Browsing by Subject "GABA"
Now showing items 1-3 of 3
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(2012)Inhibitory GABAergic neurotransmission seems to play a central role in the effects of ethanol on the central nervous system. However, the exact mechanism of ethanol action as well as the role of the GABAA subunits in this mechanism remains unclear. The imidazobenzodiazepine Ro 15-4513 acts as a partial inverse agonist of the GABAA receptors by binding to their benzodiazepine sites which contain a γ2 subunit. In addition, ethanol and Ro 15-4513 seem to bind in a competitive manner and with high affinity to δ subunit-containing extrasynaptic GABAA receptors that mediate tonic inhibition. There exists conflicting evidence about the role of the δ subunit in the mechanism of antialcohol effects of Ro 15-4513. Clinical evidence of the efficacy of γ-hydroxybutyric acid (GHB) in suppressing alcohol withdrawal syndrome has been shown, even though only little preclinical research has been done on this subject. GHB has agonistic effect on the GABAB receptors and on the putative GHB receptors. GHB seems to share a very similar pharmacological profile with ethanol and there is also some proof of their synergistic effects. However, the exact mechanism of ethanol consumption suppressing action of GHB is not exactly known. The aim of this study was to determine the role of the γ2 and δ subunits in the effects of Ro 15-4513 (0, 3 mg/kg) on voluntary ethanol drinking and on the motor coordination suppression by ethanol (1.5 g/kg). In addition the effects of nonselective benzodiazepine flurazepam (0, 6 mg/kg) and GHB (0, 100, 150 mg/kg) on ethanol drinking and the effects of GHB on motor coordination were examined. The rotarod method (∅ 6 cm, fixed speed 6 r.p.m.) was chosen to determine the motor coordination. The drinking-in-the-dark (DID) method was applied to study the drinking effects. In this method the water bottle in the home cage of each mouse was replaced with an ethanol dilution (20 % v/v) for a certain time in the beginning of the dark phase of the light/dark cycle. A knock in mouse line γ2I77-lox with a point mutation in the γ2 subunit gene was used in the experiments. The mutation decreases the affinity of the receptor for certain benzodiazepine structures like that of Ro 15-4513 in the brain. The C57BL/6J mouse line was used as control. Both Ro 15-4513 (3 mg/kg) and GHB (150 mg/kg) significantly reduced ethanol drinking. The GHB dose of 100 mg/kg failed to reach significance probably due to the relatively long drinking time (1 h) used in the experiment in comparison to the short half-life of this drug. Flurazepam (6 mg/kg) significantly enhanced ethanol drinking which as expected was not affected by the mutation of the γ2I77-lox mouse line. Ro 15-4513 (3 mg/kg) failed to reduce the ethanol-induced suppression of motor coordination probably due to a too low dose. The GHB rotarod experiments suggest that the GHB (150 mg/kg) ethanol drinking suppressing effect may have been partly caused by its sedative effects. There was no significant difference between the used mouse lines in the effects of Ro 15-4513. This would suggest that the γ2 subunit does not play a significant role in the effects of Ro 15-4513. However, in order to draw a final conclusion more experiments must be done with the γ2I77-lox as well as with the δ subunit knockout mouse line, which we were unfortunately not able to include in this study as originally planned.
