bluuebunny/crossref_metadata_2025 huggingface dataset:
Reversible Alterations of Cerebral γ‐Aminobutyric Acid in Pyrithiamine‐Treated Rats: Implications for the Pathogenesis of Wernicke′s Encephalopathy
Authors: Héroux Maryse , Butterworth Roger F.
Published: October 2006

Abstract: Treatment of rats with the central thiamine antagonist, pyrithiamine, results in severe neurological symptoms such as loss of righting reflex. Measurement of γ‐aminobutyric acid (GABA) content of brain tissue from symptomatic pyrithiamine‐treated (PT) rats revealed significant reductions in thalamus, cerebellum, and pons. GABA content of cerebral cortex, however, was unaltered. Activities of the thiamine‐dependent enzyme α‐ketoglutarate dehydrogenase (aKGDH) were reduced in parallel with the GABA changes. On the other hand, activities of the GABA‐synthetic enzyme glutamic acid decarboxylase (GAD) remained within normal limits, with the exception of a small but significant decrease in thalamus of symptomatic PT rats. Affinities and densities of high‐affinity [3H]muscimol binding sites on crude cerebral membrane preparations from symptomatic PT rats were unchanged. Thiamine administration to symptomatic animals resulted in correction of abnormal righting reflexes and in normalization of decreased GABA levels and reduced αKGDH activities in cerebellum and pons. Thalamic GABA levels and αKGDH activities, on the other hand, remained significantly lower than normal. These results suggest that the reversible symptoms of pyrithiamine treatment may result from impaired GABA synthesis in cerebellum and pons of these animals. Similar mechanisms may play a role in the pathogenesis of the reversible symptoms of Wernicke′s encephalopathy in man.
Proenkephalin A Processing in the Upper Digestive Tract: Isolation and Characterisation of Phosphorylated N‐Terminally Extended Met‐Enkephalin Arg6Phe7 Variants
Authors: Watkinson Allan , Dockray Graham J. , Young Janice , Gregory Harry
Published: October 2006

Abstract: Previous studies suggest the processing of proenkephalin A in the porcine upper digestive tract might differ from that in the brain. To characterise more precisely some of the products, we have used antibodies to Met‐enkephalin Arg6Phe7 (MERF) in radioimmunoassay to monitor the isolation of immunoreactive peptides from extracts of porcine pyloric antral muscle, antral mucosa, and duodenum. Seph‐adex G50 gel filtration of each extract produced a single broad peak of high‐molecular‐weight MERF‐immunoreactivity. On anion‐exchange chromatography the antral muscle MERF‐immunoreactivity fractionated into two major peaks, and that from the antral mucosa and duodenum each into four major peaks, suggesting tissue specific processing of proenkephalin A within the porcine gut. Reverse‐phase HPLC and Edman degradation analysis revealed that the least acidic antral muscle peptide was a 31‐residue N‐terminally extended form of MERF that is equivalent to proenkephalin A 209–239. Alkaline phosphatase digestion of the N‐terminally extended MERF variants indicated that some of these peptides were modified by phosphorylation. We conclude that there are complex patterns of proenkephalin A processing in the porcine gut, which in part are due to phosphorylation.
Lithium Selectively Inhibits Muscarinic Receptor‐Stimulated Inositol Tetrakisphosphate Accumulation in Mouse Cerebral Cortex Slices
Authors: Whitworth P. , Kendall D. A.
Published: October 2006

The in vitro effects of Li on agonist‐ and depolarization‐stimulated accumulation of inositol phosphates were determined in mouse cerebral cortex slices. Of the agents examined, only the cholinergic agonist carbachol produced a significant accumulation of inositol tetrakisphosphate (InsP4) in the absence of Li. Lithium at 5 mM enhanced the accumulation of inositol monophosphate (InsP1) and inositol bisphosphate (InsP2) due to all the stimuli used and potentiated inositol trisphosphate (InsP3) accumulation due to histamine and noradrenaline, although at lower Li concentrations, carbachol‐stimulated InsP3 accumulation was reduced. Li also enhanced InsP4 accumulation in the presence of noradrenaline, histamine, and elevated KG level but, in marked contrast, reduced carbachol‐stimulated InsP4 accumulation with an IC50 of 100 μM. There was a significant time delay between the initiation of carbachol stimulation and the beginning of the InsP4 inhibition due to Li. The phorbol ester 4β‐phorbol 12β‐myristate 13α‐acetate did not mimic the effects of Li. The results suggest that muscarinic receptor‐mediated InsP4 production might be one of the targets for the therapeutic action of Li.
[3H]Ro 16–6491, a Selective Probe for Affinity Labelling of Monoamine Oxidase Type B in Human Brain and Platelet Membranes
Authors: Cesura A. M. , Imhof R. , Takacs B. , Galva M. D. , Picotti G. B. , Prada M. Da
Published: October 2006

