bluuebunny/crossref_metadata_2025 huggingface dataset:
Amphetamine and Reserpine Deplete Brain Biogenic Amines and Alter Blow Fly Feeding Behavior
Authors: Brookhart Gary L. , Edgecomb Robert S. , Murdock Larry L.
Published: October 2006

Abstract: HPLC with electrochemical detection was used to determine the levels of p‐hydroxyphenylethanolamine (octopamine), 3,4‐dihydroxyphenylethylamine (dopamine), and 5‐hydroxytryptamine (5‐HT) in the brains of control, reserpine, and d‐amphetamine‐treated blow flies, Phormia regina Meigen. Parallel studies were carried out to assess the effects of the two drugs on fly feeding behavior, measured as mean acceptance threshold: the minimum sucrose concentration to which the average fly in a population will respond by proboscis extension when its tarsi contact the solution. In saline‐injected control flies, all three amines were found at levels of approximately 2 pmol/brain. Thirty minutes after injection with d‐amphetamine (12 μg/fly), brain octopamine was depleted by 85%, whereas dopamine and 5‐HT were depleted by 70%. Reserpine (5 μg/fly) caused 70% depletion of dopamine and >90% depletion of both octopamine and 5‐HT 24 h after injection. However, the effect of reserpine was much slower in onset (hours versus minutes) and more persistent (days versus hours) than was the effect of d‐amphetamine. With either drug, the time course of amine depletion closely matched the time course of the increase in feeding threshold observed in drug‐treated flies. These results suggest that CNS pools of the biogenic amines, octopamine, dopamine, and 5‐HT are important in governing blow fly responsiveness to food stimuli.
Effect of Dexamethasone on Prostaglandin Synthesis in Various Areas of the Rat Brain
Authors: Weidenfeld J. , Lysy J. , Shohami E.
Published: October 2006

Abstract: Glucocorticoid hormones are known to inhibit the production of prostaglandins in many cell types and tissues. The effect of these hormones on the biosynthesis of brain tissue is not yet clear. In the present study we investigated the effect of dexamethasone on the release of prostaglandin E2 (PGE2), thromboxane B2 (TXB2), and 6‐keto‐PGF1α from various brain areas of male rats. Slices from cortex, hippocampus, hypothalamus, and striatum taken from rats pretreated with dexamethasone 4 mg/kg body weight or vehicle, 18 h and 2 h prior to killing, were incubated in Krebs‐Ringer‐bicarbonate for 1 h. The accumulation of PGs in the medium was determined by radioimmu‐noassay. Pretreatment with dexamethasone significantly reduced the release of all PGs from the cortex by 40–50%. In the striatum and hippocampus only TXB2 was reduced by ∼40%. In the hypothalamus the effect of dexamethasone was not significant. When slices of the same brain areas from intactrats were incubated for 1 h in the presence of 40 μM dexamethasone, only the release of PGE2 from the cortex was reduced (by 30%). These results suggest that glucocorticoids can inhibit PG synthesis in brain tissue, and that the cortex is the most sensitive area to the inhibitory effect of the hormone.
Quantitative Autoradiographic Analysis of the Effects of Electroconvulsive Shock on Serotonin‐2 Receptors in Male and Female Rats
Authors: Biegon Anat , Israeli Malka
Published: October 2006

Abstract: Chronic electroconvulsive shock (ECS) is known to increase the level of serotonin‐2 (S2) receptors in male rat brain. Using quantitative autoradiography, we have studied the distribution pattern of these receptors in female as well as male rats and the effect of repeated ECS on the receptor level in both sexes. We find that although the distribution of S2 receptors is generally similar in males and females, they respond differently to repeated ECS. In males we found the expected increase in S2 binding, which was localized to specific cortical, hippocampal, and septal regions. In females, no increase was found in the cortex or septum and relatively small increases were found in the hippocampus. It appears that the regulation of S2 receptors by ECS is sex‐dependent.
Immunohistochemical Staining and Enzyme Activity Measurements Show myo‐Inositol‐1‐Phosphate Synthase to Be Localized in the Vasculature of Brain
Authors: Wong Yun‐Hua H. , Kalmbach Sandra J. , Hartman Boyd K. , Sherman William R.
Published: October 2006

Rabbit anti‐bovine myo‐inositol‐1‐phosphate synthase was used to examine the distribution of that enzyme in perfused and immersion‐fixed bovine brain and testis. In brain, intense and specific staining was found in the walls of all the vascular elements including cerebral capillaries. The remainder of brain parenchyma exhibited only low levels of background staining. In testis, an organ rich in the enzyme, blood vessels showed no specific staining. Instead, the enzyme was found in the seminiferous epithelium of the seminiferous tubules, perhaps localized in spermatozoa. To confirm the brain finding, the activity of myo‐inositol‐1‐phosphate synthase was measured in bovine brain microvessel preparations and brain pial vessels. In these preparations the activity of the enzyme was found on average to be 7 and 22 times enriched over that in whole brain, respectively. The activities of two other enzymes of inositol metabolism, myo‐inosose reductase and myo‐inositol‐1‐phosphatase, were also examined for their distribution in brain. Those enzymes were found to be generally distributed. The surprising finding of a vascular localization of myo‐inositol‐1‐phosphate synthase in brain raises new questions about the mechanism by which myo‐inositol is concentrated to such high cellular levels in the principal substance of that organ.
Determination of Endogenous Acetylcholine Release in Freely Moving Rats by Transstriatal Dialysis Coupled to a Radioenzymatic Assay: Effect of Drugs
Authors: Consolo Silvana , Wu Chun Fu , Fiorentini Francesco , Ladinsky Herbert , Vezzani Annamaria
Published: October 2006

