to cause the synaptic neurochemical deficits reported in DS.
Visualization of Antipsychotic Drug Binding to Living Mesolimbic Neurons Reveals D2 Receptor, Acidotropic, and Lipophilic Components
Authors: Rayport Stephen , Sulzer David
Published: July 2010

Abstract: To examine the binding of antipsychotic drugs to living neurons, we applied fluoroprobe derivatives of the D2 antagonist spiperone to mesolimbic system neurons in postnatal culture. We found that rhodamine‐N‐(p‐aminophenethyl)spiperone (rhodamine‐NAPS) stereospecifically labeled the plasma membranes of 38 ± 6% of ventral tegmental area neurons, 22 ± 7% of which were dopaminergic, and 50 ± 6% of medium‐sized putatively GABAergic nucleus accumbens neurons, with a time constant of ∼8 min. In contrast, the BODIPY derivative of NAPS rapidly labeled intracellular sites in all neurons in a punctate pattern, consistent with acidotropic uptake. Native antipsychotics also show acidotropic uptake, which we visualized by their displacement of the fluorescent weak base vital dye acridine orange from acidic intracellular compartments. We found that acidotropic uptake correlated best with the partition coefficients of the drugs. With a time constant of 23 min, rhodamine‐NAPS labeled all neurons in a pattern suggestive of lipophilic solvation. Thus, initially rhodamine‐NAPS makes possible visualization of D2 receptors on living neurons; however, acidotropic uptake and lipophilic solvation obscure receptor labeling and may account for time‐dependent factors in the action of antipsychotic drugs, as well as affect their use as radioreceptor ligands.
Resuscitation of Brain Neurons in the Presence of Ca2+ After Toxic NMDA‐Receptor Activity
Authors: Eimerl Sara , Schramm Michael
Published: July 2010

Abstract: Cultured cerebellar granule cells were subjected to toxic activation of the NMDA receptor that was terminated by MK‐801. Subsequent resuscitation experiments were mostly conducted in the presence of a physiological concentration of Ca2+. Addition of pyruvate and inorganic phosphate, in addition to glucose, which was always present, rescued ∼40% of the dying neurons. La3+ and ruthenium red were also effective resuscitating agents. The combination of pyruvate, inorganic phosphate, and ruthenium red rescued 65% of the dying neurons. Parallel studies with 45Ca indicated that La3+ and ruthenium red facilitated the decrease of 45Ca in the neurons, whereas inorganic phosphate, supported by energy‐yielding pyruvate, formed perhaps, a less harmful Ca complex inside the neurons.
Identification of the 5‐Hydroxytryptamine2C Receptor as a 60‐kDa N‐Glycosylated Protein in Choroid Plexus and Hippocampus
Authors: Abramowski Dorothee , Staufenbiel Matthias
Published: July 2010

Abstract: The rat 5‐hydroxytryptamine2C (5‐HT2C) receptor was identified as N‐glycosylated polypeptide of 60‐kDa apparent molecular mass using antibodies against its putative third and fourth (C‐terminal) cytoplasmic domain. To show that the polypeptides detected on western blots and by immunoprecipitation represent the 5‐HT2C receptor, binding studies of the 5‐HT2C ligand [3H]‐mesulergine to immunoprecipitates from extracts of pig choroid plexus were performed. We demonstrate the presence of a signal sequence that was cleaved off during membrane insertion resulting in a 38‐kDa polypeptide. During further maturation, the receptor was N‐glycosylated at two sites via a 48‐kDa intermediate. This intermediate was far more abundant in choroid plexus than in hippocampus and may represent an intracellular receptor reserve. After transfection of 5‐HT2C cDNAs into cultured cells, polypeptides were observed that differed from the ones found in vivo due to abnormal N‐glycosylation and possibly other alterations depending on the system used. Thus the 5‐HT2C receptor expressed in cell lines may also differ in function from the receptor in its native tissue.
Brain Histaminergic System in Mast Cell‐Deficient (Ws/Ws) Rats: Histamine Content, Histidine Decarboxylase Activity, and Effects of (S)α‐Fluoromethylhistidine
Authors: Sugimoto Koreaki , Maeyama Kazutaka , Alam Md. Khorshed , Sakurai Eiko , Onoue Hitoshi , Kasugai Tsutomu , Kitamura Yukihiko , Watanabe Takehiko
Published: July 2010

