Long-term daily access to alcohol alters dopamine-related synthesis and signaling proteins in the rat striatum

Chronic alcohol exposure can adversely affect neuronal morphology, synaptic architecture and associated neuroplasticity. However, the effects of moderate levels of long-term alcohol intake on the brain are a matter of debate. The current study used 2-DE (two-dimensional gel electrophoresis) proteomics to examine proteomic changes in the striatum of male Wistar rats after 8 months of continuous access to a standard off-the-shelf beer in their home cages. Alcohol intake under group-housed conditions during this time was around 3–4 g/kg/day, a level below that known to induce physical dependence in rats. After 8 months of access rats were euthanased and 2-DE proteomic analysis of the striatum was conducted. A total of 28 striatal proteins were significantly altered in the beer drinking rats relative to controls. Strikingly, many of these were dopamine (DA)-related proteins, including tyrosine hydroxylase (an enzyme of DA biosynthesis), pyridoxal phosphate phosphatase (a co-enzyme in DA biosynthesis), DA and cAMP regulating phosphoprotein (a regulator of DA receptors and transporters), protein phosphatase 1 (a signaling protein) and nitric oxide synthase (which modulates DA uptake). Selected protein expression changes were verified using Western blotting. We conclude that long-term moderate alcohol consumption is associated with substantial alterations in the rat striatal proteome, particularly with regard to dopaminergic signaling pathways. This provides potentially important evidence of major neuroadaptations in dopamine systems with daily alcohol consumption at relatively modest levels.     

Prenatal alcohol exposure alters phosphorylation and glycosylation of proteins in rat offspring liver

To gain more insights into the translational and PTM that occur in rat offspring exposed to alcohol in utero, 2‐D PAGE with total, phospho‐ and glycoprotein staining and MALDI‐MS/MS and database searching were conducted. The results, based on fold‐change expression, revealed a down‐regulation of total protein expression by prenatal alcohol exposure in 7‐day‐old and 3‐month‐old rats. There was an up‐regulation of protein phosphorylation but a down‐regulation of glycosylation by prenatal alcohol exposure in both age groups. Of 31 protein spots examined per group, differentially expressed proteins were identified as ferritin light chain, aldo‐keto reductase, tumor rejection antigen gp96, fructose‐1,6‐bisphosphatase, glycerol‐3‐phosphate dehydrogenase, malate dehydrogenase, and γ‐actin. Increased phosphorylation was observed in proteins such as calmodulin, gluthatione S‐transferase, glucose regulated protein 58, α‐enolase, eukaryotic translation elongation factor 1 β‐2, riboprotein large P2, agmatinase, ornithine carbamoyltransferase, quinolinate phosphoribosyltransferase, formimidoyltransferase cyclodeaminase, and actin. In addition, glycosylation of adenosine kinase, adenosylhomocysteine hydrolase, and 3‐hydroxyanthranilate dioxygenase was reduced. Pathways affected by these protein alterations include cell signaling, cellular stress, protein synthesis, cytoskeleton, as well as glucose, aminoacid, adenosine and energy metabolism. The activity of the gluconeogenic enzyme fructose‐1,6‐bisphosphatase was elevated by prenatal alcohol. The observations may have important physiological implications.     

Fetal alcohol exposure alters neurosteroid levels in the developing rat brain

Neurosteroids are modulators of neuronal function that may play important roles in brain maturation. We determined whether chronic prenatal ethanol exposure altered neurosteroid levels in the developing brain. Rat dams were exposed to: (i) a 5% ethanol-containing liquid diet that produces peak maternal blood alcohol levels near the legal intoxication limit (approximately 0.08 g/dL); (ii) an isocaloric liquid diet containing maltose-dextrin instead of ethanol with pair-feeding; (iii) rat chow ad libitum. Neurosteroid levels were assessed in offspring brains using radioimmunoassay or gas chromatography-mass spectrometry techniques. A prenatal ethanol exposure-induced increase in pregnenolone sulfate levels, but not dehydroepiandrosterone sulfate levels, was evident at the earliest time point studied (embryonic day 14). This effect lasted until post-natal day 5. Levels of other neurosteroids were assessed at embryonic day 20; pregnenolone levels, but not allopregnanolone levels, were elevated. Pregnenolone sulfate levels were not altered in the maternal brain. Neither pregnenolone nor pregnenolone sulfate levels were significantly altered in the fetal liver, placenta and maternal blood, indicating that the effect of ethanol is not secondary to accumulation of peripherally-produced steroids. Fetal ethanol exposure has been shown to decrease both cellular and behavioral responsiveness to neurosteroids, and our findings provide a plausible explanation for this effect.     