Abstract: The technique of intracerebral dialysis in combination with a sensitive and specific radioenzymatic method was used for recovery and quantification of endogenous extracellular acetylcholine from the striata of freely moving rats. A thin dialysis tube was inserted transversally through the caudate nuclei, and the tube was perfused with Ringer solution, pH 6.1, at a constant rate of 2 μl min−1. The perfusates were collected at 10‐min intervals. In the presence of 1 and 10 μM physostigmine, acetylcholine release was 4.5 ± 0.02 and 7.3 ± 0.3 pmol/10 min, respectively (not corrected for recovery). The latter concentration of the acetylcholinesterase inhibitor was used in all experiments. Under basal conditions, acetylcholine output was stable over at least 4 h. A depolarizing K+ concentration produced a sharp, reversible 87% increase in acetylcholine output. Both the basal and K+‐stimulated release were Ca2+ dependent. The choline uptake inhibitor hemicholinium‐3 (20 μg intracerebroventricularly) reduced striatal acetylcholine output to 35% of the basal value within 90 min. Scopolamine (0.34 mg/kg s.c.) provoked a sharp enhancement of acetylcholine release of ∼63% over basal values, whereas oxotremorine (0.53 mg/kg i.p.) transiently reduced acetylcholine release by 54%. These results indicate the physiological and pharmacological suitability of transstriatal dialysis for monitoring endogenous acetylcholine release.
Enzymic Synthesis of Leukotriene B4 in Guinea Pig Brain
Authors: Shimizu Takao , Takusagawa Yutaka , Izumi Takashi , Ohishi Nobuya , Seyama Yousuke
Published: October 2006

Abstract: Leukotriene B4 [5(S), 12(R)‐dihydroxy‐6,14‐cis‐8,10‐trans‐eicosatetraenoic acid] was obtained from endogenous arachidonic acid when slices of the guinea pig brain cortex were incubated with the calcium ionophore A 23187. Enzymes involved in its synthesis, arachidonate 5‐lipoxygenase [arachidonic acid to 5(S)‐hydroperoxy‐6‐trans‐8,11,14‐cis‐eicosatetraenoic acid and subsequently to leukotriene A4] and leukotriene A4 hydrolase (leukotriene A4 to B4), were present in the cytosol fraction. Arachidonate 5‐lipoxygenase was Ca2+‐dependent, and was stimulated by ATP and the microsomal membrane, as was noted for the enzyme from mast cells. The lipid hydroperoxides stimulated 5‐lipoxygenase by four‐ to sixfold. The leukotriene A4 hydrolase activity was rich in brain, and the specific activity (0.4 nmol/min/mg of protein) was much the same as that of guinea pig leukocytes. High activities of these enzymes were detected in the olfactory bulb, pituitary gland, hypothalamus, and cerebral cortex. Since leukotriene B4 is enzymically synthesized in the brain, possible roles related to neuronal functions or dysfunctions deserve to be examined.
An Enzyme‐Linked Immunoassay for Nerve Growth Factor (NGF): A Tool for Studying Regulatory Mechanisms Involved in NGF Production in Brain and in Peripheral Tissues
Authors: Weskamp G. , Otten U.
Published: October 2006

Abstract: A sensitive enzyme‐linked immunosorbent assay (ELISA) for nerve growth factor (NGF) has been developed. The sensitivity of this assay (0.1 pg/well) permits the quantification of endogenous immunoreactive NGF in the peripheral nervous system and the CNS. Studies on the regulatory mechanisms involved in NGF production indicate that, in addition to neurally mediated mechanisms, other stimuli, e.g., inflammation, significantly contribute to NGF production.
Effects of Chronic Ethanol Treatment on the β‐Adrenergic Receptor‐Coupled Adenylate Cyclase System of Mouse Cerebral Cortex
Authors: Saito Toshikazu , Lee John M. , Hoffman Paula L. , Tabakoff Boris
Published: October 2006

Abstract: Chronic ingestion of ethanol, which produced tolerance and physical dependence, resulted in altered function of the cerebral cortical β‐adrenergic receptor‐coupled adenylate cyclase system in mice. Although there was no change in basal adenylate cyclase activity, or in the activity of the digitonin‐solubilized catalytic unit, stimulation of adenylate cyclase activity by the nonhydrolyzable guanine nu‐cleotide analog guanylylimidodiphosphate [Gpp(NH)p] was reduced in brains of ethanol‐fed animals. Ethanol added in vitro increased adenylate cyclase activity, and this enhancement, in the presence of Gpp(NH)p, was also reduced in cortical membranes of ethanol‐fed mice. Furthermore, the maximal response to isoproterenol was decreased, and the EC50 for isoproterenol stimulation of adenylate cyclase activity was increased in ethanol‐fed animals. The results are consistent with a qualitative or quantitative defect in the function of the stimulatory guanine nucleotide‐bind‐ing protein (Ns), as well as in the β‐adrenergic receptor, after chronic ethanol exposure. In part, these changes appear to be similar to those that occur during heterologous desensiti‐zation of various receptor systems, and may be associated with dependence on or tolerance to ethanol.
Different Behaviors Among Lysosomal Enzymes in the Cerebellum of Jaundiced Gunn Rats with Cerebellar Hypoplasia
Authors: Sato Hiroshi , Keino Hiroomi , Aono Sachiko , Semba Reiji , Kashiwamata Shigeo
Published: October 2006

