Asetyylikoliinin nikotiinireseptorit välittäjäaineiden vapautumisen säätelijöinä

Nicotinic acetylcholine receptors are ion channel receptors that consist of five subunits and have an important role in modulating neurotransmitter release in the central nervous system. The literature review part of this thesis presents an overview of the structure, function and diverse subunit composition of nicotinic receptors and reviews the scientific literature on their function as modulators of neurotransmitter release. Relevant literature on the role of the nicotinic receptors of the striatum, the hippocampus and the prefrontal cortex in the modulation of the release of dopamine, glutamate, GABA, acetylcholine, noradrenalin and serotonin is reviewed. Finally, a summary for each of the brain areas and some conclusions are presented. The experimental part of this thesis consists of a series of experiments, where the ability of morphine to activate the presynaptic nicotinic receptors modulating dopamine release in the mouse striatum was investigated based on opioid-nicotine-interactions reported earlier. The possible effect of morphine was studied by measuring the release of radiolabeled dopamine from perfused synaptosomes prepared from mouse striatum. In addition, the effect of nicotine was studied to confirm the correct functioning of the method and to obtain data for comparison with the morphine results. Both nicotine and morphine elicited the release of [3H]dopamine from striatal synaptosomes. The release of [3H]dopamine elicited by morphine was blocked by nicotinic antagonists, suggesting that the effect of morphine was mediated by nicotinic receptors. Use of the selective antagonist α-conotoxin MII revealed that the effect of morphine, similar to nicotine, was mediated in part by α6β2* receptors and in part by other receptors, possibly α4β2*. In addition, the opioid antagonist naloxone blocked the effects of both nicotine and morphine, likely via direct antagonism of nicotinic receptors. However, the concentrations of morphine and naloxone needed for affecting [3H]dopamine release were very high, which suggests that the clinical relevance of the effects described here is likely to be small. The involvement of opioid receptors was deemed to be unlikely but, along with possible non-specific effects by high concentrations, could not be completely ruled out.Asetyylikoliinin nikotiinireseptorit ovat viidestä alayksiköstä muodostuvia ionikanava-reseptoreita, joiden pääasiallinen tehtävä keskushermostossa on välittäjäaineiden vapautumisen säätely. Tämän tutkielman kirjallisuuskatsauksessa esitetään yleiskuva nikotiinireseptorien rakenteesta, toiminnasta ja vaihtelevasta alayksikkökoostumuksesta sekä tarkastellaan tutkimuskirjallisuutta, joka koskee nikotiinireseptoreita välittäjäaineiden vapautumisen säätelijöinä. Katsauksessa käydään läpi oleellisin kirjallisuus striatumin, hippokampuksen ja prefrontaalisen aivokuoren nikotiinireseptorien osuudesta dopamiinin, glutamaatin, GABA:n, asetyylikoliinin, noradrenaliinin ja serotoniinin vapautumisen säätelyssä. Katsauksen lopuksi esitetään yhteenveto kunkin aivoalueen osalta ja joitakin tutkimustiedon perusteella tehtyjä johtopäätöksiä. Tutkielman kokeellinen osa käsittää koesarjan, jonka tarkoituksena oli aiemmin havaittujen opioidi-nikotiini-interaktioiden perusteella tutkia morfiinin kykyä aktivoida hiiren striatumin dopaminergisten hermopäätteiden presynaptisia dopamiinin vapautumista sääteleviä nikotiini-reseptoreita. Tämän selvittämiseen pyrittiin mittaamalla radioleimatun dopamiinin vapautumista hiiren striatumista valmistetuista perfusoiduista synaptosomeista. Morfiinin lisäksi tutkittiin nikotiinin vaikutusta menetelmän toiminnan varmistamiseksi ja vertailukohdan saamiseksi. Sekä nikotiinin että morfiinin todettiin lisäävän [3H]dopamiinin vapautumista striataalisista synaptosomeista. Nikotiiniantagonistit estivät morfiinin aiheuttamaa [3H]dopamiinin vapautumista viitaten vaikutuksen välittymiseen nikotiinireseptorien kautta. Selektiivistä antagonistia α-konotoksiini MII:a hyödyntäen todettiin morfiinin vaikutuksen välittyneen nikotiinin tapaan osittain α6β2*-tyypin reseptorien kautta ja osittain muiden, mahdollisesti α4β2*-tyypin reseptorien kautta. Lisäksi havaittiin opioidiantagonisti naloksonin estävän sekä nikotiinin että morfiinin vaikutuksen todennäköisesti nikotiinireseptoreita salpaamalla. [3H]dopamiinin vapautumiseen vaikuttavien morfiini- ja naloksonipitoisuuksien todettiin kuitenkin olevan hyvin korkeita. Yhdisteiden mahdollisten presynaptisten nikotiinireseptorien kautta välittyvien striataalista dopamiinin vapautumista säätelevien vaikutusten kliininen merkittävyys lienee näin ollen vähäinen. Vaikutusten välittymisen opioidireseptorien kautta todettiin olevan epätodennäköistä, mutta tätä tai korkeiden pitoisuuksien aiheuttamia epäspesifisiä vaikutuksia ei voitu täysin sulkea pois