Calcium signaling and neuronal vulnerability to ischemia in the striatum

Neurons express extremely different sensitivity to ischemic insults. The neuronal vulnerability is region-specific and the striatum is among the most susceptible areas to ischemic damage. Projecting GABAergic medium-sized neurons are very sensitive to energy metabolism impairment, whereas interneurons are selectively spared. However, the reasons for this differential vulnerability are largely unknown. Calcium ions (Ca2+) are important intracellular messengers enabling several physiological processes. However, excessive Ca2+ influx from the extracellular space or release from internal stores can elevate Ca2+ to levels that exceed the capacity of single neurons to appropriately buffer such overload. This capacity also appears to be a peculiar feature of single neuronal subtypes. This review will provide a brief survey of the ionic basis underlying the differential responses to in vitro ischemia of distinct striatal neuronal subtypes, mainly focusing on the role of Ca2+. The potential relevance of these findings in the development of therapeutic strategies for acute stroke will be discussed.     

Characterization of proteins in the rat striatum following acute cocaine administration

Cocaine administration in the brain alters gene expression via dopamine and glutamate receptor-mediated intracellular signaling cascades. The current study was designed to identify alterations in the total proteome in the rat dorsal striatum in response to intraperitoneal injection of cocaine (20 mg/kg). The results demonstrated that alterations of specific proteins at 20, 120, and 360 min following acute cocaine injection decreased over the time course. Proteins that were identified as having changed as a result of exposure to acute cocaine were found to be involved in a variety of functions necessary for maintaining cellular structure, metabolism, and gene expression in the dorsal striatum.     

Manganese exposure alters the expression of N‐methyl‐d‐aspartate receptor subunit mRNAs and proteins in rat striatum

Manganese is one of the ubiquitous environmental pollutants that can induce an indirect excitotoxicity caused by altered glutamate (Glu) metabolism. The present study has been carried out to investigate the effect of Mn on the expression of N‐methyl‐d ‐aspartate receptor (NR) subunit mRNAs and proteins in rat striatum when rats were in manganism. The rats were divided randomly into four groups of six males and six females each: control group (group 1) and 8, 40, and 200 μmol/kg Mn‐treated groups (groups 2–4). The control group rats were subcutaneously (s.c.) injected with normal saline. Manganese‐treated rats were s.c. injected with respectively 8, 40, and 200 μmol/kg of MnCl₂ · 6H₂O in normal saline. The administration of MnCl₂ · 6H₂O for 4 weeks significantly increased Mn concentration in the striatum. With the increase in administered MnCl₂ dosage, Glu concentration and cell apoptosis rate increased significantly. The relative intensity of NR2A mRNA decreased significantly in 8 μmol/kg Mn‐treated rats. However, relative intensities of NR1 and NR2B mRNAs decreased significantly in 40 μmol/kg Mn‐treated rats. Similarly, the relative intensity of NR2A protein showed a significant decrease in 40 μmol/kg Mn‐treated rats whereas those of NR1 and NR2B decreased significantly in 200 μmol/kg Mn‐treated rats. Therefore, the expression of NR2A mRNA and protein were much more sensitive to Mn than that of NR1 and NR2B. In conclusion, the results suggested that Mn induced nerve cell damage by increasing extracellular Glu level and altered expression of NR subunit mRNAs and proteins in rat striatum. © 2010 Wiley Periodicals, Inc. J Biochem Mol Toxicol 24:1–9, 2010; Published online in Wiley InterScience ( www.interscience.wiley.com ). DOI 10.1002/jbt.20306     

Effects of chronic antidepressant treatment on dopamine-related [3H]SCH 23390 and [3H]Spiperone binding in the rat striatum

