Browsing by Subject "microdialysis"
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(2013)Literature review: The plasma membrane DA transporter (DAT) belongs to the family of Na+/ClÙÄÉ≠ dependent neurotransmitter transporters. DAT is the primary mechanism for clearance of dopamine from the extracellular space and transporting it back to the presynaptic nerve terminals. There's a great interest in the DAT and its regulation as its substrate, dopamine, mediates a wide array of physiological functions e.g. locomotor activity, cognition and the control of motivated behaviors. With selective transport DAT limits the intensity and the duration of dopaminergic signal. Its function is regulated by several kinases, phosphatase and protein-protein interactions. The altered expression of DAT may be related to several neurological diseases such as Parkinson's disease, addiction and ADHD. To study DAT's function, several genetically modified mouse lines including DAT knockout mice, DAT knockdown mice and DAT knock in mice with elevated DAT levels have been generated. Experimental part: Glial cell line-derived neurotrophic factor (GDNF) plays important role in the survival and function of dopaminergic neurons, learning, memory and synaptic plasticity. More recently, several studies have shown that GDNF can also negatively regulate the actions of abused drugs. The aim of this study was to investigate GDNF's role and mechanism of action in plasticity and function of the dopaminergic neurons projecting to striatum. For that purpose, we used in vivo microdialysis in freely moving mice. We chose two different mouse lines: MEN2B mice with constitutive active Ret-signaling and elevated striatal dopamine concentrations, and GDND-cKO mice that lack GDND in the central nervous system. Microdialysis guide cannula was implanted in the dorsal striatum in the stereotaxic surgery and the mice were allowed to recover for 5-7 days. The concentrations of dopamine and its metabolites DOPAC and HVA and also 5-HIAA were determined from the samples by highperformance liquid chromatography. Microdialysis was performed twice for every mouse on days 1 and 4. Between microdialysis days, the mice were given amphetamine 1 mg/kg i.p. on days 2 and 3. In the microdialysis experiment, the mice received amphetamine stimulation (100 µM/60 min) via microdialysis probe. The placements of microdialysis probes were verified from fixed brain sections after the experiments. Amphetamine increased the dopamine output in both mouse lines, but there were no statistically significant differences in striatal dopamine concentrations between genotypes neither after acute nor chronic administration. However, there was a difference between the dopamine outputs in days 1 and 4 in both MEN2B and GDNF-cKO mice: The striatal dopamine concentrations were significantly lower on the second microdialysis day. This may be a sing from tolerance to the drug. However, without more research, it is not possible, by this experiment, to draw direct conclusions of GDNF's role in addiction and in plasticity in striatum. It is possible that the differences between genotypes are too small to be seen with microdialysis. Development of compensatory mechanisms in mice cannot be ruled out either. Effects may also vary between different brain areas.
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(2012)Review of the literature: The purpose of the review is to go through what is known about mechanisms of actions of different neurotrophic factors (GDNF, neurturin, CDNF and MANF) and how they are transported within the brain. Neurotrophic factors are endogenous and secreted proteins which have a pivotal role in the development and maintenance of neurons. They support the survival of neurons and they can help them to recover from different injuries. Due to these functions neurotrophic factors might be beneficial for the treatment of neurodegenerative disorders like Parkinson's disease. There are a great deal of studies that clearly show the neuroprotective and neurorestrorative function of GDNF and neurturin on dopaminergic neurons. They are also studied in clinical studies with Parkinson's patients but the results have been partly contradictory. The signalling route of GDNF and neurturin via RET tyrosinekinasereceptor is fairly well known but the other mechanisms of action of these factors needs to be studied further. CDNF and MANF constitute a novel, evolutionarily conserved family of neurotrophic factors. They are shown to have neuroprotective and neurorestrorative actions on dopaminergic neurons both in vitro and in vivo in a rodent model of Parkinson's disease. The mechanisms of action of CDNF and MANF are not quite clear at the moment. There are two different domains in their structure both of which are likely to carry different functions. The N-terminal domains of these proteins are close to saposins, lipid and membrane binding proteins, some of which are shown to have neurotrophic and anti-apoptotic effects. The C-terminal domain of MANF, in turn, is structurally close to the SAP-domain of Ku70-protein which binds Bax in the cytoplasm and thus inhibits apoptosis mediated by Bax. CDNF and MANF might protect neurons both via intracellular mechanisms and extracellularly acting like a secreted neurotrophic factor. CDNF and GDNF are transported retrogradially from striatum to substantia nigra. MANF, unlike the others, is transported from striatum to the frontal cortex. MANF and CDNF are shown to have better diffusion properties in the brain parenchyma than GDNF. Experimental part: We studied, by means of microdialysis, the effects of CDNF, MANF and GDNF on the dopaminergic neurotransmission of naive rats within the striatum. Neurotrophic factors (10 µg) and PBS as a negative control were injected into the left striatum in stereotaxic surgery. After this rats recovered one week before the first mircodialysis. The second mircodialysis was performed three weeks after the surgery. The samples were collected from the left striatum of freely moving rats. During the microdialysis neurotransmission was stimulated by replacing the perfusion solution with hypertonic potassium solution and with amphetamine solution. The concentration of dopamine, DOPAC, HVA and 5-HIAA was measured from the dialysate samples. In vivo TH-activity experiment was carried out for three rats in each group. NSD1015 was injected i.p.after which rats were decapitated and their striatums were dissected. The concentration of L-DOPA, dopamine and metabolites on the treated and untreated hemisphere were analyzed from the tissue samples. The amount of L-DOPA in the striatum after NSD1015-treatment indicates how active TH-enzyme is. There were no significant differences in the concentrations of dopamine and metabolites during the baseline. MANF and CDNF increased the release of dopamine from the nerve terminals compared to GDNF and PBS one week after the surgery. Three weeks after the surgery there was still significant increase in the release of dopamine in MANF group compared to GDNF group. Also the dopamine-DOPAC-turnover was increased significantly in MANF group compared to GDNF and PBS groups one week after the surgery. DOPAC/HVA -ratio was significantly smaller in GDNF group than in other groups one week after the surgery. These findings suggest that MANF potentiates dopaminergic neurotransmission most drasticly. The effects of MANF seem to last longer time than the effects of other neurotrophic factors. CDNF seems to increase the release of dopamine from the nerve terminals as well. The potentiation of dopaminergic neurotransmission could be due to increased biosynthesis of dopamine or due to the potentiation of the function of nerve terminals. In the results of the TH-activity experiment there was a trend according to which L-DOPA is synthesized less after the neurotrophic factor treatment that after the PBS treatment. This suggests that neurotrophic factors might decrease the activity of TH-enzyme.
