bluuebunny/crossref_metadata_2025 huggingface dataset:
Blockade of Striatal 5‐Hydroxytryptmine2Receptors Reduces the Increase in Extracellullar Concentrations of Dopamine Produced by the Amphhetamine analogue 3,4‐Methylenedioxymethamphetamine
Authors: Schmidt Christopher J. , Sullivan C. K. , Fedayal G. M.
Published: July 2010

Abstract: 5‐Hydroxytryptamine2(5‐HT2) receptor antagonists have been shown to interfere with the stimulation of striatal dopamine synthesis and release produced by the amphetamine analogue 3,4‐methylenedioxymethamphetamine (MDMA). To localize the receptors responsible for the attenuation of MDMA‐induced release, 5‐HT2receptor antagonists were infused via the microdialysis probe directly into the brains of awake, freely moving rats before the systemic administration of MDMA. Intrastriatal infusions of the selective 5‐HT2antagonist MDL 100, 907 produced a concentration‐dependent inhibition of MDMA‐induced dopamine release. Similar results were observed with intrastriatal infusions of the 5‐HT2antagonist amperozide. In contrast, infusion of MDL 100, 907 into the midbrain region near the dopaminergic cell bodies was with out effect on the MDMA‐induced elevation of extracellular dopamine in the ipsilateral striatum. Neither antagonist attenuated basal transmitter efflux nor the MDMA‐stimulated release of [3H]dopamine from striatal slices in vitro indicating that the in vivo effect of the antagonists was not due to inhibition of the dopamine uptake carrier. Intrastriatal infusion of tetrodotoxin reduced both basal and MDMA‐stimulated dopamine efflux and eliminated the effect of intrastriatal MDL 100, 907. The results indicate that 5‐HT2receptors located in the striatum augment the release of dopamine produced by high doses of MDMA. Furthermore, these 5‐HT2receptors appear to be located on nondopaminergic elements of the striatum.
Desensitization of Nicotine‐Stimulated [3H]Dopamine Release from Mouse Striatal Synaptosomes
Authors: Grady Sharon R. , Marks Michael J. , Collins Allan C.
Published: July 2010

Potential desensitization of brain nicotinic receptors was studied using a [3H]dopamine release assay. Nicotine‐stimulated [3H]dopamine release from mouse striatal synaptosomes was concentration‐dependent with an EC50of 0.33 ± 0.13 μMand a Hill coefficient of 1.44 ± 0.18. Desensitization by activating concentrations of nicotine had a similar EC50and a half‐time of 35 s. Concentrations of nicotine that evoked little release also induced a concentration‐dependent desensitization (EC50=6.9 plusmn; 3.6 nM, t1/2= 1.6‐2.0 min,nH=1.02 ± 0.01). Both types of desensitization produced a maximum 75% decrease in [3H]dopamine release. Recovery from desensitization after exposure to low or activating concentrations of nicotine was time‐dependent with half‐times of 6.1 min and 12.4 min, respectively. Constants determined for binding of [3H]nicotine to striatal membrane at 22°C included aKDof 3.7 ± 0.5 nM, Bmaxof 67.5 ± 2.2 fmol/mg, and Hill coefficient of 1.07 ± 0.06. Association of nicotine with membrane binding sites was biphasic with half‐times of 9 s and 1.8 min. The fast rate process contributed 37% of the total reaction. Dissociation was a uniphasic process with a half‐time of 1.6 min. Comparison of constants determined by the release and binding assays indicated that the [3H]‐nicotine binding site could be the presynaptic receptor involved in [3H]dopamine release in mouse striatal synaptosomes.
SR 140333, a Novel, Selective, and Potent Nonpeptide Antagonist of the NK1 Tachykinin Receptor: Characterization on the U373MG Cell Line
Authors: Oury‐Donat F. , Lefevre I. A. , Thurneyssen O. , Gauthier T. , Bordey A. , Feltz P. , Emonds‐Alt X. , Fur G. Le , Soubrie P.
Published: July 2010

The effects of a novel nonpeptide NK1 tachy‐kinin receptor antagonist, SR 140333, on the functional consequences of NK1 receptor activation in a human astrocytoma cell line, U373MG, were investigated. Radioligand binding conducted with125l‐Bolton‐Hunter substance P revealed a competitive inhibition by SR 140333 and itsRenantiomer SR 140603 withKivalues of 0.74 and 7.40 nM, respectively. The NK1‐selective agonist, [Sar9,Met(O2)11]‐substance P, stimulated the formation of inositol phosphates with an EC50of 3.8 × 10−9M.SR 140333 blocked the stimulatory effect of this agonist (10−7M) with an IC50of 1.6 × 10−9M,whereas the effect of another NK1 agonist, septide (EC50= 1.5 × 10−8M)was antagonized with an IC50of 2.1 × 10−10M.Enhancement of [3H]taurine release by [Sar9,Met(O2)11]‐substance P (EC50= 7.4 × 10−9M) was also inhibited by SR 140333 with an IC50of 1.8 × 10−9M. SR 140603 was 10‐fold less potent than SR 140333 in inhibiting inositol monophosphate formation and [3H]taurine release. The calcium mobilization induced by [Sar9,Met(O2)11]‐substance P (10−8M) was totally prevented by 10−8MSR 140333. Patchclamp experiments showed that SR 140333 depressed the outward current evoked by 5 × 10−8M[Sar9, Met(O2)11]‐substance P with an IC50of 1.3 × 10−9M.The expression of c‐foswas stimulated by [Sar9,Met(O2)11]‐substance P with an EC50of 2.5 × 10−10M, an effect that was also inhibited by SR 140333 with an IC50of 1.1 × 10−9M.The present results illustrate the sequential events of the response elicited by NK1 agonists, which were antagonized by SR 140333, demonstrating its powerful NK1 antagonist activity on a functional basis.
Regional Profile of Developmental Changes in the Sensitivity of theN‐Methyl‐D‐Aspartate Receptor to Polyamines
Authors: Subramaniam Swaminathan , McGonigle Paul
Published: July 2010