Abstract: Activities of six lysosomal enzymes in the cerebellum of jaundiced homozygous (jj) Gunn rats were examined from 5 to 20 days of life and compared with those in heterozygotes (j+). Significantly higher enzyme activities were first detected at 8 days. The jj/j+ activity ratios of all enzymes peaked at 15 days. The ratios of β‐glycerophospha‐tase, β‐mannosidase, and acid lipase were only 1.3–1.7, whereas those of arylsulfatase and cathepsin were 2.0 and 3.1, respectively. The most striking increase in activity was observed with β‐glucuronidase, the ratio of which was 8.4. These results indicate a selective increase in activities of certain lysosomal enzymes in the hypoplastic cerebellum of jj rats.
Acetylcholine Receptor Binding Properties at the Rat Neuromuscular Junction During Aging
Authors: Smith Dean O. , Chapman Michael R.
Published: October 2006

Abstract: Specific binding characteristics of acetylcholine receptors at the diaphragm neuromuscular junction of rats aged 10 (mature adult) and 28 (aged) months were assayed by measuring 125I‐α‐bungarotoxin binding. Maximal binding to intact tissue samples was greater in the older rats; this could be attributed to an age‐related increase in terminal branching. The toxin concentration at which half‐maximal binding occurred increased in the older rats. Binding kinetics were assayed in finely minced tissue samples, and the association rate constant was observed to decrease in the 28‐month animals. Retardation of the initial rate of toxin binding by d‐tubocurarine (dTC) in minced tissue was described by a two‐component nonlinear Hofstee plot; IC50 values (7.1–7.2 μM and 39.0–46.5 nM) were about the same for both age groups, but there was a significant shift toward the low‐affinity values in the aged rats. Rhodamine‐conju‐gated α‐bungarotoxin was used to visualize receptor localization. There were no major changes in receptor distribution, and nerve terminals were consistently associated with receptors and vice versa. The data indicate a shift toward lower binding affinity during aging, which may involve changes either in one of the two toxin‐binding sites on individual receptors, in dTC blocking of the channel moiety, or in receptor types.
Threonine Entry into Rat Brain After Diet‐Induced Changes in Plasma Amino Acids
Authors: Tews Jean K. , Greenwood John , Pratt Oliver E. , Harper Alfred E.
Published: October 2006

Abstract: Passage of amino acids across the blood‐brain barrier is modified by the amino acid composition of the blood. Because blood amino acid concentrations respond to changes in protein intake, we have examined associations among diet, plasma amino acid patterns, and the rate of entry of threonine into the brain. Rats were adapted for 8 h/ day for 7–10 days to diets containing 6, 18, or 50% casein before receiving a single, independently varied, final meal of a diet containing 0, 6, 18, or 50% casein. After 4–7 h, they were anesthetized and infused intravenously with [14C]threonine for 5 min before plasma and brain samples were taken for determination of radioactivity and amino acid content. Plasma and brain threonine concentrations decreased as protein content increased in the diets to which the rats had been adapted. Plasma threonine concentrations increased twofold, from 1.6 to 3.0 mM, when rats adapted to 6% casein meals received a single 50% casein meal rather than a nonprotein meal; a fivefold increase, from 0.13 to 0.69 mM, occurred when rats had been previously adapted to 50% casein meals. Increasing the protein content of the final meal did not increase brain threonine concentrations. Highest and lowest rates of threonine entry into the brain occurred, respectively, in rats adapted to 6 and 50% casein meals. Changes in plasma threonine concentrations and threonine flux into brain reflected protein content of both pretreatment and final meals.
Biochemical and Pharmacological Characterization of [3H]GBR 12935 Binding In Vitro to Rat Striatal Membranes: Labeling of the Dopamine Uptake Complex
Authors: Andersen Peter H.
Published: October 2006

Abstract: Binding of the selective dopamine (DA) uptake inhibitor [3H]GBR 12935 to rat striatal membranes was characterized biochemically and pharmacologically. [3H]‐GBR 12935 binding at 0°C was reversible and saturable and Scatchard analysis indicated a single binding site with a KD of 5.5 nM and a Bmax of 760 pmol/mg tissue. [3H]GBR 12935 labeled two binding sites. One binding site was identified as the classic DA uptake site, since methylphenidate, cocaine, diclofensine, and Lu 19–005 potently inhibited [3H]GBR 12935 binding to it. Binding to the second site was inhibited by high concentrations of the above compounds. IC50 values for inhibition of [3H]GBR 12935 binding to the DA uptake site were proportional to IC50 values for inhibition of DA uptake. However, substrates of DA uptake, e.g., DA and 1‐methyl‐4‐phenylpyridine, and DA releasers, e.g., the amphetamines, inhibited [3H]GBR 12935 binding less than DA uptake. Rate experiments excluded the possibility that these “weak” inhibitors affected the binding by alloste‐ric coupled binding sites. The second binding site was not a noradrenergic, serotonergic, or GABAergic uptake site. Neither was it a dopaminergic, acetylcholinergic, histaminic, serotonergic, or adrenergic receptor. However, [3H]GBR 12935 was potently displaced from it by disubstituted piper‐azine derivatives, i.e., flupentixol and piflutixol. DA uptake and the DA uptake binding site of [3H]GBR 12935 were located primarily in the striatum, but the piperazine acceptor site was distributed uniformly throughout the brain. Also only the DA uptake binding site was destroyed by 6‐OH‐DA. Thus, [3H]GBR 12935 labels the classic DA uptake site in rat striatum and also a piperazine acceptor site. Substrates for DA uptake and releasers of DA inhibited [3H]GBR 12935 binding with low potency, but did not alter the rate constants for [3H]GBR 12935 binding. Therefore inhibitors of DA uptake label the carrier site and prevent the carrier process.
70‐Kilodalton Heat Shock Protein Induction in Cerebellar Astrocytes and Cerebellar Granule Cells In Vitro: Comparison with Immunocytochemical Localization After Hyperthermia In Vivo
Authors: Marini Ann M. , Kozuka Masao , Lipsky Robert H. , Nowak Thaddeus S.
Published: October 2006