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(2016)Prolyl oligopeptidase (PREP) is a serine protease which is extensively present in the mammalian system and especially abundant in the brain. Despite the long research history of PREP its physiological function has remained unclear. PREP has been suggested to regulate the functions of many bioactive peptides by hydrolysis and on the other hand to participate in several intracellular processes probably via direct protein-protein interactions. One of the functions suggested for PREP is the regulation of the brain neurotransmitter systems and based on, for instance, the location in the brain PREP has been connected to both excitatory and inhibitory neurotransmitter systems. The literature review of this thesis first describe the brain neurotransmitter systems associated to PREP in general with some examples of diseases related to their malfunctions. In addition the structure of PREP and its location in the brain, both subcellular and cellular levels, and in distinct neurotransmitter systems, are presented, after which the different proposed functions for PREP are reviewed. The aim of the experimental part of this thesis was to investigate the effects of PREP on the brain neurotransmitter concentrations in the mouse nigrostriatal pathway and also to mouse motor behavior. The main research methods were microdialysis, tissue assays and cylinder test. The study was composed of two sections with five week duration each. The first section was performed with wild-type mice expressing naturally PREP and the second section with PREP-knockout (ko) mice and their wild-type littermates. The mice were injected unilaterally above the substantia nigra with adeno associated (AAV1) hPREP viral vector or with AAV1-eGFP (green fluorescent protein) viral vector as a control treatment. The cylinder test was carried out before the injection, and two and four weeks afterwards. Microdialysis was used to study the actions of PREP on the extracellular concentrations of dopamine (DA) and its metabolites dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), gamma-aminobutyric acid (GABA) and 5-hydroxyindoleacetic acid (5-HIAA), the major metabolite of serotonin (5-HT). In addition to the baseline assay the concentrations were measured after two amphetamine treatments (10 and 30 µM) administered via the microdialysis probe. The probe guide cannulas were inserted to mice striatums 1-2 weeks before the microdialysis measurement. In the end of the experiment the tissue concentrations of DA, DOPAC, HVA, 5-HT and 5-HIAA were measured from striatum and substantia nigra. Both the microdialysis and tissue sample concentrations were quantified with high performance liquid chromatography. In the first study section the PREP enzyme activity was also determined from striatum. Neither the complete deprivation nor over-expression of PREP in the nigrostriatal pathway had clear or consistent effects on the levels of neurotransmitters studied when compared to naturally occurring PREP expression. When comparing the differences between control treated groups of PREP-ko and littermate mice, a greater amphetamine stimulated DA-levels was seen in the former group proposing negative regulatory influence of PREP. In both study sections the tissue assay results were difficult to interpret due to observed responses also with AAV1-eGFP control treatment in comparison with untreated side of the brain. This was seen as a lower DA- and DOPAC-levels in substantia nigra and thus the meaning of the changes caused by PREP treatment is hard to comprehend. The results of the cylinder test may implicate some protective effect of the PREP-ko-genotype against viral vector injections in general. Then again the existence of compensatory mechanisms is possible when using knockout animals and thus the genotype differences are hardly ever unequivocal. The results of this thesis do not suggest outright regulatory effects of PREP on the neurotransmitter functions in the mouse nigrostriatal pathway although the confirmation of this requires further studies, especially in regard to GABAergic and glutamatergic systems. Studies should include a scale of different behavioral tests of motor activity and repeated microdialysis experiment with some defining method changes. The possible function and mechanisms of PREP as a regulator of neurotransmitter intake or release is rationale to study at molecular level with applicable methods.
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(2014)Parkinson's disease causes changes in the basal ganglia GABAergic neurotransmission in addition to the well-known dopaminergic changes. These GABAergic modulations may cause somed of the symptoms not responding well to the standard dopaminergic medication. Neurotrophic factors are a group of endogenous proteins showing promise as a future treatment for Parkinson's disease. They are known to have neuroprotective and neurorestorative effects on the dopaminergic cells. Their effects to the GABAergic cells are still mostly unknown. Intrastriatal injection of GDNF to rats caused significantly slower weight gain compared to CDNF, MANF one week after stereotaxic operation (p=0,002 for CDNF vs. GDNF and p<0,001 for MANF vs. GDNF). Difference to the vehicle (phosphate buffered saline) used as a negative control was not statistically significant (p=0,055). Three weeks after the operation the differences between the treatment groups were no longer statistically significant. Because of problems with the separation in analysis, microdialysis samples remain still to be analysed. To help the analysis of GABA in the future we determined the analytical parameters of the analytical apparatus. We also defined differences in probe permeability between 1 mm and 2 mm probes and between old and new batches. GABA analysis was performed with a HPLC-fluorometric detection of o-phtaldialdehyde-derived GABA. Detection limit for old apparatus was 7,2 nM and for new apparatus 6,2 nM in a sample of 15 µl (0,11 pmol and 93 fmol respectively). Quantification limits defined were 22 nM and 19 nM (0,33 pmol and 0,28 pmol) for the old and the new apparatus, respectively. Upper limit of quantification was estimated to be 246 nM (3,7 pmol). Probes had significant differences in permeability between 1 mm and 2 mm probes, as well as between batches. The variance of permeability of 1 mm probes was estimated to be approximately twofold compared to the 2 mm probes. Furthermore the permeability of 1 mm probes varied between batches significantly. An average of permeability of the old batch was 34 % lower than that of a new batch (p<0,001).
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