Rat forebrain cortex
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Lithium is regarded as a unique therapeutic agent for the management of bipolar disorder (BD). In efforts to explain the favourable effects of lithium in BD, a wide range of mechanisms was suggested. Among those, the effect of clinically relevant concentrations of lithium on the plasma membrane was extensively studied. However, the biophysical properties of brain membranes isolated from experimental animals exposed to acute, short-term and chronic lithium have not been performed to-date. In this study, we compared the biophysical parameters and level of lipid peroxidation in membranes isolated from forebrain cortex (FBC) of therapeutic lithium-treated and/or sleep-deprived rats. Lithium interaction with FBC membranes was characterized by appropriate fluorescent probes. DPH (1,6-diphenyl-1,3,5-hexatriene) and TMA-DPH (1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulphonate) were used for characterization of the hydrophobic lipid core and Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) for the membrane-water interface. Lipid peroxidation was determined by immunoblot analysis of 4-HNE-(4-hydroxynonenal)-protein adducts. The organization of polar head-group region of FBC membranes, measured by Laurdan generalized polarization, was substantially altered by sleep deprivation and augmented by lithium treatment. Hydrophobic membrane interior characterized by steady-state anisotropy of DPH and TMA-DPH fluorescence was unchanged. Chronic lithium had a protective effect against peroxidative damage of membrane lipids in FBC. In summary, lithium administration at a therapeutic level and/or sleep deprivation as an animal model of mania resulted in changes in rat FBC membrane properties.
- MeSH
- fluidita membrány účinky léků MeSH
- krysa rodu rattus MeSH
- lipidové dvojvrstvy metabolismus MeSH
- lithium farmakologie MeSH
- membránové lipidy metabolismus MeSH
- peroxidace lipidů účinky léků MeSH
- přední mozek účinky léků metabolismus MeSH
- spánková deprivace metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
UNLABELLED: Proteomic analysis was performed in post-nuclear supernatant fraction (PNS) prepared from forebrain cortex of rats exposed to increasing doses of morphine (10-50mg/kg) for 10days and sacrificed 24h (group +M10) or 20days (group +M10/-M20) after the last dose of morphine. PNS fraction was resolved by 2D-ELFO and stained by CBB. Analysis of the difference between (+M10) and (-M10) samples of PNS by PDQuest accompanied by MALDI-TOF MS/MS indicated the significant change of 28 proteins. Importantly, the number of altered proteins was decreased to 14 after 20days of nurturing animals in the absence of morphine. This new and important finding indicating the ability of mammalian organism to return to physiological norm after removal of the drug was verified by an independent methodology - gel-free & label-free quantification and normalization procedure denominated as MaxLFQ. The 113 proteins were identified as altered by morphine in (+M10) samples when compared with (-M10) samples of PNS and this number was decreased to 19 after 20days of nurturing the animals in the absence of this drug. BIOLOGICAL SIGNIFICANCE: Forebrain cortex of rats exposed to morphine for 10days is severely altered as far as the overall protein composition is involved. Depending on the method used for protein detection and quantification, 28 (MALDI-TOF MS/MS) or 113 (MaxLFQ) altered proteins were identified. Importantly, in rats sacrificed 20days after the last dose of morphine, the number of altered proteins was decreased to 14 (MALDI-TOF MS/MS) and 19 (MaxLFQ), respectively. Our data indicate the high ability of living organism to oppose the drastic, morphine-induced change of the target tissue protein composition with the aim to return to the physiological norm after complete removal of the drug.
