bluuebunny/crossref_metadata_2025 huggingface dataset:
MK‐801 Fails to Protect Against the Dopaminergic Neuropathology Produced by Systemic 1‐Methyl‐4‐Phenyl‐1,2,3,6‐Tetrahydropyridine in Mice or Intranigral 1‐Methyl‐4‐Phenylpyridinium in Rats
Authors: Sonsalla Patricia K. , Zeevalk Gail D. , Manzino Lawrence , Giovanni Andrew , Nicklas William J.
Published: October 2006

Abstract: Previous studies from this laboratory demonstrated that (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo[a,d]cyclohepten‐5,10‐imine maleate (MK‐801), an N‐methyl‐D‐aspartate (NMDA) receptor antagonist, did not prevent neurotoxicity to dopaminergic neurons in mice produced by systemic treatment with 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP). However, Turski et al. [Nature349, 414–418 (1991)] reported that extended treatment of rats with NMDA receptor antagonists (six injections at 4‐h intervals) did prevent the loss of nigral dopaminergic neurons resulting from an intranigral infusion of 1‐methyl‐4‐phenylpyridinium (MPP+), the neurotoxic metabolite of MPTP. The present studies examined if a similar extended treatment with MK‐801 would protect mice from the neurotoxicity of systemically administered MPTP. Six intraperitoneal injections of MK‐801 given at 4‐h intervals did not protect mice against the MPTP‐induced neostriatal dopamine loss measured 1 week after treatment. In other experiments, designed to replicate and expand on the results of Turski et al. (1991), the extended treatment of rats with MK‐801 did not prevent MPP+‐induced cell loss in the infused substantia nigra pars compacta or the dopamine depletion in the ipsilateral neostriatum at 7‐11 days after MPP+ infusion. These results do not support the hypothesis that NMDA receptors are involved with MPTP/MPP+‐induced neurodegeneration.
Characterisation and Regional Localisation of Pre‐ and Postsynaptic Glycoproteins of the Chick Forebrain Showing Changed Fucose Incorporation Following Passive Avoidance Training
Authors: Bullock Sarah , Rose Steven P. R. , Zamani Reza
Published: October 2006

Abstract: To identify those glycoproteins whose synthesis or modification is necessary for memory formation, we have studied the uptake of radiolabelled fucose into synaptic plasma membranes (SPMs) and postsynaptic densities (PSDs) derived from two specific left and right forebrain loci, at two different times after training of 1‐day‐old chicks on a one‐trial passive avoidance learning task. To increase the reliability of the comparison, a double‐labelling method was used. Tissue samples from intermediate medial hyper‐striatum ventrale (IMHV) and lobus parolfactorius (LPO) were isolated at 6 and 24 h after training. At both times, training resulted in region‐specific changes, both increases and decreases, in incorporated radioactivity into pre‐ and postsynaptic glycoproteins. After 6 h, there was a relative decline in incorporation into both SPMs and PSDs of the right IMHV of trained chicks, a decline that persisted in the PSDs until 24 h. A small decline in incorporation in SPMs from the right LPO of trained chicks at 6 h was reversed by 24 h, by which time there was a 64% increase in incorporation into SPMs and a 24% increase into PSDs of the left LPO. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis analysis of left and right hemisphere samples containing LPO revealed that 6 h after training the main effect was presynaptic, including a reduction of incorporation into high molecular mass glycoproteins, of 150–180 kDa, and an increase in a lower molecular mass (41 kDa) fraction. By 24 h after training, a left hemisphere presynaptic glycoprotein of molecular mass ∼50 kDa showed the biggest increase in fucosylation. In addition, a wide group of postsynaptic glycoproteins of both hemispheres, in the ranges 150–180, 100–120, and 33 kDa now showed increases in incorporation. Some other fractions showed decreases. These results are in accord with previous data on incorporation obtained using the amnesic agent 2‐deoxyga‐lactose. They also support the hypothesis that memory formation involves the strengthening of connections between pre‐ and postsynaptic neurons of the LPO by growth or modulation of pre‐ and postsynaptic structures.
γ‐Aminobutyric AcidA Receptor Pharmacology in Rat Cerebral Cortical Synaptoneurosomes
Authors: DeLorey Timothy M. , Brown George B.
Published: October 2006

Abstract: Equilibrium binding interactions at the γ‐amino‐butyric acid (GABA) and benzodiazepine recognition sites on the GABAA receptor‐Cl− ionophore complex were studied using a vesicular synaptoneurosome (microsacs) preparation of rat brain in a physiological HEPES buffer similar to that applied successfully in recent GABAergic 36Cr flux measurements. NO 328, a GABA reuptake inhibitor, was included in the binding assays to prevent the uptake of [3H]muscimol. Under these conditions, the equilibrium dissociation constant (Kd) values for [3H]muscimol and [3H]‐diazepam binding are 1.9 μM 40 nM, respectively. Binding affinities for these and other GABA and benzodiazepine agonists and antagonists correlate well with the known physiological doses required to elicit functional activity. This new in vitro binding protocol coupled with 36C1− flux studies should prove to be of value in reassessing the pharmacology of the GABAA receptor complex in a more physiological environment.
Two Forms of GO Type G Proteins: Identification and Distribution in Various Rat Tissues and Cloned Cells
Authors: Asano Tomiko , Morishita Rika , Kato Kanefusa
Published: October 2006