Abstract: Induction of the 70‐kDa heat shock protein, hsp70, was evaluated in cultured cerebellar astrocytes and granule cell neurons subjected to a hyperthermic stress, using a monoclonal antibody and an oligonucleotide probe that selectively recognize stress‐inducible species of hsp70‐related proteins and RNAs, respectively. Immunoblots of cultures enriched in either granule cells or astrocytes, and immunocytochemical localization studies in cocultures of these cell types, demonstrated that hsp70 induction was restricted to the astrocyte population. Amino acid incorporation experiments showed little difference in the loss and recovery of overall protein synthesis activity in these two cell types following transient hyperthermic stress. RNA blot hybridizations confirmed the preferential glial induction of hsp70. In vivo immunocytochemical studies in brains of adult rats following hyperthermia were consistent with earlier observations that suggested a primarily glial and vascular localization of the heat shock response in most brain regions, although the intense immunoreactivity in the cerebellar granule cell layer suggests that there is induction of hsp70 in these neurons under in vivo conditions. These results suggest the potential value of such defined cell cultures in identifying mechanisms responsible for differences in the heat shock response of various cell types in vitro, and in revealing factors that may account for the apparent absence of the stress response in cultured cerebellar granule cell neurons.
α‐Bungarotoxin Binds to Human Acetylcholine Receptor α‐Subunit Peptide 185–199 in Solution and Solid Phase but Not to Peptides 125–147 and 389–409
Authors: Griesmann Guy E. , McCormick Daniel J. , De Aizpurua Henry J. , Lennon Vanda A.
Published: October 2006

Abstract: The nicotinic acetylcholine receptor (AChR) of human skeletal muscle has a reducible disulfide bond near the neurotransmitter binding site in each of its α‐subunits. By testing a panel of overlapping synthetic peptides encompassing the α‐subunit segment 177–208 (containing cysteines 192 and 193) we found that specific binding of 125I‐labelled α‐bungarotoxin (α‐BTx) was maximal in the region 185–199. Binding was inhibited by unlabelled α‐BTx d‐tubocurarine > atropine > carbamylcholine. Peptide 193–208 did not bind α‐BTx, whereas 177–192 retained 40% binding activity. Peptides corresponding to regions 125–147 (containing cysteines 128 and 142) and 389–409, or peptides unrelated to sequences of the AChR failed to bind α‐BTx. No peptide bound 125I‐α‐labelled parathyroid hormone. The apparent affinity (KD) of α‐BTx binding to immobilized peptides 181–199 and 185–199 was ∼25 μM and 80 μM, respectively, in comparison with α‐BTx binding to native Torpedo ACh receptor (apparent KD∼0.5 nM). In solution phase, both peptides effectively competed with solubilized native human AChR for binding of α‐BTx, and peptide 185–199 showed little evidence of dissociation after 24 h. Peptides that bound α‐BTx did so when sulfhydryls were reduced. Cysteine modification, by N‐ethylmaleimide or acetamidomethylation, abolished α‐BTx‐binding activity. The data implicate the region of cysteines 192 and 193 in the binding of neurotransmitter to the human receptor.
Cholinergic Deficit Induced by Ethylcholine Aziridinium (AF64A) Transiently Affects Somatostatin and Neuropeptide Y Levels in Rat Brain
Authors: Hörtnagl Heide , Sperk Günther , Sobal Grazyna , Maas Dagmar
Published: October 2006

Abstract: The question whether during the process of cholinergic degeneration somatostatin‐ and/or neuropeptide Y‐containing neurons in rat hippocampus and cortex react to the withdrawal of cholinergic function was addressed. After bilateral intracerebroventricular injection of the cholinotoxin ethylcholine aziridinium (AF64A; 1 or 2 nmol/ventricle) in rats, the activity of choline acetyltransferase (ChAT) started to decline in the hippocampus within 24 h. The reduction of ChAT activity reached its maximum within 4 days (34 and 55% after 1 and 2 nmol of AF64A/ventricle, respectively) and persisted during the observation period of 14 days. In the parietal cortex, ChAT activity decreased by 23% 4 days after 2 nmol of AF64A/ventricle. The loss in ChAT activity was accompanied by a transient decline in the levels of somatostatin and a transient increase in the levels of neuropeptide Y in both brain areas. In the hippocampus, the reduction in somatostatin content was most pronounced after 2 days (by 22 and 33% after 1 and 2 nmol of AF64A/ventricle, respectively). Within 14 days, somatostatin levels returned to control values. Neuropeptide Y levels increased slightly by ∼25% of control values in the hippocampus. The changes described were present in both the dorsal and ventral subfields of the hippocampus. Similar but less pronounced changes in levels of both neuropeptides were observed in the parietal cortex. The present data provide further evidence for a close neuronal interrelationship between cholinergic and somatostatin‐ and/or neuropeptide Y‐containing neurons in rat hippocampus and parietal cortex.
Modulation of Non‐N‐Methyl‐D‐Aspartate Receptors in Cultured Cerebellar Granule Cells
Authors: Gallo Vittorio , Giovannini Claudio , Levi Giulio
Published: October 2006