1. The effects of chronic administration of antidepressants on dopamine-related [3H]SCH 23390 and [3H]spiperone binding to rat striatal membranes were assessed. 2. The monoamine oxidase inhibitors phenelzine (5 or 10 mg kg-1/day) and tranylcypromine (1 mg kg-1/day) and the tricyclic desipramine (10 mg kg-1/day) were administered for 28 days by constant subcutaneous infusion using Alzet (2ML4) osmotic minipumps. 3. These treatments did not alter Kd estimates for either [3H]SCH 23390 or [3H]spiperone binding sites. The monoamine oxidase inhibitors induced a decrease in the Bmax values for both [3H]SCH 23990 and [3H]spiperone binding sites. Desipramine induced a decrease in the Bmax value for [3H]SCH 23390 binding but had no effect on the Bmax value for [3H]spiperone binding.     

Anticipatory activity rhythms under daily schedules of water access in the rat.

Rats anticipate a fixed daily feeding time by entrainment of a component of their multioscillatory circadian system. The range of stimuli capable of entraining this component is little studied. Previous studies suggest that restricted water access is not an effective entrainment stimulus, as measured by general locomotion. The present study re-examined the issue, using two other measures of activity: wheel running and activity at a food-water delivery bin. Rats restricted to 1 hr of water each day in the middle of the light and to food in the 12-hr dark period showed no anticipation of this event in the wheel-running measure, but some rats did show anticipation in the delivery bin activity measure. Rats (bin activity measure only) restricted to 1 hr of water and 1 hr of food separated by intervals of 7, 10, or 12 hr, in either the light or the dark, showed consistent anticipation of food access time but little or no anticipation of water access time. Water access time also did not sustain food anticipatory rhythms in animals whose food-water schedules were reversed. However, deprivation of water or of both food and water for 72 or 90 hr was usually associated with specific increases in bin activity at both the usual feeding and drinking times. Water access, like food, appears to provide cues capable of entraining an anticipatory circadian mechanism. Differences in the type and amount of anticipatory activity preceding these events may reflect differences in the strengths of the two entrainment cues and/or in the activity levels or specific behavioral strategies promoted by hunger and thirst.     

Subcellular distribution and postnatal development of cholinergic marker proteins in the striatum of rat

The distribution of muscarinic cholinergic receptors, choline acetyl-transferase and acetylcholinesterase activities were measured in subcellular fractions of the rat striatum on the 5th and 15th days postnatally and in adulthood. The receptor density in the striatum of 5 and 15-day-old rats was 15%, respectively, of the adult value. Similar increases of the receptors could be detected in the synaptosomal and microsomal fractions in the postnatal life of rat. The activity of choline acetyltransferase on the same days was 15% and 28%. In the subcellular fractions, the enzyme activity was the highest in the microsomal fraction on both the 5th and 15th days postnatally. The activity of acetylcholinesterase in the homogenate was 6% of the adult value in the 5-day-old rat striatum, while in the synaptosomal fraction it was 11% and 47% of the adult value on the 5th and 15th days, respectively. Our results show that the development of the muscarinic cholinergic receptors precedes that of the two cholinergic enzymes in both 5 and 15-day-old rat striatum. This may suggest an early perikaryonal synthesis and the fast translocation of receptors to the axon terminals during ontogenetic development.     

Prenatal exposure to alcohol alters the Golgi apparatus of newborn rat hepatocytes: a cytochemical study

The effect of prenatal exposure to ethanol on the Golgi apparatus of newborn rat hepatocytes has been studied cytochemically using several trans-Golgi markers (thiamine pyrophosphatase, uridine diphosphatase, inosine diphosphatase, acid phosphatase, and 5'-nucleotidase) as well as a cis-side marker (osmium impregnation). The amount of cerium phosphate formed in the cytochemical reactions was roughly quantitated by stereologic methods. The Golgi apparatus of about 40% of the hepatocytes appeared disorganized after alcohol treatment, and in the other 60%, the electron density of reaction product deposits for all phosphatases investigated was decreased. 5'-Nucleotidase was completely absent in cisternae of Golgi apparatus of treated cells. In control cells impregnated with osmium tetroxide, reduced osmium compounds were observed in most Golgi cisternae and in nearby vesicles. In contrast, only small vesicles appeared positive in treated hepatocytes. These results suggest that prenatal alcohol exposure alters some Golgi functions. Thus, the decrease in nucleoside diphosphatase and 5'-nucleotidase cytochemical activities after ethanol exposure strongly suggests that this treatment could affect glycosylation in the Golgi apparatus of newborn rat hepatocytes.     