Abstract: The NMDA receptor exhibits increased sensitivity to stimulation during early development compared with the adult. In this study, we examined modulation of the NMDA receptor by polyamines during development to see if it correlates with differences in the functional responsiveness of the NMDA receptor. [3H]MK‐801 binding was measured in discrete brain regions in the presence and absence of polyamines in 3‐, 7‐, 15‐, 25‐, and 60‐day‐old Sprague‐Dawley rats. [3H]MK‐801 binding increased between postnatal days 3 and 15, with adult levels of binding being reached between days 15 and 25. Spermidine (75 μM) caused maximal stimulation of [3H]MK‐801 binding during early development, ranging from 250% in the thalamus to 450% in the caudate putamen at postnatal day 3. This effect gradually declined to levels seen in the adult by postnatal days 15–25. During all developmental stages, the stimulation seen was greater in the caudate putamen compared with the hippocampus. Diethylenetriamine (1 μM) exhibited similar developmental and regional heterogeneity in its effects on [3H]MK‐801 binding, producing substantial stimulation of binding in the neonate, but not in the adult. The EC50andEmaxvalues for the stimulatory effect of spermidine were significantly higher at day 7 compared with the adult. Unlike spermidine and diethylenetriamine, there was no regional variation in the effects of the putative “polyamine site” inverse agonist 1,10‐diaminodecane at any age and only a slightly attenuated inhibition at postnatal day 3 compared with the adult. This lack of complementarity in the regional and developmental profiles of spermidine and diethylenetriamine, on the one hand, and 1,10‐diaminodecane, on the other, suggests that their effects on [3H]MK‐801 binding are mediated at different sites. The altered sensitivity of the NMDA receptor to polyamines during development could reflect the expression of molecular variants with different sensitivities to modulation by polyamines.
Increased GABA Release in the Human Brain Cortex as a Potential Pathogenetic Basis of Hyperosmolar Diabetic Coma
Authors: Fink K. , Zentner J. , Göhert M.
Published: July 2010

Human cerebral cortical slices preincubated with [3H]GABA, [3H]noradrenaline, or 5‐[3H]hydroxytryptamine and superfused with Krebs solution or Mg2+‐free Krebs solution were used to investigate the influence of increased D‐glucose concentrations on the release of these [3H]‐neurotransmitters evoked by high K+content or NMDA receptor activation, respectively. An increase in level of D‐glucose (normal content, 11.1 mM) by 32, 60, and/or 100 mM(a range characteristic for hyperosmolar diabetic coma) increased the [3H]GABA release and inhibited the [3H]noradrenaline release evoked by both methods of stimulation. The K+‐induced 5‐[3H]hydroxytryptamine release was also inhibited by high D‐glucose content. Blockade of GABABreceptors byp‐(3‐aminopropyl)‐p‐diethoxymethylphosphinic acid (CGP 35348) attenuated the inhibitory effect of high D‐glucose content on the K+‐evoked release of [3H]noradrenaline and 5‐[3H]hydroxy‐tryptamine, suggesting that the effect on monoamine release is, at least to a major part, the result of the increased GABA release and, as a consequence, of an increased GABA concentration at inhibitory GABABreceptors. The membrane‐impermeable sorbitol mimicked the increasing effect of D‐glucose on [3H]GABA release and its inhibitory effect on 5‐[3H]hydroxytryptamine release. However, dimethyl sulfoxide, which is known to permeate rapidly through biological membranes, had no effect at concentrations equiosmolar to D‐glucose. It is concluded that a reduction in brain cell volume caused by increased extracellular, compared with cytoplasmic, osmolarity is crucial for the changes in neuronal function observed at high D‐ glucose and sorbitol content, In view of the fact that GABA is the main inhibitory neurotransmitter in the brain, the increased GABA release may be assumed to contribute to the pathogenesis of hyperosmolar diabetic coma.
Effect of Probe Size on the Concentration of Brain Extracellular Uric Acid Monitored with Carbon Paste Electrodes
Authors: Duff Adrienne , O'Neill Robert D.
Published: July 2010

Abstract: We have investigated further the anomalously high concentration of brain extracellular uric acid detected with in vivo sampling probes reported recently. The contribution by uric acid and 5‐hydroxyindoleacetic acid (5‐HIAA) to peak 2 recorded in rat striatum with chronically implanted carbon paste electrodes (CPEs) of different sizes was estimated by comparing peak current densities and the effect of the monoamine oxidase inhibitor pargyline. The concentration of uric acid in the extracellular fluid was some 50 times greater for 320‐μm‐diameter CPEs than for 160‐μm‐diameter electrodes, where the urate level was estimated at ∼1 μM. The concentration of 5‐HIAA was similar for 320‐, 260‐, and 160‐μm‐diameter CPEs. These data provide an explanation for the previously observed differences in 5‐HIAA/urate ratios re corded with 320‐μm‐diameter CPEs and smaller carbon fibre electrodes. The results also indicate that chronically implanted sampling probes of diameter >160 μm perturb the surrounding tissue, which produces uric acid by a mechanism yet unknown, although preliminary histological data suggest that glial cells may be involved.
Functional, Metabolic, and Circulatory Changes Associated with Seizure Activity in the Postischemic Brain
Authors: Katsura Ken‐ichiro , Folbergrová Jaroslava , Gidö Gunilla , Siesjö Bo K.
Published: July 2010