Abstract: Kainic acid (KA), quisqualic acid (QUIS), and α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) stimulated D‐[3H]aspartate release from cultured cerebellar granule cells in a concentration‐dependent way. The EC50 values were 50 μM for KA (Gallo et al., 1987) and 20 μM for both QUIS and AMPA, but the efficacy of QUIS appeared to be greater than that of AMPA. The release of D‐[3H]aspartate induced by KA, QUIS, and AMPA was blocked, in a dose‐dependent way, by the new glutamate receptor antagonist 6‐cyano‐2,3‐dihydroxy‐7‐nitroquinoxaline (CNQX); IC50 values were 0.7 μM in the case of AMPA (50 μM) and 1 μM in the case of KA (50 μM). AMPA (50–300 μM) inhibited the effect of 50 μM KA on D‐[3H]aspartate release. At 300 μM AMPA, the effect of KA plus AMPA was not antagonized by the KA receptor antagonist kynurenic acid (KYN). In contrast, when KA was used at an ineffective concentration (10 μM), the addition of AMPA at concentrations below the EC50 value (10–20 μM) resulted in a synergistic effect on D‐[3H]aspartate release. In this case, the evoked release of D‐[3H]aspartate was sensitive to KYN. KA stimulated the formation of cyclic GMP, whereas QUIS, AMPA, and glutamate were ineffective. The accumulation of cyclic GMP elicited by KA (100 μM) was prevented not only by the antagonists CNQX (IC50= 1.5 μM) and KYN (IC50= 200 μM), but also by the agonists AMPA (IC50= 50 μM), QUIS (IC50= 3.5 μM), and glutamate (IC50= 100 μM). We conclude that AMPA, like QUIS, may act as a partial agonist at KA receptors. Moreover, CNQX effectively antagonizes non‐N‐methyl‐D‐aspartate receptor‐mediated responses in cultured cerebellar granule cells.
Further Studies on the Changes of Chicken Brain Gangliosides During Prenatal and Postnatal Life
Authors: Sonnino Sandro , Bassi Rosaria , Chigorno Vanna , Tettamanti Guido
Published: October 2006

Abstract: The developmental profiles of the gangliosides and those of the fatty acids and long‐chain bases of the total ganglioside mixture of the brain of chicken were followed from the 10th day of incubation to the 63rd posthatching day. One O‐acetylated polysialoganglioside that seems specific of the earlier embryonic stage and up to 21 alkali‐stable components could be recognized by high‐resolution two‐dimensional TLC procedures and quantified by computer‐assisted two‐dimensional TLC densitometry. Besides a number of gangliosides identified by co‐chromatography with reference standards, 10 were of unidentified structure, and within these 4 did not belong to the gangliotetrahexosyl series. Throughout embryonic life, the ceramide portion of gangliosides was found to contain the long‐chain base species with 18 carbons. Those with 20 carbons, ∼10% of the total, were found to be present only after hatching.
Release of Immunoreactive Insulin from Rat Brain Synaptosomes Under Depolarizing Conditions
Authors: Wei Liente , Matsumoto Hajime , Rhoads Dennis E.
Published: October 2006

Abstract: Synaptosome preparations were utilized to characterize the release and compartmentalization of immunoreactive insulin (IRI) in the adult rat brain. Depolarization of synaptosomes by elevation of the external potassium ion concentration elicited release of IRI from the synaptosomes into the incubation medium. This release was reduced or eliminated under three conditions known to prevent depolarization‐induced Ca2+ flux: elevating the external MgCl2, adding CoCl2, and eliminating external Ca2+ with EGTA. Depolarization of synaptosomes by veratridine also elicited release of synaptosomal IRI. This release was inhibited by tetrodotoxin. The amount of IRI released under depolarizing conditions represented 3–7% of that contained in the synaptosomes. High levels of IRI release also were observed upon removal of external Na+ to allow depolarization‐independent influx of external Ca2+ into the synaptosomal compartment. The Ca2+ dependency of synaptosomal IRI release suggests IRI is stored in the adult rat brain in synaptic vesicles within nerve endings from which it can be mobilized by exocytosis in association with neural activity.
Regional Distribution of α‐[3H]Amino‐3‐Hydroxy‐5‐Methylisoxazole‐4‐Propionic Acid Binding Sites in Rat Brain: Effect of Chemical Modification of SH– Groups in Tissue Sections
Authors: Baudry Michel , Monaghan Daniel , Cotman Carl , Altar C. Anthony
Published: October 2006

Abstract: Previous studies have shown that chemical modifications of sulfhydryl (SH–) groups with mercurial compounds in rat brain membrane preparations increase the binding of α‐[3H]‐amino‐3‐hydroxy‐5‐methylisoxazole‐4‐propionic acid ([3H]AMPA), a ligand for the quisqualate/AMPA type of glutamate receptors. In the present study we investigated the regional distribution of SH– group modification by quantitative analysis of autoradiographic images of [3H]AMPA binding in tissue sections. We also compared the effect of SH– group modification to that of the chaotropic ion thiocyanate (SCN−) which has been generally utilized to study [3H]AMPA binding sites. Low levels of binding sites were observed in the absence of potassium thiocyanate (KSCN), with binding predominantly found in telencephalic structures. The presence of KSCN induced a relatively uniform and large (four‐ to fivefold) increase in binding throughout the different brain structures. Pretreatment of the tissue sections with the SH– group reagent p‐chloromercuriphenylsulfonic acid produced a 0.5‐ to 1.5‐fold increase in [3H]AMPA binding. The enhanced binding displayed a regional variation with the largest increase in binding observed in the outer layer of the parietal cortex whereas the lowest increase occurred in the striatum. These results indicate that SH– group modification of tissue sections produces an increase in [3H]AMPA binding similar to that observed in detergent‐treated membrane preparations. Moreover they reveal that [3H]AMPA binding sites in different brain regions vary in their susceptibility to modification by SH– reagents, suggesting the existence in brain of a heterogeneous distribution of quisqualate/AMPA receptor subtypes.
Cholesterol Synthesis Is Down‐Regulated During Regeneration of Peripheral Nerve
Authors: Goodrum J. F.
Published: October 2006