Abstract: [3H]Ro 16–6491 [N‐(2‐aminoethyl)‐p‐chloroben‐zamide HCl], a reversible “mechanism‐based” inhibitor of monoamine oxidase (MAO) type B, binds selectively and with high affinity to the active site of MAO‐B in brain and platelet membranes. Under normal conditions, the binding of [3H]Ro 16–6491 is fully reversible. However, [3H]Ro 16–6491 could be irreversibly bound (covalently) to membranes by the addition of the reducing agent NaBH3CN to the sample and adjusting to pH 4.5 with acetic acid. No irreversible labelling occurred in the absence of NaBH3CN and at neutral pH. The presence of the irreversible MAO‐B inhibitor /‐deprenyl completely abolished the irreversible labelling of the membranes by [3H]Ro 16–6491. The selective inactivation of MAO‐B, e.g., by /‐deprenyl prevented the covalent incorporation of [3H]Ro 16–6491 whereas selective inhibition of the MAO‐A by clorgyline was without effect. The covalent linkage to membranes of unlabelled Ro 16–6491 and Ro 19–6327 (a selective and reversible MAO‐B inhibitor closely related to Ro 16–6491) after the addition of NaBH3CN at pH 4.5 irreversibly inactivated MAO‐B activity whereas MAO‐A activity was unaffected. Sodium dodecyl sulfate‐polyacrylamide gel electrophoretic analysis of labelled membranes showed that [3H]Ro 16–6491 was incorporated into a single polypeptide with a molecular mass identical to the one labelled by [3H]pargyline (58 kilodaltons). Our results indicate that the polypeptide that is covalently labelled by [3H]Ro 16–6491 corresponds to one of the two MAO‐B subunits. Therefore, [3H]Ro 16–6491 represents a selective probe for affinity labelling of MAO‐B and for the investigation of the structural composition of the active site of the enzyme. Whether the reduction with NaBH3CN at pH 4.5 of the [3H]Ro 16–6491‐MAO‐B complex results in the formation of a stable adduct with the amino acid chain of the MAO‐B or with its prosthetic group, FAD, remains to be elucidated.
Regional Distribution of Cytochrome P‐450 in the Rat Brain: Spectral Quantitation and Contribution of P‐450b,e and P‐450c,d
Authors: Warner Margaret , Köhler Christer , Hansson Tiiu , Gustafsson Jan‐Åke
Published: October 2006

Abstract: The cytochrome P‐450 (P‐450) content of different regions of the rat brain was measured after partial purification of the enzyme from homogenates, and the quantitative contribution of P‐450b,e and P‐450c,d to brain P‐450 was assessed by Western immunoblotting and immunohistochemistry using rabbit antibodies raised against purified hepatic P‐450b and P‐450c (anti‐P‐450b and anti‐P‐450c, respectively). P‐450 could be quantitated by its reduced CO difference spectrum after chromatography of homogenates on p‐chloroamphetamine‐coupled Sepharose. The yield of P‐450 from whole brain was 90 ± 19 pmol/g of tissue, which is ∼ 1% of the level in liver microsomes from control rats. The amount of P‐450 recovered from homogenates of olfactory lobes, hypothalamus, thalamus, striatum, cerebral cortex, and brainstem varied between 40 and 100 pmol/g of tissue. The cerebellum was a region of exceptionally high P‐450 content, with yields of up to 400 pmol/g, whereas the substantia nigra yielded only 16–20 pmol/g. Immunohistochemical studies with anti‐P‐450b and anti‐P‐450c revealed intense staining of a limited number of cells in the cerebellum with both antibodies and in the thalamus only with anti‐P‐450c. In the cerebellum, both anti‐P‐450b and anti‐P‐450c stained the Bergmann glial cells together with their radial processes. Individual glial cells in the granular cell layer were also stained. There was no staining of Purkinje cells. In the thalamus, anti‐P‐450b gave weak staining of certain astroglia, but with anti‐P‐450c, there was intense staining of neuronal somata. Western immunoblots with P‐450 isolated from different brain regions confirmed the distribution of P‐450b,e and P‐450c,d observed with immunohistochemistry. Of all the brain regions examined, P‐450b,e was detected only in P‐450 obtained from the cerebellum and P‐450c only in the cerebellum and thalamus. However, quantitation of the P‐450b,e and P‐450c bands on the immunoblots by 125I‐labeled protein A revealed that these forms of P‐450 account for <1% of the P‐450 in the cerebellum and thalamus. This low content of P‐450b and P‐450c was also reflected in a low level of ethoxycoumarin O‐deethylase activity in the cerebellum and thalamus. From these studies, it is concluded that there are multiple forms of P‐450 in the brain and these different forms of P‐450 are highly selectively localized to certain cells. Furthermore, most of the P‐450 in the brain remains uncharacterized.
Acetylcholinesterase in Huntington's and Alzheimer's Diseases: Simultaneous Enzyme Assay and Immunoassay of Multiple Brain Regions
Authors: Hammond Pamela , Brimijoin Stephen
Published: October 2006

Abstract: A newJy developed enzyme‐linked immunosorbent assay for acetylcholinesterase (AChE) protein was combined with conventional measures of enzyme activity in a study of 15 brain regions from six control cases (non‐neurological deaths), six cases of Alzheimer's disease, and six cases of Huntington's disease. In the control brains, the mean AChE activity varied 100‐fold from region to region (cortex lowest, striatum highest). The variation in enzyme activity was exactly paralleled by a variation in protein immunoreactivity. Overall, the homospecific activity of AChE averaged 0.26 ± 0.007 mU/pg, close to the value for electrophoretically homogeneous enzyme isolated from red blood cells. Similar homospecific activities were observed in samples from Huntington's and Alzheimer's brains. Evidently, AChE that is immunoreactive but enzymatically inactive does not accumulate in any of the three conditions examined. Huntington's brain samples showed normal total contents of AChE, but Alzheimer's brains showed significant decreases of both enzyme activity and immunore‐activity in all seven cortical regions and in two out of the eight subcortical structures examined, hippocampus and nucleus accumbens.
Induction of Intracellular Superoxide Radical Formation by Arachidonic Acid and by Polyunsaturated Fatty Acids in Primary Astrocytic Cultures
Authors: Chan Pak H. , Chen Sylvia F. , Yu Albert C. H.
Published: October 2006