Nicotinic acetylcholine receptors in experimental models of Parkinson’s disease and levodopa-induced dyskinesia : focus on α5 subunit-containing receptors

No cure exists for Parkinson’s disease (PD), a disease marked by the degeneration of dopaminergic neurons of the substantia nigra pars compacta (SNC), a loss of dopamine in the dorsal striatum, and resulting motor symptoms. Furthermore, treatment of PD with levodopa is often complicated by abnormal involuntary movements (levodopa-induced dyskinesia, LID). Novel treatment options for PD and LID are thus greatly needed. Nicotinic acetylcholine receptors represent one possible novel treatment target, given the complex control they exert over dopaminergic neurotransmission, protective effects of smoking against PD, and extensive preclinical evidence of neuroprotective and antidyskinetic effects by nicotinic receptor ligands. Nicotinic receptor subtypes essential for nigrostriatal dopaminergic neurotransmission include those containing the α5 subunit, which have not been previously studied in the context of PD. In this thesis, further preclinical investigations of the role of nicotinic receptors in PD and LID were carried out. An extensive in vivo and ex vivo characterization of the role of α5-containing receptors in mouse models of PD was performed. The effects on LID by chronic nicotine treatment in drinking water and other drug treatments were studied in vivo utilizing mouse models of both moderate and severe PD and LID. The mechanisms of action underlying LID and the antidyskinetic effects of nicotine were studied by ex vivo measurements of striatal dopamine release and corticostriatal brain-derived neurotrophic factor (BDNF). In parallel, methods for stereotactic surgery and postoperative care were significantly improved. Mice lacking α5-containing nicotinic receptors were found to be less susceptible to unilateral nigrostriatal neurodegeneration, the resulting interhemispheric motor imbalance, and LID. Striatal dopamine uptake measurements suggested reduced dopamine transporter function as a possible mechanism of neuroprotection. Nicotine was found to inhibit LID, with findings suggesting a role for α6β2* nicotinic receptors. However, neither nicotinic receptor agonists nor the clinically used drug amantadine alleviated severe LID associated with near-total dopaminergic denervation. The findings also confirmed a correlation between striatal BDNF and LID. The present findings suggest the potential usability of α5-containing nicotinic receptors as a drug target against PD and LID. The findings also confirm the preclinical potential of nicotine as an antidyskinetic drug while suggesting limited efficacy in advanced PD. In addition, the findings expand previous knowledge on the possible mechanisms of LID and the antidyskinetic effects of nicotine.Parkinsonin taudin motoriset oireet aiheutuvat mustatumakkeen dopamiinihermosolujen rappeutumisesta ja dopamiinin puutoksesta aivojuoviossa. Sairaudelle ei ole löydetty parantavaa hoitoa. Lisäksi tahdottomat liikkeet (dyskinesia) vaikeuttavat usein sairauden hoitoa levodopa-lääkeaineella. Uusia mahdollisuuksia Parkinsonin taudin ja dyskinesian hoitoon siis tarvitaan. Asetyylikoliinin nikotiinireseptorit voisivat tarjota yhden mahdollisen uuden lääkevaikutuskohteen, sillä ne säätelevät aivojen dopamiinijärjestelmää, tupakoinnin tiedetään suojaavan Parkinsonin taudilta, ja laaja prekliininen näyttö viittaa nikotiinireseptoriligandeilla olevan hermosoluja suojaavia ja dyskinesiaa lieventäviä vaikutuksia. Dopaminergisen hermovälityksen kannalta oleellisiin nikotiinireseptoriala-tyyppeihin kuuluvat α5-alayksikön sisältävät reseptorit, joiden yhteyttä Parkinsonin tautiin ei aiemmin ole tutkittu. Tässä väitöskirjassa tutkittiin nikotiinireseptorien merkitystä Parkinsonin taudissa ja levodopan aiheuttamassa dyskinesiassa. α5-alayksikön sisältävien nikotiinireseptorien merkitystä Parkinsonin taudin ja dyskinesian hiirimalleissa tutkittiin kattavin in vivo- ja ex vivo-kokein. Juomaveden kautta annetun pitkäkestoisen nikotiinikäsittelyn ja muiden lääkehoitojen vaikutuksia tutkittiin sekä kohtalaisen että pitkälle edenneen Parkinsonin taudin ja dyskinesian hiirimalleissa. Dyskinesian ja nikotiinin dyskinesiaa lievittävien vaikutusten mekanismeja tutkittiin mittaamalla ex vivo dopamiinin vapautumista aivojuoviosta sekä aivoperäisen hermokasvutekijän (BDNF) pitoisuuksia aivoissa. Samanaikaisesti stereotaktisten leikkausten ja leikkauksen jälkeisen hoidon menetelmiä kehitettiin merkittävästi. α5-poistogeenisten hiirten havaittiin olevan vähemmän herkkiä dopamiinihermojen toispuoleiselle tuhoutumiselle, siitä johtuvalle aivopuoliskojen motoriselle epätasapainolle sekä levodopan aiheuttamalle dyskinesialle. Dopamiinin soluunottoa aivojuoviossa mitanneiden kokeiden perusteella dopamiinin kuljettajaproteiinin heikentynyt toiminta saattoi olla yksi hermosoluja suojaava mekanismi. Nikotiinin todettiin lievittävän levodopan aiheuttamaa dyskinesiaa, ja α6β2* nikotiinireseptorit voivat olla tärkeitä tämän vaikutuksen välittäjiä. Nikotiinireseptorien agonistit tai kliinisesti käytetty lääkeaine amantadiini eivät kuitenkaan lievittäneet vakavaa, lähes täydelliseen dopamiinihermokatoon liittyvää dyskinesiaa. Tutkimuslöydökset myös vahvistivat yhteyden BDNF:n ja levodopan aiheuttaman dyskinesian välillä. Tutkimuksen tulokset viittaavat siihen, että α5-alayksikön sisältävät nikotiinireseptorit voisivat olla mahdollinen uusi vaikutuskohde Parkinsonin taudin hoidossa. Löydökset myös vahvistavat nikotiinin olevan mahdollinen levodopan aiheuttaman dyskinesian hoitomuoto, mutta viittaavat siihen, että hoitovaste pitkälle edenneessä Parkinsonin taudissa voi olla heikko. Lisäksi tutkimus laajentaa aiempaa tietämystä dyskinesian ja nikotiinin dyskinesiaa lievittävien vaikutusten mahdollisista mekanismeista