Abstract: The discovery of apolipoprotein E synthesis and secretion by injured peripheral nerve led to the hypothesis that endoneurial apolipoprotein E serves to salvage degenerating myelin cholesterol. This salvaged cholesterol could then be reutilized by Schwann cells during remyelination via uptake through low‐density lipoprotein receptors. As a test of this hypothesis, we measured the rate of cholesterol synthesis in rat sciatic nerve endoneurium during development and at various times following a crush injury at 50 days of age. In control nerves [14C]acetate incorporation into cholesterol and 3‐hydroxy‐3‐methylglutaryl‐CoA reductase activity were closely linked throughout development, indicating that reductase activity in nerve, as in other tissues, is a good indicator of cholesterol's synthetic rate. In the crushed nerves cholesterol synthesis fell to nearly zero during the first week after the crush. There was a partial recovery during the second to fourth weeks, but unlike that of other lipids, cholesterol synthesis remained well below control nerve values throughout most of the 15‐week post‐crush period examined. Thus, cholesterol synthesis is at very low levels during the myelination of regenerating axons. These results are consistent with a receptor‐mediated down‐regulation of cholesterol synthesis by lipoproteins, and would be expected if Schwann cells were utilizing an external source of cholesterol as postulated above.
Effects of Cold Exposure on Rat Adrenal Tyrosine Hydroxylase: An Analysis of RNA, Protein, Enzyme Activity, and Cofactor Levels
Authors: Baruchin Andrea , Weisberg Edward P. , Miner Lucinda L. , Ennis David , Nisenbaum Laura K. , Naylor Edwin , Stricker Edward M. , Zigmond Michael J. , Kaplan Barry B.
Published: October 2006

Abstract: Long‐term cold exposure (5–7 days) is known to induce concomitant increases in the levels of adrenomedullary tyrosine hydroxylase (TH) RNA, protein, and enzyme activity. In this report, we compare the time courses of these changes and investigate the effects of cold exposure on the levels of biopterin, the cofactor required for tyrosine hydroxylation. After only 1 h of cold exposure, TH mRNA abundance increased 71% compared with nonstressed controls. Increases in total cellular TH RNA levels were maximal (threefold over control values) within 3–6 h of cold exposure and remained elevated throughout the duration of the experiment (72 h). TH protein levels increased rapidly after 24 h of cold exposure and reached a maximal value threefold above that of controls at 48–72 h. Despite the relatively rapid and large elevations in TH RNA and protein content, only modest increases in TH activity were detected during the initial 48 h of cold exposure. Adrenomedullary biopterin increased rapidly after the onset of cold exposure, rising to a level approximately twofold that of the nonstressed controls at 24 h, and remained at this level throughout the duration of the stress period. Taken together, the results of this time course study indicate that cold‐induced alterations in adrenal TH activity are mediated by multiple cellular control mechanisms, which may include pre‐ and posttranslational regulation. Our findings also suggest that cold stress‐induced increases in the levels of the TH cofactor may represent another key event in the sympathoadrenal system's response to cold stress.
An Easier, Reproducible, and Mass‐Production Method to Study the Blood–Brain Barrier In Vitro
Authors: Dehouck Marie‐Pierre , Méresse Stéphane , Delorme Pierre , Fruchart Jean‐Charles , Cecchelli Roméo
Published: October 2006

Abstract: To provide an “in vitro” system for studying brain capillary function, we have developed a process ofcoculture that closely mimics the “in vivo” situation by culturing brain capillary endothelial cells on one side of a filter and astrocytes on the other. Under these conditions, endothelial cells retain all the endothelial cell markers and the characteristics of the blood–brain barrier, including tight junctions and γ‐glutamyl transpeptidase activity. The average electric resistance for the monolayers was 661 Ω cm2. The system is impermeable to inulin and sucrose but allows the transport of leucine. Arabinose treatment increases transcellular transport flux by 70%. The relative ease with which such monolayers can be produced in large quantities would facilitate the “in vitro” study of brain capillary functions.
Ganglioside‐Modulated Proteolysis by Ca2+‐Activated Neutral Proteinase (CANP): A Role of Glycoconjugates in CANP Regulation
Authors: Chakrabarti Arun K. , Dasgupta Somsankar , Banik Naren L. , Hogan Edward L.
Published: October 2006

Abstract: We examined ganglioside modulation of the activity of the millimolar Ca2+‐sensitive form (mCANP) of calcium‐activated neutral proteinase (CANP), which is enriched in myelin, from brain. GM1, GD1a, GT1a, GM2, and GM4 produced a concentration‐dependent increase of mCANP activity. GD1a stimulated the greatest increase of enzyme activity (107%), followed by GT1a, whereas GD1b was inhibitory (56%). GM1, GM2, and GM4 stimulated but less so than GD1a and GT1a. Free N‐acetylneuraminic acid, asialo‐GM1, GM3, and a ganglioside mixture containing GM1, GD3, GD1a, and GD1b had no effect. The ganglioside‐mediated modulation was not affected by trifluoperazine and chlorpromazine (phospholipid‐binding antagonists). The mCANP Ca2+ requirement was significantly reduced in the presence of stimulatory gangliosides, and this increased sensitivity varied (10–50‐fold) with ganglioside structure. Gangliosides may interact with membrane mCANP and modulate its proteolytic action.
Chicken Growth‐Associated Protein GAP‐43 Is Tightly Bound to the Actin‐Rich Neuronal Membrane Skeleton
Authors: Moss D. J. , Fernyhough P. , Chapman K. , Baizer L. , Bray D. , Allsopp T.
Published: October 2006