Abstract: The effects of arachidonic acid and other polyunsaturated fatty acids (PUFAs) on both oxidative and metabolic perturbation were studied in primary cultures of rat cerebral cortical astrocytes. In the presence of 0.1 mM arachidonic acid, the rate of the reduction of nitroblue tetrazoiium (NBT) to nitroblue formazan (NBF) was stimulated from 0.65 ± 0.10 to 1.43 ± 0.15 and from 0.092 ± 0.006 to 0.162 ± 0.009 nmol/min/mg protein in intact and broken cell preparations, respectively. The rate of superoxide radical formation, as measured by the superoxide dismutase (SOD)‐inhibitable NBT reduction was 0.042 nmol/mg protein in broken cells and was negligible in intact cells. The latter is due to the impermeability of SOD into the intact cell preparation. NBF formation in intact astrocytes stimulated by arachidonic acid was both time‐ and dosedependent. Other PUFAs, including linoleic acid, linolenic acid, and docosahexaenoic acid, were also effective in stimulating NBF formation in astrocytes, whereas saturated palmitic acid and monounsaturated oleic acid were ineffective. Similar effects of these PUFAs were observed in ma‐londialdehyde formation in cells and lactic acid accumulation in incubation medium. These data indicate that both membrane integrity and cellular metabolism were perturbed by arachidonic acid and by other PUFAs. The sites of superoxide radical formation appeared to be intracellular and may be associated with membrane phospholipid domains, because liposome‐entrapped SOD, which was taken up by intact astrocytes, reduced the level of superoxide radicals and lactic acid content, whereas free SOD was not effective.
Arachidonic Acid Inhibits Choline Uptake and Depletes Acetylcholine Content in Rat Cerebral Cortical Synaptosomes
Authors: Boksa P. , Mykita S. , Collier B.
Published: October 2006

Abstract: The effects of arachidonic acid on [3H]choline uptake, on [3H]acetylcholine accumulation, and on endogenous acetylcholine content and release in rat cerebral cortical synaptosomes were investigated. Arachidonic acid (10–150 μM) produced a dose‐dependent inhibition of high‐affinity [3H]choline uptake. Low‐affinity [3H]choline uptake was also inhibited by arachidonic acid. Fatty acids inhibited high‐affinity [3H]choline uptake with the following order of potency: arachidonic > palmitoleic > oleic > lauric; stearic acid (up to 150 μM) had no effect. Inhibition of [3H]choline uptake by arachidonic acid was reversed by bovine serum albumin. In the presence of arachidonic acid, there was an increased accumulation of choline in the medium, but this did not account for the inhibition of [3H]choline uptake produced by the fatty acid. Arachidonic acid inhibited the synthesis of [3H]acetylcholine from [3H]‐choline, and this inhibition was equal in magnitude to the inhibition of high‐affinity [3H]choline uptake produced by the fatty acid. A K+‐stimulated increase in [3H]acetylcholine synthesis was inhibited completely by arachidonic acid. Arachidonic acid also depleted endogenous acetylcholine stores. Concentrations of arachidonic acid and hemicholinium‐3 that produced equivalent inhibition of [3H]choline uptake also produced equivalent depletion of acetylcholine content. In the presence of eserine, arachidonic acid had no effect on acetylcholine release. The results suggest that arachidonic acid may deplete acetylcholine content by inhibiting high‐affinity choline uptake and subsequent acetylcholine synthesis. This raises the possibility that arachidonic acid may play a role in the impairment of cholinergic transmission seen in cerebral ischemia and other conditions in which large amounts of the free fatty acid are released in brain.
In Vitro Reactivation of Rat Cortical Tryptophan Hydroxylase Following In Vivo Inactivation by Methylenedioxymethamphetamine
Authors: Stone Donna M. , Hanson Glen R. , Gibb James W.
Published: October 2006

Abstract: The activity of tryptophan hydroxylase (EC 1.14.16.4) from rat brain was significantly decreased 1 h following a single systemic injection of 3,4‐methylenedioxy‐methamphetamine (MDMA) when assessed ex vivo by ra‐dioenzymatic assay or in vivo by the quantitation of 5‐hy‐droxytryptophan accumulation following central L‐aromatic amino acid decarboxylase inhibition. Recovery of enzymatic activity in vivo, which occurred within 24 h of low‐dose MDMA treatment, appeared not to involve synthesis of new enzyme protein, because the return of enzymatic activity was not prevented by prior cycloheximide. Acutely MDMA‐depressed cortical tryptophan hydroxylase activity could be completely restored in vitro by a prolonged (20‐24 h) anaerobic incubation in the presence of dithiothreitol and Fe2+at 25°C; partial reconstitution occurred when 2‐mercapto‐ethanol was substituted for dithiothreitol. Cortical tryptophan hydroxylase acutely inactivated by methamphetamine or p‐chloroamphetamine could be similarly reactivated. MDMA‐inactivated cortical tryptophan hydroxylase derived from rats killed later than 3 days after drug treatment could not be significantly reactivated under the conditions described above, indicating the development of irreversible enzymatic damage. Kinetic analysis of enzyme reactivation revealed an approximate doubling of enzyme Vmax with no change in enzyme affinity for either substrate, tryptophan, or pterin cofactor. These studies suggest that MDMA and its congeners inactivate central tryptophan hydroxylase by inducing oxidation of key enzyme sulfhydryl groups. The reactivation capacity of drug‐inactivated enzyme at various times after MDMA treatment may provide a means of assessing the development of MDMA‐induced neurotoxicity.
Partial Isolation of Two Classes of Dopamine β‐Hydroxylase‐Containing Particles Undergoing Rapid Axonal Transport in Rat Sciatic Nerve
Authors: Studelska Daniel R. , Brimijoin Stephen
Published: October 2006