Abstract: A Go type G protein distinct from the major species of Go was recently isolated from bovine brain and designated Go*. The cDNAs encoding two forms of mammalian Goα were also isolated and designated GoAα and GoBα. To recognize two forms of Go type G proteins, we raised antibodies in rabbits against two peptides with sequences found only in the respective proteins of murine GoAα (SNTYE‐DAAAYIQTQF) and GoBα (TEAVAHIQGQYESK). Purified anti‐GoAα antibodies reacted with the major species of Goα purified from bovine and rat brain, whereas anti‐GoBα antibodies reacted only with rat Go*α, but not with the major species of Goα or bovine Go*α. These results indicate that the major species of Goα is encoded by GoAα cDNA and Go*α is encoded by GoBα cDNA. Using these antibodies, the distribution of GoA and GoB was studied in various rat tissues and cloned cells. Both GoA and GoB were present in many tissues, but their distribution in peripheral tissues was distinct. GoAα seemed to associate mainly with neural tissues. On the other hand, relatively high concentrations of GoBα were present in the brain, pituitary gland, adipose tissue, lung, and testis. The concentrations of both GoA and GoB in the brain increased during ontogenic development, but the increase in GoB was observed at a later age. Both GoA and GoB were found in such cloned cells as PC 12, NG108‐15, C6, GA‐1, G8, and 3T3‐L1 cells. Treatment of PC 12 cells with nerve growth factor caused the extension of neuron‐like processes and the increase in the level of GoA, but not in the level of GoB.
Uptake, Metabolism, and Release of N‐[3H]‐Acetylaspartylglutamate by the Avian Retina
Authors: Williamson Lura C. , Neale Joseph H.
Published: October 2006

Abstract: N‐Acetylaspartylglutamate (NAAG) is a nervous system‐specific dipeptide that is released from retinal neurons on depolarization. In the present study, extracellular metabolism, uptake, and release of [3H]NAAG were examined in the chick retina. After in vitro incubation with NAAG radiolabeled in the glutamate moiety, [3H]‐glutamate and [3H]NAAG increased in retinal cells through time‐ and temperature‐dependent processes, which were reduced in the absence of extracellular sodium. Coincubation of cells with [3H]NAAG and aspartylglutamate or phosphate resulted in the decreased extracellular appearance of [3H]glutamate, produced by hydrolysis of radiolabeled NAAG, and a consequent increased availability of [3H]‐NAAG for transport into the retinal cells. When this tissue was incubated with radiolabeled NAAG, glutamate, glutamine, or aspartate under similar conditions, only [3H]‐NAAG served as a significant source for the appearance of intracellular [3H]NAAG. These data support the conclusion that [3H]NAAG can be transported into retinal cells, whereas [3H]glutamate transport is the predominant process after release of this amino acid from NAAG by extracellular peptidase activities. After uptake, [3H]NAAG entered a cellular pool, from which the peptide was secreted under depolarizing conditions and in a calcium‐dependent manner.
Amino Acid Neurotransmitters and Dopamine in Brain and Pituitary of the Goldfish: Involvement in the Regulation of Gonadotropin Secretion
Authors: Sloley B. Duff , Kah Olivier , Trudeau Vance L. , Dulka Joseph G. , Peter Richard E.
Published: October 2006

Abstract: An isocratic high–performance liquid chromatographic technique was developed to measure levels of γ–aminobutyric acid (GABA), glutamate, and taurine in the brain and pituitary of goldfish. Accuracy of this procedure for quantification of these compounds was established by evaluating anesthetic and postmortem effects and by selectively manipulating GABA concentrations by intraperitoneal administration of the glutamic acid decarboxylase (GAD) inhibitor 3–mercaptopropionic acid or the GABA transaminase inhibitor γ–vinyl GABA. The technique provided a simple, rapid, and reliable method for evaluating the concentrations of these amino acids without the use of complex gradient chromatographic systems. To investigate the relationship between neurotransmitter amino acids and the control of pituitary secretion of gonadotropin, the effects of injection of taurine, GABA, or monosodium glutamate on GABA, glutamate, taurine, and, in some instances, monoamine concentrations in the brain and pituitary were evaluated and related to serum gonadotropin levels. Injection of taurine caused an elevation in serum gonadotropin concentrations. In addition, injection of the taurine precursor hypotaurine but not the taurine catabolite isetheonic acid elevated serum gonadotropin levels. Intracerebroventricular injection of either GABA or taurine also elevated serum gonadotropin concentrations. Pretreatment of recrudescent fish with α–methyl–p–tyrosine reduced pituitary dopamine concentrations and also potentiated the serum gonadotropin response to taurine. Injection of monosodium glutamate caused an increase of glutamate content in the pituitary at 24 h; this was followed by a decrease at 72 h after administration. Pituitary GABA, taurine, and dopamine concentrations underwent a transient depletion after monosodium glutamate administration, and this was associated with an elevation of serum gonadotropin content. The increase in serum gonadotropin concentrations in response to a gonadotropin–releasing hormone analogue was potentiated by pretreatment with monosodium glutamate. This article demonstrates that procedures causing elevation in GABA and taurine concentrations stimulate gonadotropin release in a teleost fish.
Regulatory Role of the GTP‐Binding Protein, Go, in the Mechanism of Exocytosis in Adrenal Chromaffin Cells
Authors: Ohara‐Imaizumi Mica , Kameyama Kimihiko , Kawae Nobuyuki , Takeda Kyoko , Muramatsu Shun , Kumakura Konosuke
Published: October 2006