Abstract: We have identified the chicken equivalent of growth‐associated protein GAP‐43 in a detergent‐resistant membrane skeleton from cultures of chick neurones and embryonic chick brain. Antisera to the membrane skeleton protein, the 3D5 antigen, precipitate the translation product of chick GAP‐43 cDNA, and the 3D5 antigen is also detected by antisera against synthetic peptides from the known amino acid sequence of rat GAP‐43. The chick protein and the rat GAP‐43 are biochemically similar proteins that both serve as major targets of phosphorylation by endogenous protein kinase C. The detergent‐resistant complex in which GAP‐43 is found also contains actin ˜5% of the total protein) and a neurone‐specific cell surface glycoprotein. We suggest that the membrane skeleton of neurones may be a primary site of action of GAP‐43.
Stimulation of Phospholipase D Activity by Phorbol Esters in Cultured Astrocytes
Authors: Gustavsson Lena , Hansson Elisabeth
Published: October 2006

Abstract: The phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) was found to stimulate phospholipase D activity in cultured primary astrocytes. Both the hydrolysis and the transphosphatidylation reaction catalyzed by phospholipase D were studied in cells labeled with [3H]glycerol. Phosphatidic acid (PA) synthesis was increased after addition of 100 nM TPA. When ethanol was present in the cell culture medium, phosphatidylethanol (Peth), a product of phospholipase D‐catalyzed transphosphatidylation, was formed. The half‐maximum effective concentrations (EC50) of TPA were 25 nM for PA increase as well as for Peth formation. The formation of Peth in ethanol‐treated cells was accompanied by an inhibition of the TPA‐induced increase in labeled PA. Increasing ethanol concentrations led to an increase in [3H]Peth and a decrease in [3H]PA. A protein kinase C inhibitor, 1‐(5‐isoquinolinesulfonyl)‐2‐methylpiperazine (H7), inhibited both the synthesis of PA and the formation of Peth observed after TPA addition to the astrocytes. Dioctanoylglycerol (100 μM) stimulated the formation of Peth in the presence of ethanol. In addition to the induction of Peth formation in astrocytes, TPA induced Peth formation in ethanol‐treated neurons. The present results indicate that phospholipase D activity is stimulated by TPA in cultured primary brain cells. Modulation of phospholipase D activity by protein kinase C is a mechanism that may be important in signal transduction cascades.
Biochemical Characterization of an Isolated and Functionally Reconstituted γ‐Aminobutyric Acid/Benzodiazepine Receptor
Authors: Bristow David R. , Martin Ian L.
Published: October 2006

Abstract: We have solubilized, affinity‐purified, and functionally reconstituted the γ‐aminobutyric acid/benzodiazepine (GABA/BDZ) receptor from rat brain into natural brain lipid liposomes. The detergent, 3‐[(3‐cholamidopropyl)‐dimethylammonio] 1‐propanesulphonate, was employed for the isolation of the receptor in the presence of a whole rat brain lipid extract supplemented with cholesteryl hemisuccinate. The soluble and reconstituted protein showed a homogeneous [3H]flunitrazepam binding population and the allosteric modulation of this binding site by GABA, by the pyrazolopyridine, cartazolate, and by the depressant barbiturate, pentobarbital. The purified GABA/BDZ receptor when incorporated into liposomes has been visualized by electron microscopy and reveals rosette structures, 8–9 nm in diameter, which appear to have a central pore. Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis of the reconstituted GABA/BDZ receptor reveals three major protein bands of 41, 52–56, and 59–62 kDa, the latter two of which appear as doublets. Functional receptor reconstitution is demonstrated by the measurement of GABA‐stimulated 36Cl‐ flux into the purified GABA/BDZ receptor incorporated liposomes and its modulation by the BDZs, barbiturates, and pyrazolopyridines.
Mitochondrial Complex I Deficiency in Parkinson's Disease
Authors: Schapira A. H. V. , Cooper J. M. , Dexter D. , Clark J. B. , Jenner P. , Marsden C. D.
Published: October 2006

Abstract: The structure and function of mitochondrial respiratory‐chain enzyme proteins were studied postmortem in the substantia nigra of nine patients with Parkinson's disease and nine matched controls. Total protein and mitochondrial mass were similar in the two groups. NADH‐ubiquinone reductase (Complex I) and NADH cytochrome c reductase activities were significantly reduced, whereas succinate cytochrome c reductase activity was normal. These results indicated a specific defect of Complex I activity in the substantia nigra of patients with Parkinson's disease. This biochemical defect is the same as that produced in animal models of parkinsonism by 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and adds further support to the proposition that Parkinson's disease may be due to an environmental toxin with action(s) similar to those of MPTP.
Functional Reconstitution of Purified Gi and Go with μ‐Opioid Receptors in Guinea Pig Striatal Membranes Pretreated with Micromolar Concentrations of N‐Ethylmaleimide
Authors: Ueda Hiroshi , Misawa Hidemi , Katada Toshiaki , Ui Michio , Takagi Hiroshi , Satoh Masamichi
Published: October 2006

Abstract: Functional coupling between μ‐opioid receptors and GTP‐binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective μ‐opioid agonists stimulated low‐Km GTPase in striatal membranes, in a Na+‐dependent manner. The same μ‐opioid agonist {[D‐Ala2,N‐Me‐Phe4,Gly5‐ol]‐enkephalin (DAGO)} caused no stimulation when the membranes were exposed to islet‐activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 μM) of N‐ethylmaleimide (NEM), which abolished the ADP‐ribosylation of purified Gi (the G protein that mediates inhibition of adenylate cyclase) and Go (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the μ‐agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in membranes. The μ‐agonist stimulation of low‐Km GTPase activity in NEM‐treated membranes was recovered by reconstitution with purified Gi, or Go. The μ‐agonist stimulation of low‐Km GTPase was additive when Gi and Go were simultaneously reconstituted in NEM‐treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the μ‐opioid receptor may exist in at least two different forms, separately coupled to Gi or Go.
Role of Glycine in the N‐Methyl‐d‐Aspartate‐Mediated Neuronal Cytotoxicity
Authors: Patel Jitendra , Zinkand William C. , Thompson Carolann , Keith Richard , Salama Andre
Published: October 2006