Age-dependent modifications of mitochondrial proteins in cerebral cortex and striatum of rat brain

The protein composition of free mitochondria purified from cerebral cortex and striatum during aging was analyzed by gel electrophoresis. Mitochondria were isolated from cerebral cortex and striatum of 4-, 12-, and 24-month-old rat brain. The percent amount of mitochondrial proteins after gel-electrophoretic separation was determined densitometrically. A significant decrease in the amount of two polypeptides (with molecular weights of 20 and 16 kDa, respectively) in both brain regions during aging was found. The decrease was higher in the striatum indicating a greater vulnerability of this brain area to the aging process. The age-dependent modifications of mitochondrial proteins observed may play an important role in several mitochondrial functions, such as energy transduction and transport processes as well as in structural changes occurring with age, causing altered membrane permeability and fluidity.     

Dual contradictory roles of cAMP signaling pathways in hydroxyl radical production in the rat striatum

Studies have suggested that cAMP signaling pathways may be associated with the production of reactive oxygen species. In this study, we examined how modifications in cAMP signaling affected the production of hydroxyl radicals in rat striatum using microdialysis to measure extracellular 2,3-dihydroxybenzoic acid (2,3-DHBA), which is a hydroxyl radical adduct of salicylate. Up to 50 nmol of the cell-permeative cAMP mimetic 8-bromo-cAMP (8-Br-cAMP) increased 2,3-DHBA in a dose-dependent manner (there was no additional increase in 2,3-DHBA at 100 nmol). Another cAMP mimetic, dibutyryl cAMP (db-cAMP), caused a nonsignificant increase in 2,3-DHBA at 50 nmol and a significant decrease at 100 nmol. Up to 20 nmol of forskolin, which is a direct activator of adenylyl cyclase, increased 2,3-DHBA, similar to the effect of 8-Br-cAMP; however, forskolin resulted in a much greater increase in 2,3-DHBA. A potent inhibitor of protein kinase A (PKA), H89 (500 μM), potentiated the 8-Br-cAMP- and forskolin-induced increases in 2,3-DHBA and antagonized the inhibitory effect of 100 nmol of db-cAMP. Interestingly, the administration of 100 nmol of 8-bromo-cGMP alone or in combination with H89 had no significant effect on 2,3-DHBA levels. Doses of 100 nmol of a preferential PKA activator (6-phenyl-cAMP) or a preferential PKA inhibitor (8-bromoadenosine-3',5'-cyclic monophosphorothionate, Rp-isomer; Rp-8-Br-cAMPS), which also inhibits the cAMP-mediated activation of Epac (the exchange protein directly activated by cAMP), suppressed or enhanced, respectively, the formation of 2,3-DHBA. Up to 100 nmol of 8-(4-chlorophenylthio)-2'-O-methyladenosine-cAMP, which is a selective activator of Epac, dose-dependently stimulated the formation of 2,3-DHBA. These findings suggest that cAMP signaling plays contradictory roles (stimulation and inhibition) in the production of hydroxyl radicals in rat striatum by differential actions of Epac and PKA. These roles might contribute to the production of hydroxyl radicals concomitant with cAMP in carbon monoxide poisoning, because the formation of 2,3-DHBA was potentiated by the PKA inhibitor H89 and suppressed by Rp-8-Br-cAMPS, which inhibits PKA and Epac.     

Alteration of dopamine synthesis in rat striatum subsequent to selective type A monoamine oxidase inhibition

Abstract: Pretreatment of rat striatal slices with the selective type A monoamine oxidase (MAO) inhibitor clorgyline was found to produce significant inhibition of dopamine (DA) synthesis. DA synthesis was reduced by nearly 50%, but not until more than 90% of the type A enzyme was inhibited. In contrast, complete inhibition of the type B MAO following deprenyl treatment had no effect. It is suggested that interneuronal accumulation of DA following inhibition of type A MAO leads to feedback inhibition at the rate-limiting step in DA biosynthesis, tyrosine hydroxylation. These results are also consistent with the presence of a type A MAO within DA-containing neurons and provide evidence of a regulatory role for type A MAO in the synthesis of brain DA.     