Abstract: To elucidate the possible involvement of GTP‐binding proteins (G proteins) in the mechanism of exocytosis, we studied effects of pertussis toxin (PTX), guano‐sine 5′‐O‐(3‐thiotriphosphate) (GTP‐γ‐S), and antibodies against the G proteins (Gi and Go) on the secretory function of bovine adrenal chromaffin cells. Pretreatment of chromaffin cells with PTX resulted in an increase in acetylcholine‐evoked catecholamine release. High K+‐, histamine‐, or γ‐aminobutyric acid‐evoked catecholamine release was also potentiated by PTX pretreatment. The concentration of extracellular Ca2+ required for maximal release by 10−4M acetylcholine was decreased significantly in PTX‐treated cells. In digitonin‐permeabilized cells, PTX pretreatment resulted in a decrease of the half‐maximal concentration (Km) of Ca2+ required for exocytosis with no significant change in the maximal stimulation (Vmax). Exposure of permeabilized cells to GTP‐γ‐S (a nonhydrolyzable GTP analogue) inhibited Ca2+‐dependent exocytosis by reducing the affinity for Ca2+. The effects of PTX pretreatment were mimicked by treatment of permeabilized cells with polyclonal antibodies selective for the α subunit of the PTX‐sensitive G protein, Go. Treatment with similar antibodies against the α subunit of Gi had no effect. These findings suggest that Go directly controls the Ca2+‐triggered process in the machinery of exocytosis by lowering the affinity of the unknown target for Ca2+.
Adrenalectomy Potentiates Immediate Early Gene Expression in Rat Brain
Authors: Li Xiaohua , Song Ling , Kolasa Krystyna , Jope Richard S.
Published: October 2006

Abstract: Administration of kainate or pentylenetetrazole increased c‐fos, c‐jun, junB, and junD mRNA levels in rat brain in a dose‐dependent manner. Kainate increased these mRNA levels predominantly in the hippocampus, and pentylenetetrazole was more effective in the cortex. Adrenalectomy (3 days) was used to eliminate endogenous glucocorticoid hormones. Adrenalectomy significantly potentiated kainate‐induced increases, compared with increases caused by kainate (4 mg/kg) alone, in the hippocampal mRNA levels of c‐fos and junB by 6.5‐fold and of junD by twofold and tended to augment c‐jun mRNA. Corticosterone administration blocked the potentiated stimulation of these mRNA levels caused by adrenalectomy. Adrenalectomy also significantly increased pentylenetetrazole‐induced levels of c‐fos mRNA in the cortex. These results demonstrate that glucocorticoids modulate immediate early gene expression in the brain, raising the possibility that this interaction contributes to interneuronal and interindividual differences in responses to stimuli and to the effects of stress‐ or disease‐induced changes in glucocorticoid concentrations.
Developmental Changes in β‐Citryl‐L‐G1utamate Concentration and Its Synthetic and Hydrolytic Activities in Neuronal Cells Cultured from Chick Embryo Optic Lobes
Authors: Miyake Masaharu , Morino Hideo
Published: October 2006

Abstract: Developmental changes in the concentration of β‐citryl‐L‐glutamate (β‐CG) have been examined in the cerebrum and optic lobe of the developing chick brain and in primary cultured neuronal cells from the chick embryo optic lobes with gas chromatographic and HPLC methods originated in our studies. A sharp peak was shown by β‐CG, with a maximal concentration at 13 days of incubation in the optic lobe of the developing chick brain but decreasing markedly to adult levels. The developmental change in primary cultured neurons was similar to that in the optic lobe of the developing chick brain. Changes in synthetic and hydrolytic activities of β‐CG were studied during growth of primary cultured neurons. Incorporation of radioactivities from radiolabeled pyruvate and alanine into β‐CG increased significantly on day 3 of culture, reaching a plateau on day 6, whereas that from radioactive glutamine and glutamate increased gradually from day 3 to day 12 of culture. The hydrolyzing enzyme activity of β‐CG during neuron growth was low until day 3 of culture, when it increased significantly until day 12. Similar developmental changes were observed in the developing chick embryo optic lobes.
Isolated Bovine Spinal Motoneurons Have Specific Ganglioside Antigens Recognized by Sera from Patients with Motor Neuron Disease and Motor Neuropathy
Authors: Yoshino Hiide , Miyatani Nobuyuki , Saito Megumi , Ariga Toshio , Lugaresi Alessandra , Latov Norman , Kushi Yasunori , Kasama Takeshi , Yu Robert K.
Published: October 2006

Abstract: The gangliosides GM1 and GD1b have recently been reported to be potential target antigens in human motor neuron disease (MND) or motor neuropathy. The mechanism for selective motoneuron and motor nerve impairment by the antibodies directed against these gangliosides, however, is not fully understood. We recently investigated the ganglioside composition of isolated bovine spinal motoneurons and found that the ganglioside pattern of the isolated motoneurons was extremely complex. GM1, GD1a, GD1b, and GT1b, which are major ganglioside components of CNS tissues, were only minor species in motoneurons. Among the various ganglioside species in motoneurons, several were immunoreactive to sera from patients with MND and motor neuropathy. One of these gangliosides was purified from bovine spinal cord and characterized as N‐glycolylneuraminic acid‐containing GM1 [GM1(NeuGc)] by compositional analysis, fast atom bombardment mass spectra, and the use of specific antibodies. Among seven sera with anti‐GM1 antibody activities, five sera reacted with GM1(NeuGc) and two did not. Two other gangliosides, which were recognized by another patient's serum, appeared to be specific for motoneurons. We conclude that motoneurons contained, in addition to the known ganglioside antigens GM1 and GDlb, other specific ganglioside antigens that could be recognized by sera from patients with MND and motor neuropathy.
Molecular Properties of 5‐Hydroxytryptamine3 Receptor‐Type Binding Sites Purified from NG1O8‐15 Cells
Authors: Boess Frank G. , Lummis Sarah C. R. , Martin Ian L.
Published: October 2006