Abstract: The mast cell‐deficient [Ws/Ws (White spotting in the skin)] rat was investigated with regard to the origin of histamine in the brain. No mast cells were detected in the pia mater and the perivascular region of the thalamus of Ws/Ws rats by Alcian Blue staining. The histamine contents and histidine decarboxylase (HDC) activities of various brain regions of Ws/Ws rats were similar to those of +/+ rats except the histamine contents of the cerebral cortex and cerebellum. As the cerebral cortex and cerebellum have meninges that are difficult to remove completely, the histamine contents of these two regions may be different between Ws/Ws and +/+ rats. We assume that the histamine content of whole brain with meninges in Ws/Ws rats is <60% of that in +/+ rats. So we conclude that approximately half of the histamine content of rat brain is derived from mast cells. Next, the effects of (S)α‐fluoromethylhistidine (FMH), a specific inhibitor of HDC, on the histamine contents and HDC activities of various regions of the brain were examined in Ws/Ws rats. In the whole brain of Ws/Ws rats, 51 and 37% of the histamine content of the control group remained 2 and 6 h, respectively, after FMH administration (100 mg/kg of body weight). Therefore, we suggest that there might be other histamine pools including histaminergic neurons in rat brain.
Thermodynamic Parameters of Frog Brain κ‐Opioid Receptors
Authors: Moitra Jaideep , Öktem Huseyin Avni , Borsodi Anna
Published: July 2010

Abstract: The thermodynamic parameters for [3H]‐ethylketocyclazocine binding in frog (Rana esculenta) brain membranes have been examined. Computer‐based nonlinear regression analysis of the untransformed equilibrium displacement data showed that this ligand bound to two sites with different affinities and capacities in this tissue. KA values derived from equilibrium displacement curves have been used for calculating the changes in the standard Gibbs energy, enthalpy, and entropy during the binding process. Van't Hoff plots are bipartite, with transitions occurring at 18°C for both the high‐ and the low‐affinity sites. For the high‐affinity site, the reaction appears to be associated with a decrease in enthalpy below the transition temperature and a significant gain in entropy above this temperature. The reverse appears to be true for the low‐affinity site. We conclude that this profile fairly approximates the mixed agonist‐antagonist nature of this ligand and surmise that thermodynamic analysis could be a very useful tool for characterization of the nature of cloned opioid receptors in vitro.
Molecular Species Analysis of 1,2‐Diglycerides on Phorbol Ester Stimulation of LA‐N‐1 Neuroblastoma Cells During Proliferation and Differentiation
Authors: Lang D. , Leray C. , Mestre R. , Massarelli R. , Dreyfus H. , Freysz L.
Published: July 2010

Abstract: 1,2‐Diacyl‐sn‐glycerol (DAG) is a product of cell activation that has emerged as an important intracellular messenger whose primary function appears to be the activation of protein kinase C. They originate by the activation of phospholipases, which hydrolyze different phospholipids depending on the external stimulus and the nature of the cells, leading to the production of different molecular species. In the present study the quantitative changes in the total mass and the molecular species of DAG formed on phorbol ester (12‐O‐tetradecanoylphorbol 13‐acetate) stimulation were investigated in proliferating and retinoic acid (RA)‐differentiated human LA‐N‐1 cells. The TPA treatment of both cell types elicited an increase in the total amount of DAG. The increase was biphasic; i.e., an initial peak at 2–5 min was followed by a sustained increase that persisted for >30 min. The analysis of the molecular species of DAG and phospholipids showed that in proliferating LA‐N‐1 cells, the DAG increase corresponds to the production of mainly saturated/monounsaturated (16:0–18:1, 18:0–18:1) and saturated/saturated (16:0–16:0, 16:0–18:0) species, suggesting that they originate essentially from the hydrolysis of phosphatidylcholine. In contrast, RA‐differentiated cells responded to TPA treatment by increasing the level of saturated/polyunsaturated (16:1–22:6, 18:0–22:6, 16:0–20:4, 18:0–20:4) and monounsaturated/monounsaturated (18:1‐18:1) species, suggesting mainly a phosphatidylethanolamine origin. These findings indicate that the treatment of LA‐N‐1 cells with TPA generates different molecular species of DAG depending on their physiological state. These observations suggest in turn that different phospholipases are activated by TPA in proliferating and RA‐differentiated cells.