Abstract: The rapid bidirectional transport of dopamine β‐hydroxylase (DBH) in adrenergic axons provides a means of analyzing the life cycle of adrenergic storage vesicles. We compared the physical characteristics of DBH‐containing particles traveling to or returning from the terminal varicosities of ligated rat sciatic nerves. Density gradient centrifu‐gation and Sephacryl S1000 gel‐permeation chromatography were used to fractionate extracts from nerve segments proximal or distal to the ligatures. A series of experiments indicated the existence of at least two populations of rapidly transported DBH‐containing particles, a “light” 85‐nm particle and a larger “dense” 120‐nm particle. The 85‐nm particles were prevalent in unligated nerve, but accounted for only one‐third of the total anterogradely transported DBH activity accumulated after 18 h. The 120‐nm particles were barely detectable in unligated nerve, but they accumulated at twice the rate of the 85‐nm particles and accounted for the rest of the anterogradely transported particulate DBH activity. These two populations of particles were readily isolated from proximal nerve extracts by sucrose density gradient centrifugation. Similar‐appearing dense and light peaks of particulate DBH activity were obtained from distal nerve extracts. Much of the retrogradely transported DBH of the extracts, however, was associated with large particles (>300 nm) not resolved by Sephacryl SI000. Retrogradely transported exogenous NGF was found only in the dense sucrose gradient peak. We propose that the 85‐nm DBH‐containing particles correspond to “large dense‐cored vesicles,” and that the 120‐nm particles are derived from the dense tubules visualized in adrenergic nerves by the chromaffin reaction.
Presynaptic K‐Opioid Receptors on Noradrenergic Nerve Terminals Couple to G Proteins and Interact with the α2‐Adrenoceptors
Authors: Allgaier Clemens , Daschmann Beate , Sieverling Jens , Hertting Georg
Published: October 2006

Abstract: Stimulation‐induced noradrenaline (MA) release in rabbit hippocampus is inhibited by activation qf presynaptic α2‐adrenoceptors and k‐opioid receptors. The purpose of the present study was to investigate (a) an interference between the α2‐ and k‐mechanisms, and (b) a coupling of the opioid receptors to pertussis toxin (PT)‐sensitive guanine nucleotide‐binding proteins (G proteins), as has been previously shown for the α2‐receptors. [3H]NA release from hippocampal slices was evoked by electrical field stimulation (360 pulses/3 Hz). Inhibition of stimulation‐evoked NA release by the preferential K‐receptor agonist ethylketocyclazocine (EKC) was increased in the presence of the α2‐adrenoceptor antagonist yohimbine (0.1 or 1.0 μM). When autoinhibitionn was completely removed, EKC (1 μM) almost abolished transmitter release. Pretreatment of hippocampal tissue wiih either PT (8 μg/ml; 18 h) or N‐ethylmaleimide (NEM) (30 μM; 30 min), which has been shown to alkylate PT substrates, diminished the EKC‐produced inhibition of NA release. The K‐mecha‐nism was still impaired by these compounds when the α2‐ receptors were blocked with yohimbine. An effect of NEM on the active site of the K‐receptor seems to be unlikely, because NEM diminished the EKC‐induced inhibition of release irrespective of whether or not the opioid receptor was occupied by EKC during exposure to NEM. The present results suggest an interference of both α2‐ and k‐opioid receptor‐coupled signal transduction possibly through competition for a common pool of G proteins.
Long‐Term Opiate Exposure Leads to Reduction of the αi‐1 Sufyunit of GTP‐Binding Proteins
Authors: Attali Bernard , Vogel Zvi
Published: October 2006

Abstract: Desensitization or tolerance is a major consequence of long‐term opiate exposure. The mechanism of opiate desensitization is only poorly understood. We report that exposure of raj spinal cord‐dorsal root ganglion cocultured neurons to k‐opiate agonists is accompanied by a 60–70% reduction in the level of the αi subunit of GTP‐binding proteins. Using selective antibodies, which discriminate among the various αi subunit forms, it was found that the opiate treatment leads to a reduction in the amount of the αi‐1 subunit. The levels of αs, αo, and β subunits remain unchanged. Tnis molecular event could underlie the development of tolerance and cross‐tolerance to opiates.
Homologous Desensitization of Muscarinic Cholinergic, Histaminergic, Adrenergic, and Serotonergic Receptors Coupled to Phospholipase C in Cerebellar Granule Cells
Authors: Dillon‐Carter Ora , Chuang De‐Maw
Published: October 2006

Abstract: Cultured cerebellar granule cells express phospholipase C‐coupled muscarinic cholinergic, histaminergic, α1‐adrenergic, and serotonergic receptors. In an attempt to study desensitization of these neurotransmitter receptors, cells were prestimulated with saturating concentrations of carbachol, histamine, norepinephrine, or serotonin during the labeling of cells with myo‐[3H]inositol and then rechallenged with various receptor agonists for their ability to elicit accumulation of [3H]inositol monophosphate in the presence of lithium. Prestimulation with each of these receptor agonists was found to cause a time‐dependent desensitization to subsequent stimulation with the desensitizing agonist. Thus, prestimulation for 0.5, 4, and 18 h decreased carbachol response to 87 ± 4, 52 ± 2, and 40 ± 1% of the control, respectively; histamine response to 37 ± 2, 24 ± 2, and 18 ± 2%, respectively; norepinephrine response to 55 ± 5, 14 ± 1, and 10 ± 1%, respectively; and serotonin response to 36 ± 1, 18 ± 1, and 9 ± 2%, respectively. In all cases, the responses mediated by receptors which were not prestimulated remained virtually unchanged, thus indicating homologous desensitization. Dose‐response studies indicate that the desensitization was associated with a major reduction in the maximal extent of agonist‐induced responses. The basal accumulation was markedly enhanced following 0.5‐ and 4‐h prestimulation, but returned to near normal after 18‐h pretreatment. Biologically active phorbol ester, 4β‐phorbol 12‐myristate 13‐acetate, rapidly attenuated basal phospholipase C activity, as well as the responses mediated by carbachol, histamine, norepinephrine, and serotonin, suggesting that activation and translocation of protein kinase C might play a role in the desensitization of phospholipase C‐coupled receptors. Possible mechanisms of homologous desensitization of receptors in granule cells are discussed.
Evidence for Distinct 5‐Hydroxytryptamine2 Binding Site Subtypes in Cortical Membrane Preparations
Authors: Pierce Pamela A. , Peroutka Stephen J.
Published: October 2006