Abstract: 5‐Hydroxytryptamine3 (5‐HT3) receptor‐type binding sites were solubilised from NG108‐15 mouse neuro‐blastoma X rat glioma hybrid cells using five different detergents (n‐octyl‐β‐D‐glucoside, Triton X‐100, 3‐[3‐(cholamidopropyl)dimethylammonio]‐1‐propanesulphonate (CHAPS), sodium cholate, and deoxycholate) and the solubilisation efficiencies compared. The equilibrium binding, kinetic properties, and pharmacological profile of solubilised binding sites were similar to those of 5‐HT3 receptor‐type binding sites (5‐HT3R) in membrane preparations determined using [3H]GR65630. The solubilised binding sites were purified using an affinity column constructed by coupling the high‐affinity antagonist GR1 19566X to an Affi‐Gel 15 resin. The affinity of purified 5‐HT3R for [3H]GR65630 was reduced threefold compared to the crude soluble preparation, but the pharmacological profile was similar. The sedimentation coefficient of the purified protein (US, detergent: CHAPS) was determined by sucrose density gradient centrifugation. The apparent molecular mass of the detergent/binding site complex (370 kDa) was determined by size exclusion chromatography in the presence of n‐dodecyl‐β‐D‐maltoside. Gel electrophoresis of the purified protein revealed bands at apparent molecular masses of 36, 40, 50, and 76 kDa. Electron microscopy of the negatively stained purified protein showed the presence of round particles of 8–9 nm diameter with a 2‐nm stained pit in the centre, closely resembling the doughnut shapes described for nicotinic acetylcholine receptors.
Loss and Ca2+‐Dependent Retention of Scinderin in Digitonin‐Permeabilized Chromaffin Cells: Correlation with Ca2+‐Evoked Catecholamine Release
Authors: Vitale M. L. , Del Castillo A. Rodríguez , Trifaró J.‐M.
Published: October 2006

Abstract: Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca2+‐dependent actin‐filament‐severing protein), but not gelsolin, is among the proteins that leak out from digitonin‐permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15–40 μM) of digitonin for 5 min released scinderin into the medium. One‐minute treatment with 20 μ digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose‐dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin‐permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca2+‐binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromamn cell secretory response. Permeabilization in the presence of increasing free Ca2+ concentrations produced a concomitant enhancement in the subsequent Ca2+‐dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micro‐molar Ca2+ concentrations retained Ca2+‐binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca2+‐dependent catecholamine release observed in permeabilized chromaffin cells.
A Quantitative Study of the Ca2+/Calmodulin Sensitivity of Adenylyl Cyclase in Aplysia, Drosophila, and Rat
Authors: Yovell Yoram , Kandel Eric R. , Dudai Yadin , Abrams Thomas W.
Published: October 2006

Abstract: Studies in Aplysia and Drosophila have suggested that Ca2+/calmodulin‐sensitive adenylyl cyclase may act as a site of convergence for the cellular representations of the conditioned stimulus (Ca2+ influx) and unconditioned stimulus (facilitatory transmitter) during elementary associative learning. This hypothesis predicts that the rise in intracellular free Ca2+ concentration produced by spike activity during the conditioned stimulus will cause an increase in the activity of adenylyl cyclase. However, published values for the Ca2+ sensitivity of Ca2+/calmodulin‐sensitive adenylyl cyclase in mammals and in Drosophila vary widely. The difficulty in evaluating whether adenylyl cyclase would be activated by physiological elevations in intracellular Ca2+ levels is in part a consequence of the use of Ca2+/EGTA buffers, which are prone to several types of errors. Using a procedure that minimizes these errors, we have quantified the Ca2+ sensitivity of adenylyl cyclase in membranes from Aplysia, Drosophila, and rat brain with purified species‐specific calmodulins. In all three species, adenylyl cyclase was activated by an increase in free Ca2+ concentration in the range caused by spike activity. Ca2+ sensitivity was dependent on both calmodulin concentration and Mg2+ concentration. Mg2+ raised the threshold for adenylyl cyclase activation by Ca2+ but also acted synergistically with Ca2+ to activate maximally adenylyl cyclase.
Triiodothyronine Has Diverse and Multiple Stimulating Effects on Expression of the Major Myelin Protein Genes
Authors: Tosic Mirjana , Torch Sakina , Comte Véronique , Dolivo Michel , Honegger Paul , Matthieu Jean‐Marie
Published: October 2006

Abstract: If the importance of triiodothyronine (T3) on brain development including myelinogenesis has long been recognized, its mechanism of action at the gene level is still not fully elucidated. We studied the effect of T3 on the expression of myelin protein genes in aggregating brain cell cultures. T3 increases the concentrations of mRNA transcribed from the following four myelin protein genes: myelin basic protein (Mbp), myelin‐associated glycoprotein (Mag), proteolipid protein (Plp), and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (Cnp). T3 is not only a triggering signal for oligodendrocyte differentiation, but it has continuous stimulatory effects on myelin gene expression. Transcription in isolated nuclei experiments shows that T3 increases Mag and Cnp transcription rates. After inhibiting transcription with actinomycin D, we measured the half‐lives of specific mRNAs. Our results show that T3 increases the stability of mRNA for myelin basic protein, and probably proteolipid protein. In vitro translation followed by myelin basic protein‐specific immunoprecipitation showed a direct stimulatory effect of T3 on myelin basic protein mRNA translation. Moreover, this stimulation was higher when the mRNA was already stabilized in culture, indicating that stabilization is achieved through mRNA structural modifications. These results demonstrate the diverse and multiple mechanisms of T3 stimulation of myelin protein genes.
Stimulation of δ‐Opioid Receptors Reduces the In Vivo Binding of the Cholecystokinin (CCK)‐B‐Selective Agonist [3H]pBC 264: Evidence for a Physiological Regulation of CCKergic Systems by Endogenous Enkephalins
Authors: Ruiz‐Gayo M. , Durieux C. , Fournié‐Zaluski M. C. , Roques B. P.
Published: October 2006

