Browsing by Subject "dopamiini"

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.

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.

Kolinergiset α6*-nikotiinireseptorit ovat kiinnostavia, koska ne liittyvät mahdollisesti Parkinsonin tautiin ja nikotiiniriippuvuuteen. Ionikanavina toimivat nikotiinireseptorit ovat muodostuneet viidestä alayksiköstä, jotka esiintyvät erilaisina yhdistelminä. α6-alayksikköä sisältävät nikotiinireseptorit sijaitsevat presynaptisesti ja säätelevät dopamiinin vapautumista dopaminergisessä hermopäätteessä. α6*-nikotiinireseptorit ovat keskittyneet vain tietyille aivoalueille ja niitä esiintyy runsaasti dopaminergisissä hermosoluissa. α6*-nikotiinireseptoreita on erityisesti mesolimbisen ja nigrostriataalisen hermoradan dopaminergisissä hermosoluissa. Lisäksi niitä on paljon näkemiseen liittyvillä aivoalueilla. Nikotiini toimii asetyylikoliinin tavoin aktivoimalla α6*-nikotiinireseptoreita, mikä johtaa dopamiinin vapautumiseen hermopäätteessä. α6*-nikotiinireseptoreiden sijainnin, määrän ja toiminnan tutkimisessa on käytetty apuna muun muassa niille selektiivisiä antagonisteja, saalistavista merietanoista peräisin olevia α-konotoksiineja, erityisesti α-konotoksiini MII:ta. Nigrostriataalisella hermoradalla, joka ulottuu substantia nigrasta striatumiin, α6*-nikotiinireseptorit voivat vaikuttaa liikkeen säätelyyn. Nikotiini vapauttaa dopamiinia nigrostriataalisen hermoradan päätepisteessä, striatumissa, mikä voi lisätä liikeaktiivisuutta. Nikotiinilla on havaittu olevan hyödyllisiä vaikutuksia Parkinsonin taudin eläinmalleissa, mutta Parkinsonin tautipotilailla nikotiinihoidosta saadut tutkimustulokset ovat ristiriitaisia ja puutteellisia. α6*-nikotiinireseptoreille voitaisiin kehittää selektiivisiä agonisteja, joiden avulla lääkehoito voitaisiin kohdentaa paremmin ja vältyttäisiin mahdollisilta haittavaikutuksilta. Tupakanvieroitukseen tarvittaisiin lisää uusia selektiivisiä lääkehoitoja, joilla olisi hyvä hoitomyöntyvyys ja mahdollisimman vähän haittavaikutuksia. Mesolimbinen hermorata, joka ulottuu ventraaliselta tegmentaalialueelta nucleus accumbensiin, liittyy riippuvuuden syntyyn. Nikotiinin vaikutukset välittyvät VTA:n kautta nucleus accumbensiin, jossa vapautuu dopamiinia. Osa nikotiinin vaikutuksista välittyy myös presynaptisten α6*-nikotiinireseptorien kautta. Selektiivisistä α6*-nikotiinireseptoreiden antagonisteista voisi olla hyötyä nikotiiniriippuvuuden hoidossa, sillä niiden vaikutus vastaisi osittaisagonistin vaikutusta. α-konotoksiini PIA:n vaikutuksia nikotiinin aiheuttamaan dopamiinin vapautumiseen tutkittiin in vivo mikrodialyysimenetelmällä. α-konotoksiini PIA saalistavasta merietanasta eristetty selektiivinen α6*-nikotiinireseptoreiden antagonisti. Tutkimuksessa käytettiin vapaana liikkuvia urospuoleisia Wistar-rottia. Tutkimuksen kohteena olevat aivoalueet olivat striatum ja nucleus accumbens. Rotille asennettiin anestesiassa ohjauskanyyli joko striatumiin tai nucleus accum-bensiin stereotaktisen laitteen avulla. Mikrodialyysikokeessa koetinten tasapainotuksen jälkeen kerättiin perustason näytteet ja pistettiin saliini tai nomifensiini tai vaihdettiin Ringer-ruiskun tilalle α-konotoksiini PIA-ruisku. Puolen tunnin päästä pistettiin saliini tai nikotiini ja vaihdettiin Ringer-ruisku takaisin. Näytteitä kerättiin 15 minuutin välein yhteensä 5,5 tuntia. Lopuksi aivot otettiin talteen ja niistä tehtyjen aivoleikkeiden avulla tarkastettiin koetinten paikat. Mikrodialyysinäytteistä määritettiin HPLC-menetelmällä dopamiinin ja sen metaboliittien DO-PAC:n ja homovaniliinihapon sekä koejärjestelyssä oletettavasti muuttumattomana pysyvän 5-HIAA:n pitoisuudet. Koejärjestelyssä päätettiin käyttää tutkittavien aivoalueiden dopamiinipitoisuuden nostamiseen nomifensiinia, joka estää dopamiinin takaisinottoa hermopäätteissä. Käsittelyryhminä olivat saliini-saliini (n=striatum ja nucleus accumbens, 8+7), saliini-nomifensiini (n=8+4), saliini-nikotiini (n=3+4), nomifensiini-nikotiini (n=10+13) ja nomifensiini-nikotiini-α-konotoksiini PIA (n=8+5). Rottia jouduttiin hylkäämään eri syistä joko ennen mikrodialyysia, mikrodialyysin aikana tai sen jälkeen. Rottia hylättiin yhteensä 70 kpl. Tilastollisessa analyysissä tutkittujen käsittelyiden tai aivoalueiden välille ei saatu merkitseviä eroja, koska eläinten välinen hajonta oli liian suurta. Silmämääräisesti nomifensiini-nikotiinikäsittely nosti striatumin ja nucleus accumbensin dopamiinipitoisuuksia. α-konotoksiini PIA näytti estävän dopamiinin vapautumista striatumissa ja nucleus accumbensissa, mutta erot nomifensiini-nikotiinikäsittelyyn eivät olleet tilastollisesti merkitseviä. AUC-arvolla mitattuna α-konotoksiini PIA esti dopamiinin vapautumista striatumissa 39,6 % ja nucleus accumbensissa 31,3 %. Aivoalueiden välillä ei ollut tilastollisesti merkitseviä eroja.

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.