Abstract: 5‐Hydroxytryptamine (5‐HT) displays a sixfold higher affinity for 5‐HT2 binding sites labeled by [3H]ketanserin in rat (IC50= 200 ± 40 nM) and human (IC50= 190 ± 50 nM) cortex than for 5‐HT2 sites in bovine cortex (IC50= 1,200 ± 130 nM). The Hill slopes of the 5‐HT competition curves are 0.67 ± 0.04 in rat, 0.69 ± 0.08 in human, and 0.96 ± 0.02 in bovine cortex. Scatchard analysis of (±)‐(3H]4‐bromo‐2,5‐dimethoxyamphetamine ([3H]DOB) binding in the rat indicates a population of binding sites with a KD of 0.38 ± 0.04 nM and a Bmax of 1.5 ± 0.05 pmol/g tissue. In contrast, specific [3H]DOB binding cannot be detected in bovine cortical membranes. These data indicate that species variations exist in 5‐HT2 binding site subtypes and that [3H]ketanserin appears to label a homogeneous population of 5‐HT2 binding site subtypes in bovine cortex.
Regulation of Glycogen Content in Primary Astrocyte Culture: Effects of Glucose Analogues, Phenobarbital, and Methionine Sulfoximine
Authors: Swanson Raymond A. , Yu Albert C. H. , Sharp Frank R. , Chan Pak H.
Published: October 2006

Abstract: Compounds known to affect glycogen metabolism in vivo or in cell‐free preparations were used to investigate the regulation of glycogen content in intact astrocytes cultured from newborn rat cortex. Compounds were added with fresh medium to culture dishes, and astrocyte glucose and glycogen content determined 24 h later. Increasing the medium glucose concentration from 7.5 mM to 30 mM increased cell glycogen content 80%. Addition of 2‐deoxyglucose or 3‐O‐methyl glucose (2.5–10 mM) also increased cell glycogen content, 50–100%, suggesting a regulatory rather than mass action effect of glucose on astrocyte glycogen content. The phosphorylase b inhibitors 2,2′,4,4′,5,5′‐hexabromobiphenyl and riboflavin had no effect on astrocyte glycogen content, consistent with negligible phosphorylase b activity in normal astrocytes. Phenobarbital and l‐methionine‐DL‐sulfoximine (MSO) are both known to induce astrocyte glycogen accumulation in vivo. The addition of phenobarbital (2 mM) had no effect on the glycogen content of cultured astrocytes, suggesting an indirect mechanism for the in vivo effect. MSO at 1 mM, however, induced a 300% increase in glycogen content. The time course of glucose and glycogen content after MSO administration suggests this increase to be the result of slowed glycogenolysis rather than accelerated glycogen synthesis.
Dopamine Autoreceptors Modulate the Phosphorylation of Tyrosine Hydroxylase in Rat Striatal Slices
Authors: Salah Raymond S. , Kuhn Donald M. , Galloway Matthew P.
Published: October 2006

Abstract: The hypothesis that dopamine (DA) autoreceptors modulate the phosphorylation of tyrosine hydroxylase (TH; EC 1.14.16.2) was investigated in rat striatal slices. Tissue was prelabeled with 32P inorganic phosphate, and TH recovered by immunoprecipitation with anti‐TH rabbit serum. The TH monomer was resolved on sodium dodecyl sulfate polyacrylamide gels, and the extent of phosphorylation was determined by scanning densitometry of autoradiographs. Depolarization of striatal slices with 55 mM K+ markedly increased the incorporation of 32P into several proteins, including the TH monomer (Mr= 60,000). A similar increase in TH phosphorylation occurred in response to the adenylate cyclase activator forskolin and the cyclic AMP analog dibutyryl cyclic AMP. An increase in TH phosphorylation was not observed in response to the D1‐selective agonist SKF 38393. The D2‐selective DA autoreceptor agonist pergolide decreased the phosphorylation of TH below basal levels and blocked the increase in phosphorylation elicited by 55 mM K+. The inhibitory effect of pergolide was antagonized by the D2‐selective antagonist eticlopride. Changes observed in the phosphorylation of TH were mirrored by changes in tyrosine hydroxylation in situ. These observations support the hypothesis that a reduction in TH phosphorylation is the mechanism by which DA autoreceptors modulate tyrosine hydroxylation in nigrostriatal nerve terminals.
Effect of Impact Trauma on Neurotransmitter and Nonneurotransmitter Amino Acids in Rat Spinal Cord
Authors: Demediuk Paul , Daly Michael P. , Faden Alan I.
Published: October 2006