Rat subthalamic stimulation : Evaluating stimulation-induced dyskinesias, choosing stimulation currents and evaluating the anti-akinetic effect in the cylinder test

In experimental deep brain stimulation of the subthalamic nucleus (STN HFS), stimulation currents just below the appearance threshold of stimulation-induced dyskinesias has often been used. The behavioral effect of STN HFS can be measured by the reversal of forelimb use asymmetry produced by hemiparkinsonism can be measured with the cylinder test among other tests. We used 18 Wistar rats with 6-hydroxydopamine induced hemiparkinsonism to test a customized scale to rate the severity of stimulation-induced dyskinesia; we then used these ratings to choose low and high stimulation currents. Subsequent cylinder tests showed that stimulation at the higher current, inducing mild and short-lived dyskinesias, was required for robust improvement in forelimb use, contradicting the use of currents below stimulation-induced dyskinesia threshold. It was also beneficial to separately count both all touches and first touches with the cylinder wall; this provided additional sensitivity and robustness to our results. Scoring stimulation-induced dyskinesias can be used as a quantitative measure of dyskinesias and to choose stimulation currents. Cylinder test scoring separately for both first and all touches can improve both sensitivity and reliability. STN HFS at a current producing short-lived dyskinesias was required for robust improvement in forelimb use asymmetry. (C) 2019 The Authors. Published by Elsevier B.V.Peer reviewe