Methamphetamine neurotoxicity decreases phasic, but not tonic, dopaminergic signaling in the rat striatum

Neurotoxic doses of methamphetamine (METH) are known to cause depletions in striatal dopamine (DA) tissue content. However, the effects of METH-induced insults on dopaminergic neurotransmission are not fully understood. Here, we employed fast-scan cyclic voltammetry at a carbon-fiber microelectrode in the anesthetized rat striatum to assess the effects of a neurotoxic regimen of METH on phasic and tonic modes of dopaminergic signaling and underlying mechanisms of DA release and uptake. Extracellular DA was electrically evoked by stimulation of the medial forebrain bundle mimicking tonic and phasic firing patterns for dopaminergic cells and was monitored simultaneously in both the dorsomedial and dorsolateral striatum. Kinetic analysis of evoked recordings determined parameters describing DA release and uptake. Striatal DA tissue content was quantified by high performance liquid chromatography with electrochemical detection. METH-pretreatment (four doses of 7.5 or 10.0 mg/kg s.c.) induced DA depletions of ～ 40% on average, which are reported in both striatal subregions. METH pre-treatment significantly decreased the amplitude of signals evoked by phasic, but not tonic, stimulation. Parameters for DA release and uptake were also similarly reduced by ～ 40%, consistent with effects on evoked phasic-like responses and DA tissue content. Taken together, these results suggest that METH-pretreatment selectively diminishes phasic, but not tonic, dopaminergic signaling in the dorsal striatum.     

CagA of Helicobacter pylori alters the expression and cellular distribution of host proteins involved in cell signaling

To expand our knowledge of Helicobacter pylori virulence mechanisms, we used iTRAQ (isobaric tagging reagents for relative and absolute quantification)-based proteomic analysis to investigate the effect of H. pylori on gastric AGS tissue culture cells. In particular, we were interested in finding out which effects of H. pylori were dependent on the cytotoxins CagA and VacA. Protein analysis was restricted to detergent-resistant membranes (DRMs), because both toxins were described previously to localize in lipid raft-like domains. Using H. pylori wild type and two isogenic mutants, DeltacagA and DeltavacA, we identified a total of 21 proteins that were either increased or decreased in the DRMs due to bacterial infection. The effect on three of these proteins, ezrin, syndecan-4 and Rab11-FIP1, were furthermore dependent on CagA. Because these proteins have been implicated in cell migration, adhesion and polarity, they might act as important mediators of CagA cytotoxicity.     

Toll-like receptor signaling alters the expression of regulator of G protein signaling proteins in dendritic cells: implications for G protein-coupled receptor signaling

Conserved structural motifs on pathogens trigger pattern recognition receptors present on APCs such as dendritic cells (DCs). An important class of such receptors is the Toll-like receptors (TLRs). TLR signaling triggers a cascade of events in DCs that includes modified chemokine and cytokine production, altered chemokine receptor expression, and changes in signaling through G protein-coupled receptors (GPCRs). One mechanism by which TLR signaling could modify GPCR signaling is by altering the expression of regulator of G protein signaling (RGS) proteins. In this study, we show that human monocyte-derived DCs constitutively express significant amounts of RGS2, RGS10, RGS14, RGS18, and RGS19, and much lower levels of RGS3 and RGS13. Engagement of TLR3 or TLR4 on monocyte-derived DCs induces RGS16 and RGS20, markedly increases RGS1 expression, and potently down-regulates RGS18 and RGS14 without modifying other RGS proteins. A similar pattern of Rgs protein expression occurred in immature bone marrow-derived mouse DCs stimulated to mature via TLR4 signaling. The changes in RGS18 and RGS1 expression are likely important for DC function, because both proteins inhibit G alpha(i)- and G alpha(q)-mediated signaling and can reduce CXC chemokine ligand (CXCL)12-, CC chemokine ligand (CCL)19-, or CCL21-induced cell migration. Providing additional evidence, bone marrow-derived DCs from Rgs1(-/-) mice have a heightened migratory response to both CXCL12 and CCL19 when compared with similar DCs prepared from wild-type mice. These results indicate that the level and functional status of RGS proteins in DCs significantly impact their response to GPCR ligands such as chemokines.     