Abstract: The present study was undertaken to explore how transient ischemia in rats alters cerebral metabolic capacity and how postischemic metabolism and blood flow are coupled during intense activation. After 6 h of recovery following transient forebrain ischemia 15 min in duration, bicuculline seizures were induced, and brains were frozen in situ after 0.5 or 5 min of seizure discharge. At these times, levels of labile tissue metabolites were measured, whereas the cerebral metabolic rate for oxygen (CMRO2) and cerebral blood flow (CBF) were measured after 5 min of seizure activity. After 6 h of recovery, and before seizures, animals had a 40–50% reduction in CMRO2, and CBF. However, because CMRO2 rose threefold and CBF fivefold during seizures, CMRO2 and CBF during seizures were similar in control and postischemic rats. Changes in labile metabolites due to the preceding ischemia encompassed an increased phosphocreatine/ creatine ratio, as well as raised glucose and glycogen concentrations. Seizures gave rise to minimal metabolic perturbation, essentially comprising reduced glucose and glycogen contents and raised lactate concentrations. It is concluded that although transient ischemia leads to metabolic depression and a fall in CBF, the metabolic capacity of the tissue is retained, and drug‐induced seizures lead to a coupled rise in metabolic rate and blood flow.
Structural Determinants of the Alzheimer's Amyloid β‐Peptide
Authors: Soto Claudio , Brañes María Cecilia , Alvarez Jaime , Inestrosa Nibaldo C.
Published: July 2010

Abstract: The hallmark event of Alzheimer's disease (AD) is the deposition of amyloid as insoluble fiber masses in extracellular neuritic plaques and around the walls of cerebral blood vessels. The main component of amyloid is a hydrophobic peptide, named amyloid β‐peptide (βA4), which results from the processing of a much longer membrane amyloid precursor protein (APP). This review focuses on the structural features of βA4 and the factors that determine βA4 insolubilization. Theoretical and experimental studies of the primary structure of βA4 have shown that it is composed of a completely hydrophobic C‐terminal domain, which adopts β‐strand structure, and an N‐terminal region, whose sequence permits different secondary structures. In fact, this region can exist as an α‐helical or β‐strand conformation depending on the environmental condition (pH and hydrophobicity surrounding the molecule). The effects of pH and hydrophobicity on βA4 structure may elucidate the mechanisms determining its aggregation and amyloid deposition in AD.
The Effects of l‐Glutamate and trans‐(±)‐1‐Amino‐1,3‐Cyclopentanedicarboxylate on Phosphoinositide Hydrolysis Can Be Pharmacologically Differentiated
Authors: Littman Louis , Robinson Michael B.
Published: July 2010

Abstract: The excitatory amino acid analogues l‐glutamate (l‐Glu), l‐aspartate (l‐Asp), d‐Asp, and trans‐(±)‐1‐amino‐1,3‐cyclopentanedicarboxylate (trans‐ACPD) stimulate the hydrolysis of phosphoinositides (PI). In the present studies, the effects of noncompetitive and competitive inhibitors on PI hydrolysis stimulated by excitatory amino acid analogues were examined. When agonist and inhibitor were added simultaneously to hippocampal tissue, the noncompetitive inhibitor l‐2‐amino‐3‐phosphonopropionate (l‐AP3) did not block the effects of l‐Glu, l‐Asp, or d‐Asp at concentrations that block the effects of trans‐ACPD by more than 80%. When tissue was pre‐incubated with l‐AP3, the effects of l‐Glu, l‐Asp, or d‐Asp were blocked (IC50 values between 65 and 210 µM). Unlike l‐AP3, l‐aspartate‐β‐hydroxamate (l‐AβHA) inhibited PI hydrolysis stimulated by trans‐ACPD, l‐Glu, l‐Asp, or d‐Asp when agonist and inhibitor were added simultaneously in hippocampus; its effects were not time‐dependent. In cerebellum, both l‐AP3 and l‐AβHA had agonist activity. Inhibition by the recently identified competitive inhibitor (+)‐α‐methyl‐4‐carboxyphenylglycine [(+)‐MCPG] of PI hydrolysis was also examined. (+)‐MCPG blocked PI hydrolysis stimulated by trans‐ACPD, l‐Asp, or d‐Asp in both hippocampus and cerebellum (IC50 values between 220 and 1,700 µM). The effects of (+)‐MCPG were consistent with a competitive mechanism of action. (+)‐MCPG (up to 3 mM) blocked PI hydrolysis stimulated by l‐Glu by less than 25% in both hippocampus and cerebellum.
σ‐Receptor Regulation of [3H]Arachidonic Acid Release from Rat Neonatal Cerebellar Granule Cells in Culture
Authors: Starr Jonathan B. , Werling Linda L.
Published: July 2010

Abstract: σ receptors have been identified in many brain areas and are especially abundant in those regions known to be involved in control of movement. σ receptors have been located autoradiographically in the granule cell layer of cerebellum in adult rat brain. In the current study, we identified σ receptors in rat neonatal granule cells in culture using radioligand binding. The tritium labeled form of the putative σ antagonist haloperidol bound with high affinity to membranes prepared from these cells, and ligands selective for σ receptors competed well against [3H]haloperidol binding. The excitatory amino acid N‐methyl‐d‐aspartate and the direct phospholipase A2 activator melittin stimulated the release of [3H]arachidonic acid from cerebellar granule cells. The N‐methyl‐d‐aspartate‐stimulated, but not the melittin‐stimulated, release was inhibited in a concentration‐dependent manner by the σ‐selective agonist (+)‐pentazocine. In addition, the novel σ1 agonist BD737 inhibited N‐methyl‐d‐aspartate‐stimulated release. Pentazocine inhibition was almost completely reversed by the σ antagonists NPC‐16377 and opipramol. A 1 µM concentration of the phencyclidine receptor‐selective ligand MK‐801 inhibited ∼65% of N‐methyl‐d‐aspartate‐stimulated release. These results suggest that σ receptors may play a role in modulating arachidonic acid release in cerebellar granule cells.
Localized 13C NMR Spectroscopy in the Human Brain of Amino Acid Labeling from d‐[1‐13C]Glucose
Authors: Gruetter Rolf , Novotny Edward J. , Boulware Susan D. , Mason Graeme F. , Rothman Douglas L. , Shulman Gerald I. , Prichard James W. , Shulman Robert G.
Published: July 2010