- MeSH
- abstinenční syndrom MeSH
- krysa rodu rattus MeSH
- morfin aplikace a dávkování farmakologie MeSH
- přední mozek chemie účinky léků MeSH
- proteiny analýza MeSH
- proteomika metody MeSH
- tandemová hmotnostní spektrometrie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
Lithium (Li) is a typical mood stabilizer and the first choice for treatment of bipolar disorder (BD). Despite an extensive clinical use of Li, its mechanisms of action remain widely different and debated. In this work, we studied the time-course of the therapeutic Li effects on ouabain-sensitive Na+/K+-ATPase in forebrain cortex and hippocampus of rats exposed to 3-day sleep deprivation (SD). We also monitored lipid peroxidation as malondialdehyde (MDA) production. In samples of plasma collected from all experimental groups of animals, Li concentrations were followed by ICP-MS. The acute (1 day), short-term (7 days) and chronic (28 days) treatment of rats with Li resulted in large decrease of Na+/K+-ATPase activity in both brain parts. At the same time, SD of control, Li-untreated rats increased Na+/K+-ATPase along with increased production of MDA. The SD-induced increase of Na+/K+-ATPase and MDA was attenuated in Li-treated rats. While SD results in a positive change of Na+/K+-ATPase, the inhibitory effect of Li treatment may be interpreted as a pharmacological mechanism causing a normalization of the stress-induced shift and return the Na+/K+-ATPase back to control level. We conclude that SD alone up-regulates Na+/K+-ATPase together with increased peroxidative damage of lipids. Chronic treatment of rats with Li before SD, protects the brain tissue against this type of damage and decreases Na+/K+-ATPase level back to control level.
- MeSH
- antimanika farmakologie terapeutické užití MeSH
- hipokampus účinky léků metabolismus MeSH
- kompetitivní vazba účinky léků MeSH
- krysa rodu rattus MeSH
- lithiumkarbonát farmakologie MeSH
- malondialdehyd metabolismus MeSH
- ouabain metabolismus MeSH
- peroxidace lipidů účinky léků MeSH
- potkani Wistar MeSH
- přední mozek účinky léků enzymologie metabolismus MeSH
- sodíko-draslíková ATPasa metabolismus MeSH
- spánková deprivace farmakoterapie enzymologie metabolismus MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Chronic exposure of mammalian organism to morphine results in adaption to persistent high opioid tone through homeostatic adjustments. Our previous results indicated that in the frontal brain cortex (FBC) of rats exposed to morphine for 10 days, such a compensatory adjustment was detected as large up-regulation of adenylylcyclases I (8-fold) and II (2.5-fold). The other isoforms of AC (III-IX) were unchanged. Importantly, the increase of ACI and ACII was reversible as it disappeared after 20 days of morphine withdrawal. Changes of down-stream signaling molecules such as G proteins and adenylylcyclases should respond to and be preceded by primary changes proceeding at receptor level. Therefore in our present work, we addressed the problem of reversibility of the long-term morphine effects on μ-, δ- and κ-OR protein levels in FBC. METHODS: Rats were exposed to increasing doses of morphine (10-40 mg/kg) for 10 days and sacrificed either 24 h (group +M10) or 20 days (group +M10/-M20) after the last dose of morphine in parallel with control animals (groups -M10 and -M10/-M20). Post-nuclear supernatant (PNS) fraction was prepared from forebrain cortex, resolved by 1D-SDS-PAGE under non-dissociated (-DTT) and dissociated (+DTT) conditions, and analyzed for the content of μ-, δ- and κ-OR by immunoblotting with C- and N-terminus oriented antibodies. RESULTS: Significant down-regulation of δ-OR form exhibiting Mw ≈ 60 kDa was detected in PNS prepared from both (+M10) and (+M10/-M20) rats. However, the total immunoblot signals of μ-, δ- and κ-OR, respectively, were unchanged. Plasma membrane marker Na, K-ATPase, actin and GAPDH were unaffected by morphine in both types of PNS. Membrane-domain marker caveolin-1 and cholesterol level increased in (+M10) rats and this increase was reversed back to control level in (+M10/-M20) rats. CONCLUSIONS: In FBC, prolonged exposure of rats to morphine results in minor (δ-OR) or no change (μ- and κ-OR) of opioid receptor content. The reversible increases of caveolin-1 and cholesterol levels suggest participation of membrane domains in compensatory responses during opioid withdrawal. GENERAL SIGNIFICANCE: Analysis of reversibility of morphine effect on mammalian brain.