Abstract: Cholecystokinin (CCK) and enkephalins appear to be colocalized in several brain structures, and a physiological interaction between these peptides has been suggested by a large number of pharmacological studies. In this work we have shown, by in vivo binding experiments, that the endogenous enkephalins, protected from degrading enzymes by mixed inhibitors such as kelatorphan and N‐[(R,S)‐2‐benzyl‐3‐[(S)2‐amino‐4‐methylthiobutyldithio]‐1‐oxopropyl]‐L‐phenylalanine benzyl ester (RB 101), a systemically active prodrug, modulate CCK release in mouse brain, leading to an overall increase in the extracellular levels of CCK. This was quantified by measuring the effects of both inhibitors on the in vivo binding of [3H]propionyl‐Tyr(SO3H)‐gNle‐mGly‐Trp‐(N‐Me)Nle‐Asp‐Phe‐NH2 ([3H]‐pBC 264), a selective and highly potent CCK‐B agonist. Thus, intracerebroventricular injection of kelatorphan produced a dose‐dependent inhibition of the in vivo binding of [3H]pBC 264 with a maximal effect (40%) at 50 nmol. A similar response was observed after intravenous injection of RB 101 (40 mg/kg). The specific binding of [3H]pBC 264 was also inhibited (25%) by intravenous injection of the selective δ‐opioid agonist H‐Tyr‐D‐Cys(StBu)‐Gly‐Phe‐Leu‐Thr(OtBu)‐OH (BUBUC; 2 mg/kg) but not by the μ‐agonist H‐Tyr‐D‐Ala‐Gly‐(N‐Me)Phe‐Gly‐ol (5 mg/kg), suggesting a preferential involvement of δ‐opioid receptors in the modulation of CCK release. This was confirmed by using the selective δ‐opioid antagonist naltrindole, which prevented the inhibitory effects of BUBUC and of enkephalin‐degrading enzyme inhibitors on [3H]pBC 264 binding. These results are discussed in terms of homeostatic regulation of the nociceptive threshold to opioids by endogenous CCK.
Generation of Arachidonic Acid and Diacylglycerol Second Messengers from Polyphosphoinositides in Ischemic Fetal Brain
Authors: Kunievsky Baruch , Bazan Nicolas G. , Yavin Ephraim
Published: October 2006

Abstract: Intracerebral administration of [3H]arachidonic acid ([3H]ArA) into 19–20‐day‐old rat embryos, resulted in a rapid incorporation of label into brain lipids. One hour after injection, 55.6 pmn 8.2, 18.0 pmn 3.4, and 13.7 pmn 1.3% of the total radioactivity was associated with phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine, respectively. Approximately 10% of radioactivity was found acylated in neutral lipids of which free ArA comprised only 1.5 pmn 0.2% of the total radioactivity. Complete restriction of the maternal—fetal circulation for ltm40 min did not affect the rate of [3H]ArA incorporation (t1/2= 2 min) into fetal brain lipids, suggesting an effective acylation mechanism that proceeds irrespective of the impaired blood flow. After a short restriction period (5 min), the radioactivity in diacylglycerol was elevated by 50%. After a longer restriction period (20 min), the radioactivity in the free fatty acid and diacylglycerol fractions increased to values of 130 and 87%, respectively. Polyphosphoinositides prelabeled with either [3H]ArA or 32P were rapidly degraded after 5 min of ischemia. After 20 min, the decrease in phosphatidylinositol‐4‐phosphate and phosphatidylinositol‐4,5‐bisphosphate radioactivity was 47 and 70%, respectively. Double labeling of phospholipids with [14C]palmitic acid and [3H]ArA indicated a preferential loss of [3H]ArA within the polyphosphoinositide species after 20 min, but not after 5 min of ischemia. The specific activity of [14C]palmitate remained unchanged. The current data suggest phospholipase C—mediated diacyiglycerol formation at the beginning of the insult followed by a phospholipase A2‐mediated ArA liberation at a later time, both enzymes presumably acting preferentially on polyphosphoinositide species.
Cytosolic Free Calcium and Gene Expression During Chemical Hypoxia
Authors: Carroll Joanne M. , Toral‐Barza Lourdes , Gibson Gary
Published: October 2006

Abstract: Understanding the cellular response to hypoxia may help elucidate the role of altered oxidation in neuronal death or abnormal cell function. In PC 12 cells, 30 min of chemical hypoxia (i.e., KCN) reduced ATP concentrations by 92%, but diminished viability by only 10%. Ten minutes of hypoxia increased cytosolic free calcium ([Ca2+]i) 2.5‐fold above control, but after 30 min of hypoxia, [Ca2+]i was slightly below that of nonhypoxic cells. Short periods of hypoxia also exaggerated the K+‐induced elevation of [Ca2+]i, but by 30 min these ATP‐depleted cells reestablished a calcium gradient that was equal to nonhypoxic, K+‐depolarized cells. Thus, 30 min of severe ATP depletion left [Ca2+]i and viability relatively unaffected. Nerve growth factor caused slight, but significant, improvements in ATP and viability of hypoxic cells, but had no effect on [Ca2+]i. Although [Ca2+]i was equivalent in control and hypoxic cells after 30 or 60 min, hypoxia abolished the K+‐stimulated elevation of [Ca2+]i. The nerve growth factor induction of c‐fos, an indicator of the genomic response, was diminished by sim 80%. Thus, hypoxic PC 12 cells with greatly reduced ATP stores maintained normal [Ca2+]i, but their ability to respond to external stimulation was impaired. Further, the reduced oxidation that occurs in the brain in a variety of pathological conditions may interfere with the cellular response to stimulation and growth factors.
Myelin Gangliosides of Human Peripheral Nervous System: An Enrichment of GM1 in the Motor Nerve Myelin Isolated from Cauda Equina
Authors: Ogawa‐Goto K. , Funamoto N. , Ohta Y. , Abe T. , Nagashima K.
Published: October 2006