Abstract: Cerebral metabolism of d[1‐13C]glucose was studied with localized 13C NMR spectroscopy during intravenous infusion of enriched [1‐13C]glucose in four healthy subjects. The use of three‐dimensional localization resulted in the complete elimination of triacylglycerol resonance that originated in scalp and subcutaneous fat. The sensitivity and resolution were sufficient to allow 4 min of time‐resolved observation of label incorporation into the C3 and C4 resonances of glutamate and C4 of glutamine, as well as C3 of aspartate with lower time resolution. [4‐13C]Glutamate labeled rapidly reaching close to maximum labeling at 60 min. The label flow into [3‐13C]glutamate clearly lagged behind that of [4‐13C]glutamate and peaked at t = 110–140 min. Multiplets due to homonuclear 13C‐13C coupling between the C3 and C4 peaks of the glutamate molecule were observed in vivo. Isotopomer analysis of spectra acquired between 120 and 180 min yielded a 13C isotopic fraction at C4 glutamate of 27 ± 2% (n = 4), which was slightly less than one‐half the enrichment of the C1 position of plasma glucose (63 ± 1%), p < 0.05. By comparison with an external standard the total amount of [4‐13C]glutamate was directly quantified to be 2.4 ± 0.1 µmol/ml‐brain. Together with the isotopomer data this gave a calculated brain glutamate concentration of 9.1 ± 0.7 µmol/ml, which agrees with previous estimates of total brain glutamate concentrations. The agreement suggests that essentially all of the brain glutamate is derived from glucose in healthy human brain.
Neuroprotective Effect of Hypothermia in Cortical Cultures Exposed to Oxygen‐Glucose Deprivation or Excitatory Amino Acids
Authors: Bruno V. M. G. , Goldberg M. P. , Dugan L. L. , Giffard R. G. , Choi D. W.
Published: July 2010

Abstract: We examined the effect of moderate hypothermia (30°C) on neuronal injury in murine cortical cell cultures. Lowering the temperature during and after a period of oxygen‐glucose deprivation reduced both the release of glutamate to the bathing medium and accompanying neuronal degeneration. Hypothermia immediately after brief exposure to high concentrations of NMDA or glutamate also reduced the resulting neuronal degeneration. This protective effect was not eliminated when MK‐801 and 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione were added immediately after washout of the exogenously added excitotoxin, suggesting that it was mediated by actions additional to reduction of endogenous late glutamate release. Hypothermia applied only during exposure to NMDA or glutamate, whether brief or prolonged, did not reduce subsequent cytosolic calcium accumulation or neuronal degeneration, suggesting that the postsynaptic induction of NMDA receptor‐mediated excitotoxicity is not sensitive to temperature reduction. However, hypothermia during prolonged S‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid or kainate exposure did reduce neuronal degeneration.
Role of Histidine Residues in the Adenosine A2a Receptor Ligand Binding Site
Authors: Askalan Rand , Richardson Peter J.
Published: July 2010

Abstract: The pH dependency of the binding of ligands to adenosine A2a receptors in rat striatal membranes was examined. For those agonists sensitive to adenosine deaminase a solubilised membrane preparation was used. A two‐ to fourfold increase in affinity was observed for CGS‐21680, 5′‐N‐ethylcarboxamidoadenosine, adenosine, 3′‐deoxyadenosine, 5′‐deoxyadenosine, inosine, and N6‐methoxypurine riboside on lowering the ambient pH from 7.0 to 5.5. In contrast, no such pH dependency was observed with 2′‐deoxyadenosine, although 2′‐methoxyadenosine binding was pH dependent. This effect on the affinity of CGS‐21680 was reduced by diethylpyrocarbonate and restored by hydroxylamine and implied a pK value of 7.0 for the histidine residue involved. No such dependence was observed with cyclopentyltheophylline or dimethylpropargylxanthine. It is concluded that one of the histidines conserved in the adenosine receptor binding site acts as a hydrogen bond donor to the oxygen of the 2′‐hydroxyl group of adenosine agonists.
[3H]Paroxetine Binding Is Altered in the Hippocampus but Not the Frontal Cortex or Caudate Nucleus from Subjects with Schizophrenia
Authors: Dean Brian , Opeskin Kenneth , Pavey Geoffrey , Naylor Lee , Hill Christine , Keks Nicholas , Copolov David L.
Published: July 2010

Abstract: [3H]Paroxetine binding to particulate membrane from tissue, obtained at autopsy, from the hippocampus, frontal cortex, and caudate nucleus from subjects who had or had not had schizophrenia was measured. The density of [3H]paroxetine binding to membranes from subjects who had or had not had schizophrenia did not differ. Similarly, the affinity of [3H]paroxetine binding in the frontal cortex and caudate nucleus was not different. By contrast, the affinity of [3H]paroxetine binding to hippocampal membrane from subjects who had schizophrenia was significantly lower than the affinity of binding for the nonschizophrenic subjects (0.40 ± 0.06 vs. 0.26 ± 0.02; p < 0.05). As [3H]paroxetine binds to the serotonin transporter, these data suggest that the serotonin transporter is altered in the hippocampus in subjects with schizophrenia.
Validation of a Noninvasive Method to Measure Brain Temperature In Vivo Using 1H NMR Spectroscopy
Authors: Corbett Ronald J. T. , Laptook Abbot R. , Tollefsbol Greg , Kim Ben
Published: July 2010