Implementation of improved postoperative care decreases the mortality rate of operated mice after an abundant 6-hydroxydopamine lesion of nigrostriatal dopaminergic neurons

A mouse model of Parkinson’s disease with an abundant lesion of nigrostriatal dopaminergic neurons can be achieved by stereotactic injection of 6-hydroxydopamine into the medial forebrain bundle. However, postoperative mortality can be excessively high without intensive postoperative care. Here, we show that improvements in stereotactic operations and postoperative care result in significant benefits for both animal well-being and research efficiency. Adopting a wide combination of mostly previously described improvements resulted in a decrease of postoperative mortality from 71% to 14% and an increase in successful abundant dopaminergic lesions from 46% to 81%. The techniques adopted are described in detail. In addition, we describe a simple protocol for gradual preoperative handling which can be utilized to decrease animal stress, aggressive and aversive behaviors, and to facilitate postoperative care and other subsequent handling. We propose that the implementation of these improvements greatly decreases the risk of animal suffering and that the improvements are worth adopting in any research group utilizing abundant 6-hydroxydopamine-induced dopaminergic lesions in mice. Suggestions for further improvement are also presented

Genetic lack of histamine upregulates dopamine neurotransmission and alters rotational behavior but not levodopa-induced dyskinesia in a mouse model of Parkinson’s disease

The brain histaminergic and dopaminergic systems closely interact, and some evidence also suggests significant involvement of histamine in Parkinson’s disease (PD), where dopaminergic neurons degenerate. To further investigate histamine-dopamine interactions, particularly in the context of PD, a genetic lack of histamine and a mouse model of PD and levodopa-induced dyskinesia were here combined. Dopaminergic lesions were induced in histidine decarboxylase knockout and wildtype mice by 6-hydroxydopamine injections into the medial forebrain bundle. Post-lesion motor dysfunction was studied by measuring drug-induced rotational behavior and dyskinesia. Striatal tissue from both lesioned and naïve animals was used to investigate dopaminergic, serotonergic and histaminergic biomarkers. Histamine deficiency increased amphetamine-induced rotation but did not affect levodopa-induced dyskinesia. qPCR measurements revealed increased striatal expression of D1 and D2 receptor, DARPP-32, and H3 receptor mRNA, and synaptosomal release experiments in naïve mice indicated increased dopamine release. A lack of histamine thus causes pre- and postsynaptic upregulation of striatal dopaminergic neurotransmission which may be reflected in post-lesion motor behavior. Disturbances or manipulations of the histaminergic system may thus have significant consequences for dopaminergic neurotransmission and motor behavior in both healthy and disease conditions. The findings also represent new evidence for the complex interplay between dopamine and histamine within the nigrostriatal pathway.Peer reviewe

Distinct neural responses to chord violations: A multiple source analysis study.

The human brain is constantly predicting the auditory environment by representing sequential similarities and extracting temporal regularities. It has been proposed that simple auditory regularities are extracted at lower stations of the auditory cortex and more complex ones at other brain regions, such as the prefrontal cortex. Deviations from auditory regularities elicit a family of early negative electric potentials distributed over the frontal regions of the scalp. In this study, we wished to disentangle the brain processes associated with sequential vs. hierarchical auditory regularities in a musical context by studying the event-related potentials (ERPs), the behavioral responses to violations of these regularities, and the localization of the underlying ERP generators using two different source analysis algorithms. To this aim, participants listened to musical cadences constituted by seven chords, each containing either harmonically congruous chords, harmonically incongruous chords, or harmonically congruous but mistuned chords. EEG was recorded and multiple source analysis was performed. Incongruous chords violating the rules of harmony elicited a bilateral ERAN, whereas mistuned chords within chord sequences elicited a right-lateralized MMN. We found that the dominant cortical sources for the ERAN were localized around Broca's area and its right homolog, whereas the MMN generators were localized around the primary auditory cortex. These findings suggest a predominant role of the auditory cortices in detecting sequential scale regularities and the posterior prefrontal cortex in parsing hierarchical regularities in music