Abstract: Myelins of the PNS were isolated from human motor and sensory nerves of cauda equina, and their ganglioside compositions were compared. The predominant ganglioside in the human PNS myelins, both from motor and sensory nerves, was LM1 (sialosylneolactotetraosylceramide). Sialosyl‐nLc6Cer and disialosyl‐nLc4Cer, GD3, GM3, and GDlb were detected as common components of the two nerve myelins. Furthermore, it was revealed that the motor nerve myelin contained GM1 (about 15% of total gangliosides), whereas sensory nerve myelin contained only a trace amount of GM1 (less than 5%), by TLC analyses together with TLC immunostaining using anti‐GM 1 antibody. As for the disialoganglioside fraction, the content of GD1 a, as well as that of GM1, differed in motor and sensory nerves. Thus, the different contents of the ganglioseries gangliosides in human motor and sensory nerve myelins were demonstrated.
Endopeptidase 24‐16 in Murines: Tissue Distribution, Cerebral Regionalization, and Ontogeny
Authors: Dauch P. , Masuo Y. , Vincent J. P. , Checler F.
Published: October 2006

Abstract: The tissue distribution, cerebral regionalization, and ontogeny of endopeptidase 24–16 were established in murines by means of its quenched fluorimetric substrate, Mcc‐Pro‐Leu‐Gly‐Pro‐D‐Lys‐Dnp, and its selective dipep‐tide blocker, Prolie. Endopeptidase 24–16 was particularly abundant in the liver and kidney, and the lowest specific activity was detected in the heart. In the brain, a 16‐fold difference in specific activity was observed between the poorest and the richest cerebral areas. Endopeptidase 24–16 appeared in high concentrations in the olfactory bulb and tubercule, cingulate cortex, medial striatum, and globus pallidus, and was particularly weak in the CA1, CA2, and CA3 parts of the hippocampal formation and in the cerebellum. Endopeptidase 24–16 content in thirteen thalamic nuclei indicated a rather homogeneous distribution. This homogeneity was not observed in the hypothalamus, where pronounced variations occurred between enriched zones such as suprachiasmatic and arcuate nuclei and relatively poor areas such as periventricular and supraoptic nuclei. Endopeptidase 24–16 appeared to be developmentally regulated in the mouse brain; it was already detected at the fetal stage, increased transiently after birth, then regularly declined until adulthood.
Investigations About a Direct Neurotensin‐Dopamine Interaction by Nuclear Magnetic Resonance Study, Synaptosomal Uptake of Dopamine, and Binding of Neurotensin to Its Receptors
Authors: Nouel D. , Costentin J. , Lugrin D. , Kitabgi P. , Pie N. , Davoust D.
Published: October 2006

Abstract: Interactions between dopamine and neurotensin can occur at various levels of the dopaminergic pathways. By using different approaches in vitro, we investigated the proposed hypothesis that neurotensin might bind to dopamine in the synaptic cleft. Nuclear magnetic resonance spectra of neurotensin were not modified by the addition of dopamine, and no nuclear Overhauser effect was detected. Synaptosomal uptake of [3H]dopamine in the presence of neurotensin did not lead to any modifications of the kinetic constants of the uptake. Neurotensin binding was not modified by the addition of dopamine. These results did not confirm the suggestion that neurotensin can form a complex with dopamine.
Functional Reconstitution of α‐Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionate (AMPA) Receptors from Rat Brain
Authors: Bahr Ben A. , Vodyanoy Vitaly , Hall Randy A. , Suppiramaniam Vishnu , Kessler Markus , Sumikawa Katumi , Lynch Gary
Published: October 2006

Abstract: Glutamate receptors belonging to the subclass specifically activated by α‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid (AMPA) were solubilized from rat forebrain membranes with Triton X‐100 and partially purified through a series of three chromatographic steps. Specific [3H]AMPA binding increased 30–60‐fold during the isolation procedure. A protein band recognized by antibodies against specific amino acid sequences of the glutamate receptor‐A subunit was enriched with each purification step; the molecular mass of this band (105 kDa) corresponded to that of cloned AMPA receptor subunits. Photoaffinity labeling of forebrain membranes with 6‐cyano‐7‐[3H]hitroquinoxaline‐2,3‐dione, a specific antagonist of the AMPA receptor, labeled a single band that comigrated with the immunolabeled protein. On reconstitution of the partially purified material into bilayer patches, singlechannel current fluctuations were elicited by 300 nM AMPA and blocked by 1 μM 6,7‐dinitroquinoxaline‐2,3‐dione.
Nootropic Drugs Positively Modulate α‐Amino‐3‐Hydroxy‐5‐Methyl‐4‐Isoxazolepropionic Acid‐Sensitive Glutamate Receptors in Neuronal Cultures
Authors: Copani A. , Genazzani A. A. , Aleppo G. , Casabona G. , Canonico P. L. , Scapagnini U. , Nicoletti F.
Published: October 2006

Abstract: Micromolar concentrations of piracetam, aniracetam, and oxiracetam enhanced α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)‐stimulated 45Ca2+ influx in primary cultures of cerebellar granule cells. Nootropic drugs increased the efficacy but not the potency of AMPA and their action persisted in the presence of the voltage‐sensitive calcium channel blocker nifedipine. Potentiation by oxiracetam was specific for AMPA receptor‐mediated signal transduction, as the drug changed neither the stimulation of 45Ca2+ influx by kainate or N‐methyl‐d‐aspartate nor the activation of inositol phospholipid hydrolysis elicited by quisqualate or (±)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylic acid. Piracetam, aniracetam, and oxiracetam increased the maximal density of the specific binding sites for [3H]AMPA in synaptic membranes from rat cerebral cortex. Taken collectively, these results support the view that nootropic drugs act as positive modulators of AMPA‐sensitive glutamate receptors in neurons.
Characterization of the P‐Type and V‐Type ATPases of Cholinergic Synaptic Vesicles and Coupling of Nucleotide Hydrolysis to Acetylcholine Transport
Authors: Hicks Barry W. , Parsons Stanley M.
Published: October 2006