Abstract: The goal of this study was to evaluate the potential of using the difference between the 1H NMR frequencies of water and N‐acetylaspartic acid (NAA) to measure brain temperature noninvasively. All water‐suppressed and non‐water‐suppressed 1H NMR spectra were obtained at a field strength of 4.7 T using a surface coil. Experiments performed on model solutions revealed a decrease in the difference between NMR frequencies for NAA and water as a linear function of increasing temperature from 14 to 45°C. Changing pH in the range 5.5–7.6 produced no discernible trends for concurrent changes in the slope and intercept of the linear relationship. There were minor changes in slope and intercept for solutions containing 80 or 100 mg of protein/ml versus no protein, but these changes were not considered to be of sufficient magnitude to deter the use of this approach to measure brain temperature. The protein content of swine cerebral cortex was found to remain constant from newborn to 1 month old (78 ± 12 mg/g; n = 41). Therefore, data collected for the model solution containing 80 mg of protein/ml were used as a calibration curve to calculate brain temperature in eight swine during control, hypothermia, ischemia, postischemia, or death, over a temperature range of 23–40°C. A plot of 61 temperatures determined from 1H NMR versus temperatures measured from an optical fiber probe sensor implanted 1 cm into the cerebral cortex showed excellent linear agreement (slope = 1.00 ± 0.03, r2 = 0.96). We conclude that 1H NMR spectroscopy presents a practical means of making noninvasive measurements of brain temperature with an accuracy of better than ± 1°C.
Altered Neurotrophin mRNA Levels in Peripheral Nerve and Skeletal Muscle of Experimentally Diabetic Rats
Authors: Fernyhough Paul , Diemel Lara T. , Brewster Wendy J. , Tomlinson David R.
Published: July 2010

Abstract: The levels of neurotrophin mRNA in sensory ganglia, sciatic nerve, and skeletal muscle were measured in the streptozotocin‐diabetic rat using northern blotting. Periods of diabetes of 4, 6, and 12 weeks significantly elevated brain‐derived neurotrophic factor (BDNF) mRNA levels in soleus muscle compared with age‐matched controls, the increase being highest at 6 weeks. At all time periods studied, the levels of nerve growth factor (NGF) mRNA in soleus muscle were decreased by 21–47%. Following 12 weeks of diabetes, BDNF mRNA levels were increased approximately two‐to threefold in L4 and L5 dorsal root ganglia (DRG), and in sciatic nerve, NGF mRNA levels were raised 1.65‐fold. Intensive insulin treatment of diabetic rats for the final 4 weeks of the 12‐week period of diabetes reversed the up‐regulation of BDNF mRNA in DRG and muscle and NGF mRNA in sciatic nerve. All diabetes‐induced changes in neurotrophin mRNA were not paralleled by similar alterations in the levels of β‐actin mRNA in muscle and nerve, or of GAP‐43 mRNA in DRG and nerve. It is proposed that the up‐regulation of neurotrophin mRNA is an endogenous protective and/or repair mechanism induced by insult and, as such, appears as an early marker of peripheral nerve and muscle damage in experimental diabetes.
Inhibition of Neutral Amino Acid Transport Across the Human Blood‐Brain Barrier by Phenylalanine
Authors: Shulkin Barry L. , Betz A. Lorris , Koeppe Robert A. , Agranoff Bernard W.
Published: July 2010

Abstract: The delivery of large neutral amino acids (LNAAs) to brain across the blood‐brain barrier (BBB) is mediated by the L‐type neutral amino acid transporter present in the membranes of the brain capillary endothelial cell. In experimental animals, the L‐system transporter is saturated under normal conditions, and therefore an elevation in the plasma concentration of one LNAA will reduce brain uptake of others. In this study, we used positron emission tomography (PET) to determine the effect of elevated plasma phenylalanine concentrations on the uptake of an artificial neutral amino acid, [11C]‐aminocyclohexanecarboxylate ([11C]ACHC), in human brain. PET scans were performed on six normal male subjects after an overnight fast and again 60 min after oral administration of 100 mg/kg of phenylalanine. The plasma phenylalanine concentration increased by an average of 11‐fold between the first and second scans. This increase produced a reduction in [11C]ACHC uptake in all brain regions but not in scalp. The mean ± SD influx rate constant for whole brain decreased after phenylalanine ingestion from 0.036 ± 0.002 to 0.019 ± 0.004 ml/g/min. Kinetic analysis of the effect of plasma phenylalanine concentration on the rate of [11C]ACHC uptake is compatible with a model of competitive inhibition so that large increases in the concentration of one LNAA in plasma will reduce the brain uptake of other LNAAs across the human BBB.
A UDP‐Sugar Pyrophosphatase Is Developmentally Regulated in the Rat Retina
Authors: Martina J. A. , Daniotti J. L. , Maccioni H. J. F.
Published: July 2010

Abstract: Rat retina tissue contains relatively high amounts of GD3 in relation to ganglio‐series gangliosides even in the adult stages. This was attributed in part to an activity ratio between the enzyme that converts GM3 to GD3 [sialyltransferase II (ST‐II)] and the enzyme that converts GM3 to GM2 [N‐acetylgalactosaminyltransferase (GalNAc‐T)] favorable to ST‐II. Here we report the presence in the rat retina tissue of an activity that hydrolyzes one of the substrates of GalNAc‐T, the donor sugar nucleotide UDP‐GalNAc. Chromatographic analyses of the products of degradation indicate that the activity corresponds to a UDP‐sugar pyrophosphatase/phosphodiesterase I. The activity is developmentally regulated, increasing after day 4 of postnatal development to reach values ∼10‐fold higher in the adult tissue. The activity sediments with the microsomal membranes, also hydrolyzes UDP‐Gal, does not hydrolyze CMP‐NeuNAc, requires Mn2+, and does not require detergent. Kinetic data showed that the same activity hydrolyzes UDP‐GalNAc and UDP‐Gal, each one acting as competitive inhibitor for the hydrolysis of the other (Km and Ki for UDP‐GalNAc, 48 and 33 µM, respectively; Km and Ki for UDP‐Gal, 5 and 12 µM, respectively). In another set of experiments, it was found that the activities of the GalNAc‐T and the enzyme that converts GM2 to GM1 [galactosyltransferase II (Gal T‐II)] increased about threefold from birth to day 4 and then decreased to stabilize by day 6 in values that were similar to those at birth and about one‐half those of ST‐II. Thus, at late stages the low activities of GalNAc‐T and Gal T‐II relative to ST‐II and the high activity of the UDP‐sugar nucleotide pyrophosphatase that would limit the availability of the UDP‐GalNAc and UDP‐Gal for GM2 and GM1 synthesis would be concurrent factors leading to the low proportion of ganglio‐series gangliosides relative to GD3 that characterizes the rat retina tissue.
Protein Kinase C Modulates Calcium Sensitivity of Nitric Oxide Synthase in Cerebellar Slices
Authors: Okada Daisuke
Published: July 2010