Effect of amantadine on the rate of dopamine synthesis in rat corpus striatum.

Abstract— The effect of amantadine on the rate of dopamine synthesis in rat corpus striatum was determined by three methods. (1) Measuring the rate of decline of endogenous dopamine following inhibition of synthesis with a-methyltyrosine (α-MT); (2) Measuring the rate of conversion of [3,5-³H]tyrosine to ³H-labelled catechols under conditions of an initial rate; and (3) measuring the levels of homovanillic acid (HVA), the principal metabolite of brain dopamine. Endogenous dopamine levels were 68-1 n-mole/g with a control synthesis rate of about 21 n-mole/g/h as determined using either α-MT or [3,5-³H]tyrosine. Amantadine had no effect on synthesis at doses up to 100 mg/kg using α-MT and [3,5-³H]tyrosine. HVA levels were unaffected after 30 mg/kg drug, but were elevated 48%(P < 005) after 100 mg/kg of drug. By contrast apomorphine reduced and haloperidol increased synthesis as determined by all three methods. It is concluded that amantadine has no marked effect on dopamine synthesis in rat corpus striatum.     

Regulation of psychostimulant-induced signaling and gene expression in the striatum

Amphetamine (AMPH) and cocaine are indirect dopamine agonists that activate multiple signaling cascades in the striatum. Each cascade has a different subcellular location and duration of action that depend on the strength of the drug stimulus. In addition to activating D1 dopamine-Gs-coupled-protein kinase A signaling, acute psychostimulant administration activates extracellular-regulated kinase transiently in striatal cells; conversely, inhibition of extracellular-regulated kinase phosphorylation decreases the ability of psychostimulants to elevate locomotor behavior and opioid peptide gene expression. Moreover, a drug challenge in rats with a drug history augments and prolongs striatal extracellular-regulated kinase phosphorylation, possibly contributing to behavioral sensitization. In contrast, AMPH activates phosphoinositide-3 kinase substrates, like protein kinase B/Akt, only in the nuclei of striatal cells but this transient increase induced by AMPH is followed by a delayed decrease in protein kinase B/Akt phosphorylation whether or not the rats have a drug history, suggesting that the phosphoinositide-3 kinase pathway is not essential for AMPH-induced behavioral sensitization. Chronic AMPH or cocaine also alters the regulation of inhibitory G protein-coupled receptors in the striatum, as evident by a prolonged decrease in the level of regulator of G protein signaling 4 after non-contingent or contingent (self-administered) drug exposure. This decrease is exacerbated in behaviorally sensitized rats and reversed by re-exposure to a cocaine-paired environment. A decrease in regulator of G protein signaling 4 levels may weaken its interactions with metabotropic glutamate receptor 5, Galphaq, and phospholipase C beta that may enhance drug-induced signaling. Alteration of these protein-protein interactions suggests that the striatum responds to psychostimulants with a complex molecular repertoire that both modulates psychomotor effects and leads to long-term neuroadaptations.     

Epigenetic status of Gdnf in the ventral striatum determines susceptibility and adaptation to daily stressful events

Stressful events during adulthood are potent adverse environmental factors that can predispose individuals to psychiatric disorders, including depression; however, many individuals exposed to stressful events can adapt and function normally. While stress vulnerability may influence depression, the molecular mechanisms underlying the susceptibility and adaptation to chronic stress within the brain are poorly understood. In this study, two genetically distinct mouse strains that exhibit different behavioral responses to chronic stress were used to demonstrate how the differential epigenetic status of the glial cell-derived neurotrophic factor (Gdnf) gene in the ventral striatum modulates susceptibility and adaptation to chronic stress. Our results suggest that the histone modifications and DNA methylation of the Gdnf promoter have crucial roles in the control of behavioral responses to chronic stress. Our data provide insights into these mechanisms, suggesting that epigenetic modifications of Gdnf, along with genetic and environmental factors, contribute to behavioral responses to stress.