Abstract: Both phosphointermediate‐ and vacuolar‐type (P‐and V‐type, respectively) ATPase activities found in cholinergic synaptic vesicles isolated from electric organ are immunoprecipitated by a monoclonal antibody to the SV2 epitope characteristic of synaptic vesicles. The two activities can be distinguished by assay in the absence and presence of vanadate, an inhibitor of the P‐type ATPase. Each ATPase has two overlapping activity maxima between pH 5.5 and 9.5 and is inhibited by fluoride and fluorescein isothiocyanate. The P‐type ATPase hydrolyzes ATP and dATP best among common nucleotides, and activity is supported well by Mg2+, Mn2+, or Co2+ but not by Ca2+, Cd2+, or Zn2+. It is stimulated by hyposmotic lysis, detergent solubilization, and some mitochondrial uncouplers. Kinetic analysis revealed two Michaelis constants for MgATP of 28 μM and 3.1 mM, and the native enzyme is proposed to be a dimer of 110‐kDa subunits. The V‐type ATPase hydrolyzes all common nucleoside triphosphates, and Mg2+, Ca2+, Cd2+, Mn2+, and Zn2+ all support activity effectively. Active transport of acetylcholine (ACh) also is supported by various nucleoside triphosphates in the presence of Ca2+ or Mg2+, and the Km for MgATP is 170 μM. The V‐type ATPase is stimulated by mitochondrial uncouplers, but only at concentrations significantly above those required to inhibit ACh active uptake. Kinetic analysis of the V‐type ATPase revealed two Michaelis constants for MgATP of ∼ 26 μM and 2.0 mM. The V‐type ATPase and ACh active transport were inhibited by 84 and 160 pmol of bafilomycin A1/mg of vesicle protein, respectively, from which it is estimated that only one or two V‐type ATPase proton pumps are present per synaptic vesicle. The presence of presumably contaminating Na+, K+‐ATPase in the synaptic vesicle preparation is demonstrated.
Effect of Pertussis Toxin on Radioligand Binding to Rat Brain Adenosine A1 Receptors
Authors: van der Ploeg Ingeborg , Parkinson Fiona E. , Fredholm Bertil B.
Published: October 2006

Abstract: In a previous study we showed that in vivo treatment with pertussis toxin could inhibit some, but not all, effects of adenosine in the rat hippocampus. In this study we investigated the effect of pertussis toxin on the binding of adenosine analogues to A1 receptors in rat brain. Intraventricular injection of pertussis toxin (10 μg into the lateral ventricle) did not affect A1 receptor binding in any brain region studied, as evaluated by autoradiography. In vitro treatment of brain sections (10 μm) with pertussis toxin for 5 h, under conditions when >80% of the G proteins were ADP ribosylated, did not alter radioligand binding to adenosine A1 receptors. GTP (10 μM) virtually abolished the high‐affinity agonist binding to the A1 receptor. On the other hand, in solubilized cortical membrane preparations, pertussis toxin pretreatment induced a complete shift of the A1 receptors to the low‐affinity state. This suggests that the ability of pertussis toxin to affect G proteins coupled to A1 receptors in brain depends not only on the distribution of the toxin but also on the configuration of receptors and G proteins.
Characterization of [3H]CP‐96,501 as a Selective Radioligand for the Serotonin 5‐HT1B Receptor: Binding Studies in Rat Brain Membranes
Authors: Koe B. Kenneth , Lebel Lorraine A. , Fox Carol B. , Macor John E.
Published: October 2006

Abstract: 3‐(1,2,5,6‐Tetrahydro‐4‐pyridyl)‐5‐n‐propoxyindole (CP‐96,501) was found to be a more selective ligand at the serotonin 5‐HT1B receptor than the commonly used 5‐HT1B agonist, 3‐(1,2,5,6‐tetrahydro‐4‐pyridyl)‐5‐methoxyindole (RU 24969). In rat brain membranes, the tritiated derivative, [3H]CP‐96,501, was found to bind with a high affinity (KD, 0.21 nM) to a single binding site (nH, 1.0). The receptor density of this site (Bmax, 72 fmol/mg of protein) matched that of the 5‐HT1B receptor determined with [3H]5‐HT. Competition curves of 16 serotonergic compounds in [3H]CP‐96,501 binding also indicated a single binding site. The rank order of their binding affinities with this new radioligand showed a high degree of correlation with their affinities at the 5‐HT1B receptor determined with [3H]5‐HT or [125I]iodocyanopindolol. Serotonergic compounds displayed competitive inhibition of [3H]CP‐96,501 binding. In the presence of 5′‐guanylylimidodiphosphate [Gpp(NH)p], [3H]CP‐96,501 binding was reduced, while the potency of CP‐96,501 to displace [125I]iodocyanopindolol binding was also decreased. These findings are consistent with the agonist nature of CP‐96,501. The results of this study suggest that [3H]CP‐96,501 is a useful agonist radioligand for the 5‐HT1B receptor.
Comparison of [3H]Choline and d‐[3H]Aspartate Efflux from Guinea Pig and Human Neocortex
Authors: Beani L. , Bianchi C. , Antonelli T. , Caló G. , Morari M. , Ferioli V. , Gaist G.
Published: October 2006