Abstract: N‐Methyl‐D‐aspartate (NMDA) administration exacerbates neurological dysfunction after traumatic spinal cord injury in rats, whereas NMDA antagonists improve outcome in this model. These observations suggest that release of excitatory amino acids contributes to secondary tissue damage after traumatic spinal cord injury. To further examine this hypothesis, concentrations of free amino acids were measured in spinal cord samples from anesthetized rats subjected to various degrees of impact trauma to the T9 spinal segment. Levels of excitatory and inhibitory neurotransmitter amino acids [γ‐aminobutyric acid (GABA), glutamate, aspartate, glycine, taurine] and levels of nonneurotransmitter amino acids (asparagine, glutamine, alanine, threonine, serine) were determined at 5 min, 4 h, and 24 h posttrauma. Uninjured surgical (laminectomy) control animals showed modest but significant declines in aspartate and glutamate levels, but not in other amino acids, at all time points. In injured animals, the excitatory amino acids glutamate and aspartate were significantly decreased by 5 min posttrauma, and remained depressed at 4 h and 24 h as compared with corresponding laminectomy controls. In contrast, the inhibitory amino acids, glycine, GABA, and taurine, were decreased at 5 min post‐injury, had partially recovered at 4 h, and were almost fully recovered at 24 h. The nonneurotransmitter amino acids were unchanged at 5 min posttrauma and significantly increased at 4 h, with partial recovery at 24 h. At 4 h postinjury, severe trauma caused significantly greater decreases in aspartate and glutamate than did either mild or moderate injury. These findings are consistent with the postulated role of excitatory amino acids in CNS trauma.
Presynaptic Cholinergic Mechanisms in the Rat Cerebellum: Evidence for Nicotinic, but Not Muscarinic Autoreceptors
Authors: Lapchak P. A. , Araujo D.M. , Quirion R. , Collier B.
Published: October 2006

Abstract: The present study shows that N‐[3H]meth‐ylcarbamylcholine ([3H]MCC) binds to a single population of high‐affinity/low‐density (KD= 5.0 nM; BmdK= 8.2 fmol/ mg of protein) nicotinic binding sites in the rat cerebellum. Also, there exists a single class of high‐affinity binding sites (KD= 4.8 nM BmaK= 24.2 fmol/mg of protein) in the cerebellum for the M1 specific muscarinic ligand [3H]‐pirenzepine. In contrast, the M2 ligand, [3H]AF‐DX 116. ap‐pears to bind to two classes of binding sites, i.e., a high‐affinity (KD= 3 nM)/low‐capacity (BMax= 11.7 fmol/mg of protein) class, and a second class of lower affinity (KD= 28.4 nM) and higher capacity (Bmax= 36.3 fmol/mg of protein) sites. The putative M3 selective ligand [3H]4‐diphenyIacetoxy‐N‐methylpiperidine also binds to two distinct classes of binding sites in cerebellar homogenates, one of high affinity (KD= 0.5 nM)/low capacity (Bmax= 19.5 fmol/mg of protein) nd one of low affinity (KD= 57.5 nA/)/high capacity (Bmax 140.6 fmol/mg of protein). In experiments which tested the effects of cholinergic drugs on acetylcholine release from cerebellar brain slices, the nicotinic agonist MCC enhanced spontaneous acetylcholine release in a concentration‐dependent manner, and the maximal increase in acetylcholine release (59.0‐68.0%) occurred at 10−7M. The effect of MCC to increase acetylcholine release was Ca2+‐dependent and te‐trodotoxin‐insensitive, suggesting an action on cholinergic terminals. Also, the MCC‐induced increase in acetylcholine release was effectively antagonized by dihydro‐β‐erythroidine, d‐tubocurarine, and k‐bungarotoxin, but was insensitive to either atropine or α‐bungarotoxin. Tests of the effects of muscarinic drugs, such as oxotremorine (nonselective agonist), atropine (nonselective antagonist), and AF‐DX 116 (M2 selective antagonist), on spontaneous and evoked acetylcholine release revealed the absence of muscarinic autoreceptor‐mediated regulation of acetylcholine release from cerebellar brain slices. These results support the hypothesis that in the rat cerebellum some nicotinic sites, as labeled by [3H]MCC. are localized to cholinergic terminals where they mediate positive feedback control of acetylcholine release.
Effects of Apomorphine on In Vivo Release of Dopamine and Its Metabolites in the Prefrontal Cortex and the Striatum, Studied by a Microdialysis Method
Authors: Ozaki Norio , Nakahara Daiichiro , Miura Hideki , Kasahara Yomishi , Nagatsu Toshiharu
Published: October 2006

Abstract: The effects of apomorphine (0.1‐2.5 mg/kg) on release of endogenous dopamine and extracellular levels of 3.4‐dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA) in the prefrontal cortex and the striatum were examined in vivo by a microdialysis method. Apomorphine significantly reduced release of dopamine and the extracellular levels of dopamine metabolites, DOPAC and HVA, not only in the striatum, but also in the prefrontal cortex. These findings indicate that dopamine autoreceptors modulate in vivo release of dopamine in the prefrontal cortex.
Developmental Regulation of Microtubule‐Associated Protein 2 Expression in Regions of Mouse Brain
Authors: Crandall James E. , Fischer Itzhak
Published: October 2006

Abstract: The relative levels of microtubule‐associated protein 2 (MAP2) were determined during postnatal development of the mouse in six different discrete brain regions: cerebellum, cortex, hippocampus, olfactory bulb, brainstem, and hypothalamus. Brain homogenates were electrophoresed on sodium dodecyl sulfate‐containing gels and analyzed by im‐munoblotting with MAP2‐specific antibodies. The levels of MAP2 in each region were determined using radiolabeled secondary antibodies and densitometric quantification of the autoradiograms over a range that was determined to have a linear response. The results indicated that in all regions and at all ages there was only one high‐molecular‐weight polypeptide of MAP2, which did not change in electrophoretic mobility after dephosphorylation. In most regions, the levels of MAP2 increased during the first 2 postnatal weeks. However, there were differences in the time course and relative levels of MAP2 between regions. In addition, all regions of the brain expressed the low‐molecular‐weight form of MAP2 (MAP2c) that was present at birth as a heterogeneous group of polypeptides with an apparent molecular weight of 70K. Most of the heterogeneity of MAP2c, however, was eliminated after dephosphorylation. The levels of MAP2c decreased dramatically after 2 weeks postnatally, except for the olfactory bulb, where the levels of MAP2c remained relatively high even in adults.
Lipid Hydroperoxides Inhibit Reacylation of Phospholipids in Neuronal Membranes
Authors: Zaleska Malgorzata M. , Wilson David F.
Published: October 2006