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(2010)Part I: Parkinson's disease is a slowly progressive neurodegenerative disorder in which particularly the dopaminergic neurons of the substantia nigra pars compacta degenerate and die. Current conventional treatment is based on restraining symptoms but it has no effect on the progression of the disease. Gene therapy research has focused on the possibility of restoring the lost brain function by at least two means: substitution of critical enzymes needed for the synthesis of dopamine and slowing down the progression of the disease by supporting the functions of the remaining nigral dopaminergic neurons by neurotrophic factors. The striatal levels of enzymes such as tyrosine hydroxylase, dopadecarboxylase and GTP-CH1 are decreased as the disease progresses. By replacing one or all of the enzymes, dopamine levels in the striatum may be restored to normal and behavioral impairments caused by the disease may be ameliorated especially in the later stages of the disease. The neurotrophic factors glial cell derived neurotrophic factor (GDNF) and neurturin have shown to protect and restore functions of dopaminergic cell somas and terminals as well as improve behavior in animal lesion models. This therapy may be best suited at the early stages of the disease when there are more dopaminergic neurons for neurotrophic factors to reach. Viral vector-mediated gene transfer provides a tool to deliver proteins with complex structures into specific brain locations and provides long-term protein over-expression. Part II: The aim of our study was to investigate the effects of two orally dosed COMT inhibitors entacapone (10 and 30 mg/kg) and tolcapone (10 and 30 mg/kg) with a subsequent administration of a peripheral dopadecarboxylase inhibitor carbidopa (30 mg/kg) and L- dopa (30 mg/kg) on dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens of freely moving rats using dual-probe in vivo microdialysis. Earlier similarly designed studies have only been conducted in the dorsal striatum. We also confirmed the result of earlier ex vivo studies regarding the effects of intraperitoneally dosed tolcapone (30 mg/kg) and entacapone (30 mg/kg) on striatal and hepatic COMT activity. The results obtained from the dorsal striatum were generally in line with earlier studies, where tolcapone tended to increase dopamine and DOPAC levels and decrease HVA levels. Entacapone tended to keep striatal dopamine and HVA levels elevated longer than in controls and also tended to elevate the levels of DOPAC. Surprisingly in the nucleus accumbens, dopamine levels after either dose of entacapone or tolcapone were not elevated. Accumbal DOPAC levels, especially in the tolcapone 30 mg/kg group, were elevated nearly to the same extent as measured in the dorsal striatum. Entacapone 10 mg/kg elevated accumbal HVA levels more than the dose of 30 mg/kg and the effect was more pronounced in the nucleus accumbens than in the dorsal striatum. This suggests that entacapone 30 mg/kg has minor central effects. Also our ex vivo study results obtained from the dorsal striatum suggest that entacapone 30 mg/kg has minor and transient central effects, even though central HVA levels were not suppressed below those of the control group in either brain area in the microdialysis study. Both entacapone and tolcapone suppressed hepatic COMT activity more than striatal COMT activity. Tolcapone was more effective than entacapone in the dorsal striatum. The differences between dopamine and its metabolite levels in the dorsal striatum and nucleus accumbens may be due to different properties of the two brain areas.
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(2010)The blood-brain barrier protects brain from xenobiotics that are in blood. Different in vivo and in vitro methods have been developed for studying blood brain barrier and those can be found in the literature. There are only few computational models pharmacokinetics of compounds in the brain. In this study permeability factors, which were measured in vitro or in vivo, were collected from literature. Additionally two different pharmacokinetic computer models of blood-brain barrier were described. One of which is called microdialysis model and the other efflux model. Microdialysis model is a very simple two compartmental model, the compartments being the blood and the brain. Five substances were simulated according to the values measured in vivo in rat. The model did not correlate well with the in vivo results, because of the simplicity of the model as the model missed the compartment of brain tissue and the kinetics of transporters. Efflux model has three compartments, blood, blood brain barrier endothelial cells and brain. The model was used to study the impact of the of efflux transporter at the luminal barrier of endothelial cells and passive permeability to the steady-state concentration of a compound in the brain extracellular fluid with theoretical simulations. The relation between free drug concentrations in blood and brain extracellular fluid (Kp,uu) was studied. The impact of Michaelis-Menten kinetics of efflux transporter to the concentration of compound was shown in the results. The efflux model is suitable for theoretical simulations. It is possible to add new active transporters. With theoretical simulations the results from in vitro and in vivo studies can be combined and the different factors can be studied in one simulation.
Now showing items 1-4 of 4