Abstract: The outflow of [3H]choline ([3H]Ch) evoked by electrical field stimulation and the efflux of d‐[3H]Asp induced by 35 mM KCl and 1–10 μM ouabain were studied in human and guinea pig cortical slices, kept under identical experimental conditions. [3H]Ch outflow was significantly lower whereas d‐[3H]Asp efflux was significantly higher in humans than in guinea pigs. This suggests a different proportion of the two neuronal systems in these two species. Blockade of muscarinic autoreceptors with atropine increased, whereas stimulation of α2 receptors with norepinephrine (NE) reduced, the evoked [3H]Ch outflow to the same extent in human and guinea pig cortical slices. Conversely, NE did not affect ouabain‐induced d‐[3H]Asp efflux, suggesting that an α2‐mediated control is not operative in the glutamatergic cortical structures. Desmethylimipramine, 2–5 μM, was able to increase [3H]Ch outflow through atropine‐like mechanisms only in the human. This drug at 20–50 μM inhibited [3H]Ch and d‐[3H]Asp efflux in both species, through mechanisms unrelated to its monoamine reuptake blocking properties. Thus, similarities and differences can be detected between humans and guinea pigs with regard to (a) the relative potency of the cholinergic and acidic amino acidergic signals and (b) the modulation of neurotransmitter outflow by drugs acting on auto‐ and heteroreceptors.
Fractionation of Primary Cultured Cerebellar Neurons: Distribution of Sialyltransferases Involved in Ganglioside Biosynthesis
Authors: Iber Heinrich , van Echten Gerhild , Sandhoff Konrad
Published: October 2006

Abstract: Primary cultured neurons were fractionated using sucrose density gradients. The activities of four sialyltransferases (GM3, GD3, GDla, and GTla synthase) involved in ganglioside biosynthesis were assayed in the collected fractions. The distribution of GM3 synthase coincided with that of mannosidase II, an enzyme assumed to be a cis‐Golgi marker. Both enzymes were mainly associated with the more dense fraction. GDl a and GTla synthase activities, on the other hand, were mainly recovered in the less dense fraction. Moreover, they were colocalized with thiamine pyrophos‐phatase, an enzyme assumed to be a marker of the late Golgi (trans‐Golgi and trans‐Golgi network). GD3 synthase activity was equally distributed between both fractions. These results are integrated in a model of ganglioside biosynthesis.
Regional Distribution of α2A‐and α2B‐Adrenoceptor Subtypes in Postmortem Human Brain
Authors: De Vos Hilde , Vauquelin Georges , De Keyser Jacques , De Backer Jean‐Paul , Van Liefde Isabelle
Published: October 2006

Abstract: The newly available and highly selective radiolabeled antagonist [3H]RX 821002 was used to examine the distribution of α2 adrenoceptors in human brain. High densities of α2 adrenoceptors were found in the hippocampus, frontal cortex, thalamus, amygdala, pons, and medulla oblongata. Intermediate densities were observed in the striatum (nucleus accumbens, nucleus caudatus, and putamen), globus pallidus, and substantia nigra. The KD values for [3H]RX 821002 were similar in all regions (ranging from 2.8 to 7.5 nM). On the basis of their different affinities for prazosin and oxymetazoline, the α2 adrenoceptors have been divided into α2A and α2B subtypes. To examine the α2A/α2B‐adrenoreceptor ratio in the different brain regions, we performed oxymetazoline and prazosin/[3H]RX 821002 competition binding experiments. In frontal cortex membranes, the competition curves with prazosin were steep, indicating a single class of binding sites, whereas the competition curves with oxymetazoline were shallow and fitted by computer best to a two‐site model. However, in the presence of GTP, the high‐affinity sites for oxymetazoline were partially converted into low‐affinity sites, indicating that this agonist interacts with high‐ and low‐affinity states of the α2 adrenoceptors. This implies that oxymetazoline is not very suitable for discriminating the α2A‐and α2B‐receptor subtypes in radioligand binding studies. Therefore, prazosin/[3H]RX 821002 competition binding experiments were used to investigate the distribution of the α2‐adrenoceptor subtypes in human brain. The α2A‐receptor subtype was detected in all brain regions examined. In contrast, α2B receptors were only observed in striatum and globus pallidus.
MK‐801 Increases Extracellular 5‐Hydroxytryptamine in Rat Hippocampus and Striatum In Vivo
Authors: Whitton P. S. , Biggs C. S. , Pearce B. R. , Fowler L. J.
Published: October 2006

Abstract: The effect of MK‐801 (0.25 or 0.5 mg/kg) on the extracellular concentration of 5‐hydroxytryptamine (5‐HT) and 5‐hydroxyindoleacetic acid (5‐HIAA) in rat hippocampus and striatum was studied using intracerebral dialysis. The dialysate 5‐HT concentration was dose‐dependently increased by MK‐801 in both regions. In the hippocampus, at the higher drug dose a slow increase in the 5‐HIAA level was observed, and this became significant 3 h after treatment. In contrast to this, the extracellular 5‐HIAA content in the striatum was significantly decreased 150 min after administration of both doses of MK‐801. The data are discussed in the light of the known behavioural effects of MK‐801 and possible N‐methyl‐d‐aspartic acid receptor regulation of 5‐HT release.
5‐Hydroxytryptamine Receptors
Authors: Peroutka Stephen J.
Published: October 2006

5‐Hydroxytryptamine (5‐HT) receptors consist of at least three distinct types of molecular structures (Table 1): guanine nucleotide binding protein (G protein)‐coupled receptors, ligand‐gated ion channels, and transporters. Prior to the introduction of molecular biological techniques, the classification of 5‐HT receptors was based predominantly on the pharmacological properties of the receptors. For example, “5‐HT1” receptors were defined as membrane binding sites that displayed nanomolar affinity for [3H]5‐HT (Peroutka and Snyder, 1979). Subsequently, Bradley et al. (1986) defined “5‐HT1‐like” receptors by their susceptibility to antagonism by methiothepin and/or methysergide, resistance to antagonism by 5‐HT2 antagonists, and potent agonism by 5‐carboxamidotryptamine (5‐CT). Thus, these classification systems are dependent upon the availability of selective pharmacological agents.