- MeSH
- 2D gelová elektroforéza MeSH
- abstinenční syndrom * MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- krysa rodu rattus MeSH
- morfin aplikace a dávkování škodlivé účinky MeSH
- potkani Wistar MeSH
- přední mozek metabolismus MeSH
- receptory opiátové delta metabolismus MeSH
- receptory opiátové kappa metabolismus MeSH
- receptory opiátové mu metabolismus MeSH
- western blotting MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- srovnávací studie MeSH
Opioid addiction is characterized by compulsive drug seeking and taking behavior, which is thought to result from persistent neuroadaptations. However, there is a lack of information about the changes at both the cellular and molecular levels occurring after cessation of drug administration. The aim of our study was to determine alterations of both phosphoproteome and proteome in selected brain regions of the rats (brain cortex, hippocampus, striatum, and cerebellum) 3 months after cessation of 10-day morphine treatment. Phosphoproteome profiling was performed by Pro-Q® Diamond staining. The gel-based proteomic approach accompanied by label-free quantification (MaxLFQ) was used for characterization of proteome changes. The phosphoproteomic analysis revealed the largest change in the hippocampus (14); only few altered proteins were detected in the forebrain cortex (5), striatum (4), and cerebellum (3). The change of total protein composition, determined by 2D electrophoresis followed by LFQ analysis, identified 22 proteins with significantly altered expression levels in the forebrain cortex, 19 proteins in the hippocampus, 12 in the striatum and 10 in the cerebellum. The majority of altered proteins were functionally related to energy metabolism and cytoskeleton reorganization. As the most important change we regard down-regulation of 14-3-3 proteins in rat cortex and hippocampus. Our findings indicate that i) different parts of the brain respond in a distinct manner to the protracted morphine withdrawal, ii) characterize changes of protein composition in these brain parts, and iii) enlarge the scope of evidence for adaptability and distinct neuroplasticity proceeding in the brain of drug-addicted organism.
- MeSH
- abstinenční syndrom genetika metabolismus MeSH
- časové faktory MeSH
- corpus striatum účinky léků metabolismus MeSH
- fosforylace fyziologie MeSH
- hipokampus účinky léků metabolismus MeSH
- krysa rodu rattus MeSH
- morfin škodlivé účinky MeSH
- mozeček účinky léků metabolismus MeSH
- mozková kůra účinky léků metabolismus MeSH
- poruchy spojené s užíváním opiátů genetika metabolismus MeSH
- potkani Wistar MeSH
- proteomika metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: The functional activity of trimeric guanine-nucleotide-binding proteins (G-proteins) represents an essential step in linking and regulation of the opioid receptor (mu-, delta- and kappa-OR)-initiated signaling pathways. Theoretical basis and/or molecular mechanism(s) of opioid tolerance and addiction proceeding in the central nervous system were not studied in the forebrain cortex of mammals with respect to quantitative analysis of opioid-stimulated trimeric G-protein activity. MATERIAL/METHODS: G-protein activity was measured in PercollR-purified plasma membranes (PM) isolated from the frontal brain cortex of control and morphine-treated rats by both high-affinity [32P]GTPase and [35S]GTPgammaS binding assays. Exposition to morphine was performed by intra-muscular application of this drug. Control animals were injected with sterile PBS. RESULTS: Both mu-OR (DAMGO)- and delta-OR (DADLE)-responses were clearly desensitized in PM isolated from morphine-treated rats; kappa-OR (U-69593)- and baclofen (GABAB-R)-stimulated [35S]GTPgammaS binding was unchanged, indicating the specificity of the morphine effect. Under such conditions, the amount of G-protein alpha subunits was unchanged. The order of efficacy DADLE>DAMGO>U-69593 was the same in control and morphine-treated PM. Behavioral tests indicated that morphine-treated animals were fully drug-dependent and developed tolerance to subsequent drug addition. CONCLUSIONS: Prolonged exposure of rats to high doses of morphine results in decrease of the over-all output of OR-stimulated G-protein activity in the forebrain cortex but does not decrease the amount of these regulatory proteins. These data support the view that the mechanism of the long-term adaptation to high doses of morphine is primarily based on desensitization of OR-response preferentially oriented to mu-OR and delta-OR.