Abstract: The unsaturated fatty acids that rapidly accumulate during ischemia are thought to participate in inducing irreversible brain injury, especially because they are highly susceptible to peroxidation when the tissue is reoxygenated. Our hypothesis was that peroxidation products of unsaturated fatty acids interfere with the reacylation of synaptic phospholipids, a process essential to membrane repair. To test this hypothesis, we have examined the effect of fatty acid hydroperoxides on incorporation of [1‐14C]arachidonic acid into synapto‐somal phospholipids. Rat forebrain synaptosomes were incubated with arachidonic or linoleic acid hydroperoxides and [14C]arachidonate, and then lipids were extracted and separated by TLC. Both hydroperoxides inhibited [14C]arachidonate incorporation into phospholipids in a concentration‐dependent manner, with 50% inhibition occurring at less than 25 μM hydroperoxide, in both the absence and presence of exogenous lysophospholipids. The inhibition was of the non‐competitive type. It is concluded that (a) low levels of fatty acid hydroperoxides inhibit the reacylation of synaptosomal phospholipids, and (b) this inhibition may constitute an important mechanism whereby peroxidative processes contribute to irreversible brain damage.
Reduced Glycine Stimulation of [3H]MK‐801 Binding in Alzheimer's Disease
Authors: Procter A. W. , Wjong E. H. F. , Stratmann G. C , Lowe S. L. , Bowen D. M.
Published: October 2006

The novel N‐methyl‐D‐aspartate recjeptor channel ligand (+)‐[3H]5‐methyl‐J0, l1‐dihydro‐5H‐dibenzo[a, d]‐cyclohepten‐5, 10‐imine maleate ([3H]MK‐801) has been utilized to label this receptor in human brain tissiie. Characteristics of [3H]MK‐801 binding to well‐washeq membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of the and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding Was observed in the frontal cortex under glutamate‐ and glycine‐stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine‐insensitive glycine recognition site is impaired.
Polyclonal Antisera to the Individual Neurofilament Triplet Protdins: A Characterization Using ELISA and Immunoblotting
Authors: Karlsson J.‐E. , Rosengren L. E. , Haglid K. G.
Published: October 2006

In this article, the preparation and characterization of polyclonal rabbit antisera against the individual polypeptides of bovine neurofilament (68, 150, and 200 kilodaltons) is described. Selected antisera against the 68‐ add 150‐kilo‐dalton neurofilament polypeptides were specific for the corresponding antigen in homogenates of bovine, rat, and human brain as judged by immunoblots. The antisera against the 200‐kilodalton neurofilament polypeptide crossjreacted to some extent with the 150‐kilodalton neurofilament polypeptide, especially with the human antigen. The most specific antisera were used to develop an enzyme‐linked immunosorbent assay (ELISA), and the cross‐reactivities between the antisera and the different bovine and rat neurofilament polypeptides were determined. Contrary to the results In the immunoblots, the antiserum against the 200‐kilodalton neurofilament polypeptide was subunit‐specific, as was the 150‐kilodalton antiserum. The 68‐kilodalton antiserum displayed a minute cross‐reactivity against bovine 150‐ and 200‐kilodalton neurofilaments, but it cross‐reacted somewhat more with the rat 150‐ and 200‐kilodalton antigens. Even so, the subunit specificity of the antisera is high enough to enable the development of a quantitative ELISA for determination of the individual bovine or rat neurofilament polypeptides in a mixture. This study is the necessary preparation for such an assay.
Isolated Light Chain of Tetanus Toxin Inhibits Exocytosis: Studies in Digitonin‐Permeabilized Cells
Authors: Bittner Mary A. , Habig William H. , Holz Ronald W.
Published: October 2006

Previous work indicates that the heavy chain of tetanus toxin is responsible for the binding of the toxin to the neuronal membrane and its subsequent internalization. In the present study, the light chain of tetanus toxin mimicked the holotoxin in inhibiting Ca2+‐dependent secretion of [3H]norepinephrine from digitonin‐permeabilized adrenal chromaffin cells. Preincubation of tetanus toxin with monoclonal antibodies to the light chain prevented the inhibition by tetanus toxin. Preincubation of tetanus toxin with nonimmune ascites fluid or with monoclonal antibodies directed against the C fragment (the C‐terminal of the heavy chains or the heavy‐chain portion of the B fragment did not prevent inhibition by tetanus toxin. The data indicate that the light chain is responsible for the intracellular blockade of exostosis.
Discrete Distributions of Adenosine Receptors in Mammalian Retina
Authors: Blazynski Christine
Published: October 2006

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyl‐adenosine and the mixed A1‐A2 agonist N‐[3H]ethylcarbox‐amidoadenosine ([3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer. The binding of [3H]NECA was observed primarily over the retinal pigmented epithelium and the outer and inner segments of photoreceptors. [3H]NECA labeling was not affected either by including a low concentration of unlabeled A1 agonist or by pretreating tissue with N‐ethylmaleimide to inhibit ligand binding at A1 sites. While virtually all of the [3H]NECA binding was displaced by an excess of unlabeled NECA, displacement with antagonist or a large excess of cyclohexyladenosine revealed that ∼30% of the [3H]NECA binding was at non– A1, A2 sites. The majority of the binding in the outer retina thus labeled A2 receptor sites. The unique localizations of the two classes of adenosine receptors suggest different functions in visual processing.
Rapid Stereoselective Hydrolysis of (+)‐Cocaine in Baboon Plasma Prevents Its Uptake in the Brain: Implications for Behavioral Studies
Authors: Gatley S. J. , MacGregor R. R. , Fowler J. S. , Wolf A. P. , Dewey S. L. , Schlyer D. J.
Published: October 2006