Abstract: Current evidence indicates that glutamate acting via the N‐methyl‐d‐aspartate (NMDA) receptor/ion channel complex plays a major role in the neuronal degeneration associated with a variety of neurological disorders. In this report the role of glycine in NMDA neurotoxicity was examined. We demonstrate that NMDA‐mediated neurotoxicity is markedly potentiated by glycine and other amino acids, e.g., d‐serine. Putative glycine antagonists HA‐966 and 7‐chlorokynurenic acid were highly effective in preventing NMDA neurotoxicity, even in the absence of added glycine. The neuroprotective action of HA‐966 and 7‐chlorokynurenic acid, but not that of NMDA antagonists 3‐(2‐carboxypiperazine‐4‐yl)propylphosphonate and MK‐801, could be reversed by glycine. These results indicate that glycine, operating through a strychnine‐insensitive glycine site, plays a central permissive role in NMDA‐mediated neurotoxicity.
4‐Aminopyridine Stimulates B‐50 (GAP43) Phosphorylation and [3H]Noradrenaline Release in Rat Hippocampal Slices
Authors: Heemskerk F. M. J. , Schrama L. H. , Gianotti C. , Spierenburg H. , Versteeg D. H. G. , De Graan P. N. E. , Gispen W. H.
Published: October 2006

Abstract: In situ phosphorylation of the presynaptic protein kinase C substrate B‐50 was investigated in rat hippocampal slices incubated with the convulsant drug 4‐aminopyridine (4‐AP). Phosphorylation of B‐50 was significantly enhanced 1 min after the addition of 4‐AP (100 μM). This increase by 4‐AP was concentration dependent (estimated EC50 30–50). Concomitant with the changes in B‐50 phosphorylation, 4‐AP also dose‐dependently stimulated [3H]noradrenaline ([3H]NA) release from the slices. 4‐AP stimulated [3H]NA release within 5 min to seven times the control level. The B‐50 phosphorylation induced by 4‐AP remained elevated after removal of the convulsant, this in contrast to B‐50 phosphorylation induced by depolarization with K+. A similar persistent increase was observed for [3H]NA release after a 5‐min incubation period with 4‐AP. These results give more insight into the molecular mechanisms underlying 4‐AP‐induced epileptogenesis and provide further evidence for the correlation between B‐50 phosphorylation and neurotransmitter release in the hippocampal slice.
Axonal Transport of Synaptic Vesicles and Muscarinic Receptors: Effect of Protein Synthesis Inhibitors
Authors: Levy C. , Scherman D. , Laduron P. M.
Published: October 2006

Abstract: The effect of cycloheximide, a protein synthesis inhibitor, was studied on the axonal transport of noradrenergic synaptic vesicles and presynaptic muscarinic receptors, identified by in vitro binding of [3H]dihydrotetrabenazine and [3H]quinuclidinylbenzilate, respectively, in rat sciatic nerve. Cycloheximide (1.5 mg/kg) administered subcutaneously 2 h before ligation decreased by ˜50% the accumulation of vesicles and receptors in the proximal segment above the ligature placed on the nerve; its action was detectable after a lag period of 10 h and disappeared 96 h after administration. Double ligatures were placed on the nerve at various time intervals between the first (distal) and the second (proximal) ligature, and the accumulation of vesicles and receptors proximal to the second ligature was measured; the first ligature diminished the accumulation above the second ligature. At an interval of 96 h between the first and the second ligature, cycloheximide completely prevented the accumulation of vesicles and receptors proximal to the second ligature. The effects of double ligatures and the response to cycloheximide treatment can best be explained on the assumption that an important proportion of synaptic vesicles and presynaptic receptors is being recycled in the nerve cell bodies after retrograde transport.
Short‐ and Long‐Term Alterations of Gene Expression in Limbic Structures by Repeated Electroconvulsive‐Induced Seizures
Authors: Leviel Vincent , Fayada Catherine , Guibert Bernard , Chaminade Michel , Machek Georges , Mallet Jacques , Biguet Nicole Faucon
Published: October 2006

Abstract: Rats were submitted to a series of 10 daily electroconvulsive shocks (ECS). A first group of animals was killed 1 day after the last seizure and a second group 30 days later. Tyrosine hydroxylase (TH) activity was measured using an in vitro assay in the nucleus caudatus, anterior cortex, amygdala, substantia nigra, ventral tegmental area, and locus ceruleus. The mRNA corresponding to this enzyme (TH‐mRNA) was evaluated using a cDNA probe at the cellular level in the ventral tegmental area, substantia nigra, and locus ceruleus. Met‐enkephalin (MET)‐immunoreactivity and the mRNA coding for the preproenkephalin (PPE‐mRNA) were assayed in striatum and the central nucleus of the amygdala. The day after the last ECS an increase of TH activity was observed in the ventral tegmental area, locus ceruleus, and substantia nigra in parallel with a similar increase in the amygdala and striatum; in the anterior cortex TH activity remained unchanged. TH‐mRNA was increased in the locus ceruleus, evidencing the presence in this structure of a genomic activation. The amounts of MET and PPE‐mRNA were unaffected in the striatum but increased in the amygdala. Thirty days after the last ECS we observed a decrease of TH activity in the amygdala and of TH‐mRNA amount in the ventral tegmental area. In the locus ceruleus TH‐mRNA remained higher in treated animals than in controls whereas TH activity returned to control levels. These results demonstrate that a series of ECS induces an initial increase of the activity of mesoamygdaloid catecholaminergic neurons followed by a sustained decrease through alterations of TH gene expression which could mediate the clinical effect of the treatment.