- MeSH
- abstinenční syndrom patofyziologie MeSH
- baklofen farmakologie MeSH
- buněčná membrána účinky léků enzymologie MeSH
- časové faktory MeSH
- chování zvířat účinky léků MeSH
- enkefalin, Ala(2)-MePhe(4)-Gly(5)- farmakologie MeSH
- fyziologická adaptace účinky léků MeSH
- guanosin 5'-O-(3-thiotrifosfát) metabolismus MeSH
- guanosindifosfát metabolismus MeSH
- krysa rodu rattus MeSH
- morfin aplikace a dávkování farmakologie MeSH
- mozková kůra účinky léků metabolismus MeSH
- potkani Wistar MeSH
- proteiny vázající GTP - alfa-podjednotky metabolismus MeSH
- receptory opiátové delta agonisté metabolismus MeSH
- receptory opiátové mu agonisté metabolismus MeSH
- tolerance léku MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The knowledge about proteome changes proceeding during protracted opioid withdrawal is lacking. Therefore, the aim of this work was to analyze the spectrum of altered proteins in the rat hippocampus in comparison with the forebrain cortex after 6-month morphine withdrawal. We utilized 2D electrophoretic workflow (Pro-Q® Diamond staining and Colloidal Coomassie Blue staining) which was preceded by label-free quantification (MaxLFQ). The phosphoproteomic analysis revealed six significantly altered hippocampal (Calm1, Ywhaz, Tuba1b, Stip1, Pgk1, and Aldoa) and three cortical proteins (Tubb2a, Tuba1a, and Actb). The impact of 6-month morphine withdrawal on the changes in the proteomic profiles was higher in the hippocampus-14 proteins, only three proteins were detected in the forebrain cortex. Gene Ontology (GO) enrichment analysis of differentially expressed hippocampal proteins revealed the most enriched terms related to metabolic changes, cytoskeleton organization and response to oxidative stress. There is increasing evidence that energy metabolism plays an important role in opioid addiction. However, the way how morphine treatment and withdrawal alter energy metabolism is not fully understood. Our results indicate that the rat hippocampus is more susceptible to changes in proteome and phosphoproteome profiles induced by 6-month morphine withdrawal than is the forebrain cortex.
- Publikační typ
- časopisecké články MeSH
Opioid addiction is recognized as a chronic relapsing brain disease resulting from repeated exposure to opioid drugs. Cellular and molecular mechanisms underlying the ability of organism to return back to the physiological norm after cessation of drug supply are not fully understood. The aim of this work was to extend our previous studies of morphine-induced alteration of rat forebrain cortex protein composition to the hippocampus. Rats were exposed to morphine for 10 days and sacrificed 24 h (groups +M10 and -M10) or 20 days after the last dose of morphine (groups +M10/-M20 and -M10/-M20). The six altered proteins (≥2-fold) were identified in group (+M10) when compared with group (-M10) by two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). The number of differentially expressed proteins was increased to thirteen after 20 days of the drug withdrawal. Noticeably, the altered level of α-synuclein, β-synuclein, α-enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was also determined in both (±M10) and (±M10/-M20) samples of hippocampus. Immunoblot analysis of 2D gels by specific antibodies oriented against α/β-synucleins and GAPDH confirmed the data obtained by 2D-DIGE analysis. Label-free quantification identified nineteen differentially expressed proteins in group (+M10) when compared with group (-M10). After 20 days of morphine withdrawal (±M10/-M20), the number of altered proteins was increased to twenty. We conclude that the morphine-induced alteration of protein composition in rat hippocampus after cessation of drug supply proceeds in a different manner when compared with the forebrain cortex. In forebrain cortex, the total number of altered proteins was decreased after 20 days without morphine, whilst in hippocampus, it was increased.
- MeSH
- abstinenční syndrom patologie MeSH
- časové faktory MeSH
- hipokampus účinky léků patologie MeSH
- krysa rodu rattus MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- morfin škodlivé účinky MeSH
- mozková kůra účinky léků patologie MeSH
- opioidní analgetika škodlivé účinky MeSH
- poruchy spojené s užíváním opiátů patologie MeSH
- potkani Wistar MeSH
- proteomika MeSH
- regulace genové exprese účinky léků MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- lidé MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