Abstract: The possible modulation of nitric oxide (NO) synthase (NOS) activity by protein kinase C (PKC) was investigated. Incubation of rat cerebellar slices with the specific metabotropic glutamate receptor agonist, (±)‐1‐aminocyclopentane‐trans‐1,3‐dicarboxylate (trans‐ACPD) increased cyclic GMP concentration two‐fold. The increase was dose‐dependently blocked by the protein kinase inhibitors staurosporine and calphostin C. Phorbol 12‐myristate 13‐acetate (PMA), a PKC activator, increased cyclic GMP concentration without glutamate receptor activation. The cyclic GMP increases induced by PMA and trans‐ACPD were independent of extracellular calcium blocked by Nω‐nitro‐l‐arginine, a specific NOS inhibitor, and were not additive. Measurement of citrulline formation in cerebellar slices confirmed that NOS was activated by trans‐ACPD and the activation was blocked by calphostin C. These results suggest that metabotropic glutamate receptor activates NOS through PKC. The calcium dependency of NOS activation was assessed in slices incubated with PMA and okadaic acid. NOS in both PMA‐treated and untreated slices had similar activities at 100 nM free calcium, whereas at 25–70 nM free calcium, NOS in PMA‐treated slices was more active than that in untreated slices. These results suggest that PKC regulates NO release in resting neurons by modulating the sensitivity of NOS at low calcium concentrations.
Monoclonal Antibodies to the Human γ2 Subunit of the GABAA/Benzodiazepine Receptors
Authors: Fernando Lawrence P. , Khan Zafar U. , McKernan Ruth M. , De Blas Angel L.
Published: July 2010

Abstract: The large intracellular loop (IL) of the γ2 subunit of the cloned human γ‐aminobutyric acidA (GABAA) receptor (γ2IL) was expressed in bacteria as glutathione‐S‐transferase and staphylococcal protein A fusion proteins. Mice were immunized with the fusion proteins (one protein per animal), and monoclonal antibodies were obtained. Six monoclonal antibodies reacted with the γ2IL moiety of the fusion proteins. Three of these monoclonal antibodies also immunoprecipitated a high proportion of the GABAA/benzodiazepine receptors from bovine and rat brain and reacted with a wide 44,000–49,000‐Mr peptide band in immunoblots of affinity‐purified GABAA receptors. These monoclonal antibodies are valuable reagents for the molecular characterization of the GABAA receptors in various brain regions.
The 84‐kDa Form of Human Matrix Metalloproteinase‐9 Degrades Substance P and Gelatin
Authors: Backstrom Jon R. , Tökés Zoltán A.
Published: July 2010

Abstract: Matrix metalloproteinase‐9 (MMP‐9) is secreted from cells and, once activated, is thought to degrade collagen in the extracellular matrix. Because collagen is not readily localized where neurons have been shown to produce MMP‐9 in the human brain, the ability of this enzyme to degrade bioactive peptides was investigated with representative tachykinin peptides [substance P (SP), neurokinin A, neurokinin B, and kassinin]. Latent MMP‐9 (94 kDa) was purified from the human cell line HL‐60 and converted to an intermediary active form (84 kDa) with p‐aminophenylmercuric acetate. This active form of MMP‐9 degraded SP with a kcat/Km of 170 mM−1 min−1, which is 30‐fold greater than the previously reported value for a representative collagen‐derived peptide. The major digestion products were identified as SP1–6 and SP7–11, which were derived from cleavage of the Gln6‐Phe7 bond. Minor products were also generated from cleavage of the Gly9‐Leu10 bond. The other representative tachykinin peptides were cleaved at rates >10‐fold slower than that of SP. The 84‐kDa peptidase was also active as a gelatinase. Longer treatment of MMP‐9 with p‐aminophenylmercuric acetate caused the conversion of the 84‐kDa enzyme to the established 68‐kDa active form; however, the rate of SP degradation did not increase. Because MMP‐9 is produced by neurons of the CNS, these results suggest a possible regulatory role for the enzyme in intercellular communication by altering the availability of bioactive peptides.
Effect of Ethanol Treatment on Rate and Equilibrium Constants for [3H]Muscimol Binding to Rat Brain Membranes: Alteration of Two Affinity States of the GABAA Receptor
Authors: Negro Montserrat , Chinchetru Miguel A. , Fernández Arsenio , Calvo Pedro
Published: July 2010

Abstract: Equilibrium binding curves were biphasic in control and ethanol‐treated rats. [3H]Muscimol binds to sites of high (KDA of ∼10 nM) and low (KDB of ∼0.3–0.4 µM) affinity. Chronic ethanol treatment produced a decrease in BmaxA value, and the hyperbolic binding profiles were progressively affected by the chronic and in vitro ethanol treatments, with most of this effect corresponding to the high‐affinity site. IC50 and Ki values were calculated for several competing ligands, using membranes from both control and ethanol‐treated animals. The association and dissociation curves were also biphasic, using a radioligand concentration precluding a significant occupancy of the low‐affinity sites, which suggests the existence of two forms or affinity states of the monoliganded receptor. Chronic ethanol treatment did not produce changes in the values of the dissociation rate constants (fast and slow phases). By contrast, we report for the first time a decrease in the values of the association rate constants, with this decrease being higher for the slow phase. Consequently, the dissociation equilibrium constants are two times higher in chronically ethanol‐treated animals for both phases.
Pituitary Adenylate Cyclase‐Activating Polypeptide (PACAP) Regulation of Sympathetic Neuron Neuropeptide Y and Catecholamine Expression
Authors: May Victor , Braas Karen M.
Published: July 2010