The naturally occurring enantiomer of cocaine, (–)‐cocaine, has been previously labeled with 11C on the N‐methyl group and used in conjunction with positron emission tomography to show that cocaine is rapidly taken up in the striata of human and baboon brain. In the present study, the behaviorally inactive (+)‐cocaine was similarly labeled, with a view to its use for measuring the nonspecific binding of cocaine. No brain uptake was seen, although transport of cocaine into the brain is not expected to be stereoselective. The explanation for the lack of uptake was determined to be very rapid metabolism of (+)‐cocaine in the blood. By 30s after administration of labeled (+)‐cocaine, it was undetectable in plasma. In vitro studies demonstrated that (+)‐cocaine is 50% debenzoylated to (+)‐ecgonine methyl ester within 5 s of exposure to baboon plasma but not to washed erythrocytes. The hydrolysis of (–)‐cocaine is at least 1,000 times slower. Serum butyrylcholinesterase (EC 3.1.1.8) appears to be responsible for this hydrolysis, as evidenced by its inhibition by physostigmine and catalysis by commercially available pseudocholinesterase from horse and human blood.
Forskolin Mediates the Survival of Nerve Growth Factor‐Dependent Sympathetic Neurons of Chick Embryo by a Cyclic AMP‐Independent Mechanism
Authors: Wakade Arun R. , Bhave Sanjiv V. , Malhotra Ravindra K. , Wakade Taruna D.
Published: October 2006

Forskolin has become an invaluable tool for exploring the involvement of cyclic AMP in a variety of cellular functions. The diterpine directly activates the catalytic subunit of adenylate cyclase, causing a marked increase in cyclic AMP content. Because of this well‐characterized action, practically all the observed effects of forskolin are commonly attributed to cyclic AMP‐dependent processes. We show here that forskolin exerts a neurotrophic action that is almost identical to that of nerve growth factor (NGF) and phorbol 12,13–dibutyrate (PDB) but independent of cyclic AMP. Sympathetic neurons of the chick embryo supported in culture for 2 days by NGF, forskolin plus 3‐isobutyl‐l‐methylxanthine (IBMX), or PDB had almost identical levels of cyclic AMP (between 9 and 12 pmol/mg protein). Neurons supported in culture for 2 days by NGF or PDB when challenged with forskolin plus IBMX showed almost a 15‐fold increase in cyclic AMP, but those supported by forskolin plus IBMX and then exposed to the same combination of drugs did not show an increase in cyclic AMP, exhibiting a marked downregulation. Exposure of neurons to forskolin for 2 h was ineffective in supporting long‐term survival, suggesting that an initial increase in cyclic AMP formation is not sufficient but the continued presence of the drug is essential for survival. Effects of forskolin on the survival of these neurons could be observed even in the presence of dideoxyadenosine, an inhibitor of adenylate cyclase. Neurons supported by PDB for 2 days in culture when exposed to NGF for the first time did not show any increase in cyclic AMP, providing clear evidence that NGF does not affect this second messenger in its target cells. Similarly, neurons supported by NGF for 2 days when exposed to PDB did not show an increase in cyclic AMP. All these results rule out the involvement of cyclic AMP not only in the neurotrophic action of forskolin but also in that of NGF and PDB, and suggest that forskolin supports neuronal survival by some, as yet, unknown mechanism.
Effect of Peroxide, Sodium, and Calcium on Brain Mitochondrial Respiration In Vitro: Potential Role in Cerebral Ischemia and Reperfusion
Authors: Vlessis Angelo A. , Widener Linda L. , Bartos Dagmar
Published: October 2006

Mitochondrial pyruvate‐supported respiration was studied in vitro under conditions known to exist following ischemia, i.e., elevated extramitochondrial Ca2+, Na+, and peroxide. Ca2+ alone (7–10 nmol/mg) decreased state 3 and increased state 4 respiration to 81 and 141% of control values, respectively. Sodium (15 mM) and/or tert‐butyl hydroperoxide (tBOOH; up to 2,000 nmol/mg protein) alone had no effect on respiration; however, Na+ or tBOOH in combination with Ca2+ dramatically altered respiration. Respiratory inhibition induced by Ca2+ and tBOOH does not involve pyruvate dehydrogenase (PDH) inhibition since PDH flux increased linearly with tBOOH concentration (R= 0.96). Calcium potentiated tBOOH‐induced mitochondrial NAD(P)H oxidation and shifted the redox state of cytochrome b from 67 to 47% reduced. Calcium (5.5 nmol/mg) plus Na+ (15 mM) decreased state 3 and increased state 4 respiratory rates to 55 and 202% of control values, respectively. Sodium‐ as well as tBOOH‐induced state 3 inhibition required mitochondrial Ca2+ uptake because ruthenium red addition before Ca2+ addition negated the effect. The increase in state 4 respiration involved Ca2+ cycling since ruthenium red immediately returned state 4 rates back to control values. The mechanisms for the observed Ca2+‐, Na+‐, and tBOOH‐induced alterations in pyruvate‐supported respiration in vitro are discussed and a multifactorial etiology for mitochondrial respiratory dysfunction following cerebral ischemia in vivo is proposed.