Abstract: Two forms of pituitary adenylate cyclase‐activating polypeptide (PACAP), the 38‐ and 27‐amino‐acid forms (PACAP38 and PACAP27, respectively), which share amino acid sequence homology with vasoactive intestinal peptide (VIP), were evaluated for their abilities to regulate sympathetic neuron catecholamine and neuropeptide Y (NPY) expression. PACAP38 and PACAP27 potently and efficaciously stimulated NPY and catecholamine secretion in primary cultured superior cervical ganglion (SCG) neurons; 100‐ to 1,000‐fold higher concentrations of VIP were required to modulate secretion, suggesting that SCG neurons express the PACAP‐selective type I receptor. PACAP38 elicited a sustained seven‐ to ninefold increase in the rate of NPY secretion and three‐fold stimulation in the rate of catecholamine release. PACAP38 and PACAP27 produced parallel neuronal NPY and catecholamine release, but cellular levels of NPY and catecholamines were differentially regulated. Sympathetic neuron NPY content was decreased, whereas cellular total catecholamine levels were elevated by the PACAP peptides; total NPY and catecholamine levels (secreted plus cellular content) were increased. In concert with the increased total peptide and transmitter production, pro‐NPY and tyrosine hydroxylase mRNA levels were elevated. Furthermore, PACAP38 was more efficacious than PACAP27 in regulating pro‐NPY and tyrosine hydroxylase mRNA. SCG neuronal expression of mRNA encoding the type I PACAP receptor further supported the studies demonstrating that sympathetic neuronal levels of NPY and catecholamine content and secretion and mRNA are differentially regulated by the PACAP peptides.
Induction of Neuronal Apoptosis by Excitotoxins Associated with Long‐Lasting Increase of 12‐O‐Tetradecanoylphorbol 13‐Acetate‐Responsive Element‐Binding Activity
Authors: Finiels Françoise , Robert Jean‐Jacques , Samolyk Marie‐Laure , Privat Alain , Mallet Jacques , Revah Frédéric
Published: July 2010

Abstract: We show that excitotoxic cell death, which is associated with pathological neurodegenerative processes, can display morphological and biochemical features characteristic of apoptosis, a mode of cell death typical of physiological neuronal elimination during development. Cortical neurons cultured in the absence of serum, stimulated with NMDA, glutamate, or quisqualate after 3–5 days in vitro, showed significant degeneration. This death was blocked by 1 µM MK‐801, indicating that it was mediated by the activation of NMDA receptors. Dying cells displayed an apoptotic morphology, characterized by cytoplasm and chromatin condensation. No internucleosomal DNA degradation was observed, confirming that morphological changes of apoptosis can be dissociated from DNA laddering. Inhibitors of protein or RNA synthesis abolished cell death, and the protective effect of cycloheximide was similar when the drug was applied 2 h before or 8 h after glutamate. These experiments suggest the participation of active gene transcription in the mechanism of death. We thus analyzed the modulation of transcription factors in dying cells using electrophoretic mobility shift assays. The level of factors binding to the 12‐O‐tetradecanoylphorbol 13‐acetate‐responsive element (TRE) displayed a late and sustained increase preceding neuronal death, which was not found for factors complexing the Sp1 P, Oct, and USF binding sites. These results raise the possibility that apoptosis is one of the mechanisms of death in the pathologies linked to excitotoxicity and that activation of TRE‐binding factors could play a role in these processes.
Microglial Cathepsin B: An Immunological Examination of Cellular and Secreted Species
Authors: Ryan R. E. , Sloane B. F. , Sameni M. , Wood P. L.
Published: July 2010

Abstract: The cysteine proteinase cathepsin B (CB) was isolated from immortalized murine BV‐2 microglial cells and examined via sodium dodecyl sulfate‐polyacrylamide gel electrophoresis and immunoblotting to establish physicochemical properties of CB from what is generally considered the resident CNS macrophage. Microglial proteases have been implicated in several pathological processes occurring in the CNS, including neurodegeneration. Murine microglial CB was observed to consist of two major single‐chain species of 32 and 34 kDa, with pls of 5.5‐5.2 and 5.1‐4.5, respectively. In addition, a minor 24‐kDa CB species was also observed in some microglial preparations. The major CB isozymes in microglia differed from those observed in murine liver and brain, which consisted of both single‐ and double‐chain CB variants of 31 and 24–25 kDa/5 kDa, respectively, with pl values of 5.5‐4.5. A microglial pro‐CB of 37 kDa was also isolated, which could be processed to the 34‐kDa single‐chain CB species. Cystatin was observed to prevent pro‐CB processing, whereas E‐64 and leupeptin were only partially inhibitory. The 37‐kDa pro‐CB species was observed to undergo processing into the 34‐kDa CB species when incubated at pH 5.5 but remained stable with respect to molecular mass when incubated at pH 7.0. In contrast, the 34‐kDa single‐chain CB species was observed to autodegrade when incubated at pH 7.0, whereas incubation at pH 5.5 did not affect the integrity of the species as monitored by immunoblotting. Both pro‐CB and 32‐kDa single‐chain CB species were observed extracellularly following lipopolysaccharide activation of BV‐2 microglial cells.
Cytochalasins Protect Hippocampal Neurons Against Amyloid β‐Peptide Toxicity: Evidence that Actin Depolymerization Suppresses Ca2+ Influx
Authors: Furukawa Katsutoshi , Mattson Mark P.
Published: July 2010

Abstract: Increasing data suggest that the amyloid β‐peptide (Aβ), which accumulates in the brains of Alzheimer's victims, plays a role in promoting neuronal degeneration. Cell culture studies have shown that Aβ can be neurotoxic and recent findings suggest that the mechanism involves destabilization of cellular calcium homeostasis. We now report that cytochalasin D, a compound that depolymerizes actin microfilaments selectively, protects cultured rat hippocampal neurons against Aβ neurotoxicity. Cytochalasin D was effective at concentrations that depolymerized actin (10–100 nM). The elevation of [Ca2+]i induced by Aβ, and the enhancement of [Ca2+]i responses to glutamate in neurons exposed to Aβ, were markedly attenuated in neurons pretreated with cytochalasin D. The protective effect of cytochalasin D appeared to result from a specific effect on actin filaments and reduction in calcium influx, because cytochalasin E, another actin filament‐disrupting agent, also protected neurons against Aβ toxicity; the microtubule‐disrupting agent colchicine was ineffective; cytochalasin D did not protect neurons against the toxicity of hydrogen peroxide. These findings suggest that actin filaments play a role in modulating [Ca2+]i responses to neurotoxic insults and that depolymerization of actin can protect neurons against insults relevant to the pathogenesis of Alzheimer's disease.
Characterization of the Excitoprotective Actions of N‐Methyl‐d‐Aspartate in Cultured Cerebellar Granule Neurons
Authors: Damschroder‐Williams Pat , Irwin Robert P. , Lin Sui‐Zhen , Paul Steven M.
Published: July 2010

Abstract: Exposure of cultured cerebellar granule neurons to subtoxic concentrations of N‐methyl‐d‐aspartate (NMDA) has been shown previously to result in a neuroprotective state, as measured by subsequent exposure to toxic concentrations of glutamate. In the present study, we have further characterized the excitoprotective actions of NMDA in these neurons. NMDA‐induced excitoprotection was concentration dependent (EC50∼30 µM) and time dependent, with maximal protection observed following 16 h of preexposure to NMDA. NMDA‐induced excitoprotection did not require continuous exposure to NMDA, as a 4‐h preincubation was sufficient to induce full excitoprotection when measured 8 h later. Maximal protection was manifest as a “right shift” in the concentration‐response relationship for glutamate toxicity of approximately three orders of magnitude (EC50∼30 µM in untreated neurons compared with ≥50 mM in NMDA‐treated neurons). After removal of NMDA, complete reversal of the excitoprotective state was observed by 48 h (t1/2≈24 h). The ability of NMDA to induce excitoprotection was observed in neurons maintained for up to 14 days in vitro (DIV) [postnatal day (PND) 22], but was absent at 21 and 32 DIV (PND 29–40), despite little to no difference in the toxicity of glutamate at any DIV examined. Preexposure of cerebellar granule neurons to a maximally excitoprotective concentration of NMDA (50 µM) failed to alter the density of NMDA receptors measured by the specific binding of [3H]MK‐801. Moreover, the immediate elevation in intracellular free calcium concentration ([Ca2+]i) induced by glutamate exposure and measured by microfluorimetry and the Ca2+‐sensitive indicator fura‐2 was similar in NMDA‐pretreated and untreated neurons. As reported previously, NMDA‐induced excitoprotection in cerebellar granule neurons was, however, reversed by coincubation with the protein synthesis inhibitor cycloheximide. Taken together, these data suggest that NMDA receptor‐mediated excitoprotection in cerebellar granule neurons is mediated via both a transcriptionally directed and a developmentally regulated postreceptor mechanism(s).
Three-Dimensional Analysis of Zygomatic-Maxillary Complex Fracture Patterns
Authors: Pau Candace Y. , Barrera Jose E. , Kwon Jaehwan , Most Sam P.
Published: July 2010

Zygomatic-maxillary (ZMC) complex fractures are a common consequence of facial trauma. In this retrospective study, we present a novel method of ZMC fracture pattern analysis, utilizing three-dimensional visualization of computed tomography (CT) images to record displacement of the malar eminence in a three-dimensional coordinate plane. The pattern of fracture was then correlated with treatment outcome. Facial CT scans were obtained from 29 patients with unilateral ZMC fractures and 30 subjects without fractures and analyzed. Briefly, displacement of the malar eminence (ME) on the fractured side was measured in medial-lateral (x), superior-inferior (y), and anterior-posterior (z) dimensions, as well as Euclidean distance, by comparison to ME location on the unfractured side. Baseline natural variance in asymmetry was accounted for by comparing ME location on the left and right sides in subjects without fractures. Patients who required open reduction and internal fixation (ORIF) to repair the ZMC fracture alone had significantly greater cumulative ME displacements than patients who did not require ORIF ( p = 0.02). Additionally, patients with a high fracture score of 3, 4, or 5 (assigned based on severity displacement in each dimension) had significantly higher rates of ORIF than patients with a low fracture score of 0, 1, or 2 ( p = 0.05). Severe displacement in one or more dimensions was associated with higher rates of ORIF than seen in patients with only neutral or mild displacements in all dimensions ( p = 0.05). Severe x displacement was most strongly correlated with surgical intervention ( p = 0.02). Overall, orbital floor repair was less strongly associated with most displacement measures than ZMC repair alone; however, patients requiring orbital floor repair had greater Euclidean ME displacements than patients who did not require orbital floor repair ( p = 0.02). Fracture severity, as determined by multiple parameters in this novel evaluation system, is associated with higher rates of ORIF in patients with unilateral ZMC fractures. Determination of ZMC fracture pattern may thus be informative when considering treatment options.
Self-dual Einstein spaces, heavenly metrics, and twistors
Authors: Alexandrov Sergei , Pioline Boris , Vandoren Stefan
Published: July 2010

Four-dimensional quaternion-Kähler metrics, or equivalently self-dual Einstein spaces M, are known to be encoded locally into one real function h subject to Przanowski’s heavenly equation. We elucidate the relation between this description and the usual twistor description for quaternion-Kähler spaces. In particular, we show that the same space M can be described by infinitely many different solutions h, associated with different complex (local) submanifolds on the twistor space, and therefore to different (local) integrable complex structures on M. We also study quaternion-Kähler deformations of M and, in the special case where M has a Killing vector field, show that the corresponding variations in h are related to eigenmodes of the conformal Laplacian on M. We exemplify our findings on the four-sphere S4, the hyperbolic plane H4, and on the “universal hypermultiplet,” i.e., the hypermultiplet moduli space in type IIA string compactified on a rigid Calabi–Yau threefold.