Browsing by master's degree program "Master's Programme in Neuroscience"

Accumulating evidence indicates that the plasticity-inducing effects of conventional antidepressant drugs like fluoxetine are mediated by their direct binding to TrkB. TrkB is the receptor of the brain-derived neurotrophic factor (BDNF), a neurotrophic factor of critical importance for neuron survival and synaptic plasticity. In addition, it has recently been reported that LSD and psilocybin, two psychedelic compounds with therapeutic potential, also bind to TrkB with higher affinity than antidepressants. It has been proposed that the differences in binding affinity between conventional antidepressants and psychedelics may help explain the much faster and longer-lasting antidepressant effects of psychedelics. Psychedelics and classical antidepressants bind to the transmembrane domain of TrkB dimers, where they act as positive allosteric modulators by potentiating the action of endogenous BDNF. The transmembrane binding sites of LSD and fluoxetine, despite being partially overlapping, are distinct and induce different conformational changes when bound to TrkB dimers. However, it is still unknown whether there are differences in the TrkB dimerization dynamics and neurotrophic signalling pathways induced by psychedelics when compared to conventional antidepressants. In this study, we investigated whether psychedelics and classical antidepressants promote TrkB dimerization and neurotrophic signalling in a differential manner. The effects of psychedelics on the TrkB dimerization dynamics and neurotrophic signalling associated with plasticity were studied treating N2a cells and primary cortical neuronal cultures with LSD or fluoxetine. Dimerization of the TrkB receptor in the presence of experimental compounds is assayed by protein-fragment complementation assay (PCA). Results show a significant dimerization in cells treated with LSD, whereas non-significant response in the ones treated with fluoxetine. The phosphorylation state of the neuronal TrkB receptor in three different tyrosines (Y515, Y706, and Y816) was checked as a marker of its activation by Western blot. Primary cortical cultures were treated with classical antidepressant fluoxetine (10uM) or psychedelic LSD (100nM) for 1 hour, when their effects on TrkB phosphorylation were compared. This experiment showed a significant increase of phosphorylation in TrkB Y816 after LSD treatment in cortical neuronal cultures, while fluoxetine treatment showed no significant effect. This indicates that LSD is able to activate the BDNF-TrkB signalling pathway associated with PLCg1 recruitment and induction of plasticity at an early time point and with a much lower concentration than fluoxetine, which would support LSD’s much more potent antidepressant and plasticity-inducing effects when compared to fluoxetine’s. Together, these results suggest that psychedelics that bind to TrkB, like LSD, are more potent than classical antidepressants in inducing TrkB-BDNF signalling. Overall, this study provides further evidence that TrkB is a critical mediator of psychedelics’ actions on neurotrophic signalling preceding their plasticity-enhancing and antidepressant effects and sheds more light on the common and differential mechanisms used by psychedelics and conventional antidepressants to produce their therapeutic effects.

Glioblastoma is the most aggressive and lethal tumor in the central nervous system. One of the main challenges of current treatment reside in its high intratumoral heterogeneity. Within one tumor, there could be several glioblastoma subtypes which harbor distinct molecular signatures and associated with different clinical properties. For instance, mesenchymal-like glioblastoma patients have the shortest survival time and this subtype is highly resistant to radiation. The current subtype classification heavily relies on transcriptomic analysis, which is not available for every patient due to its high cost. Here, I utilized a polycation reagent to treat neural-progenitor-like and mesenchymal-like glioblastoma cells with increasing concentrations and evaluate their cell viability using luminescence-based CellTiter-GLO assay. I found that neural-progenitor-like glioblastomas cell growth was significantly inhibited at intermediate concentrations of the polymer, while mesenchymal-like glioblastomas were less affected. With polymer concentration increasing, the inhibition effect displays a dose-dependent trend. My results demonstrated that neural-progenitor-like glioblastomas are more sensitive to the polymer than mesenchymal-like ones. The different sensitivity towards the polymer between these two subtypes suggests polymer could be used as a potential reagent to distinguish glioblastoma subtypes.

Abstract: The EEG measurement protocol is standardized and in use globally. The skull is measured to ensure that the electrodes are placed in the correct position. Measurements are necessary because skull sizes and shapes are different. Studies for placing electroencephalograph (EEG) electrodes on a human head are typically introduced theoretically before students are granted the opportunity to practice. Due to the limited availability of EEG equipment and supervisory staff, students encounter shortened practical training sessions and lengthy waiting periods transitioning from theory to practical components. The main aim of this project was to create a learning environment with game technologies to help students study electrode placement during the idle time between theory lessons and practical training. We set out to determine whether students experienced some learning gain and if they had a positive experience with the learning environment. We simultaneously assessed if fuzzy feedback is preferred over exact feedback. Additionally, the aim was to make use of a design-based approach with the information from a User Experience Questionnaire (UEQ) the EEG-simulator. Our group developed and tested a digital learning application that provides a 3D model of a human head, on which learners can practice placing EEG electrodes. We followed a user-centric design science approach to ensure our application appeals to our target audience. We used an observational post-test only design with two experimental groups and a control group. We applied a widely accepted user experience questionnaire to ascertain which of our two feedback systems elicited the best user experience. We also qualitatively analyzed diaries the students kept, as they worked with the learning environment, to better understand future development options for further maximizing the environment’s learning benefit. The overall application was well-received, and students opined that the application significantly enhanced their practical session experience. Although the post-test evaluation showed no difference between the two experimental groups, the user experience questionnaire showed that the fuzzy feedback system was preferred over the exact feedback. Furthermore, it was evident that students who had not used the learning environment struggled more to come to terms with the practical session. The personal experience recording by the students revealed several suggested improvements to the learning environment. We conclude that, with further development, this EEG placement learning application could address the idle period between demonstration lessons and practical training. We also venture to state that fuzzy feedback is preferred because of the high-fidelity mimicry of real teacher feedback. The last part of the research was to develop the EEG simulator so that it will increase theory learning with a simulator, that works, and this is ongoing. We have developed the last EEG simulator version with AR (augmented reality) mobile version that can be used with any smart devices. The future work is to test EEG application and does application influence student's theory learning process.

Meningeal lymphatics vessels (mLVs), the recently characterized lymphatics in the central nervous system (CNS), provide a link between the adaptive immune system and the CNS. mLVs could be important for the activation of T cell-mediated adaptive immune response, by draining antigens from the brain to the deep cervical lymph nodes, where they are presented to T cells. In traumatic brain injury (TBI), we hypothesized that the activation of self-reactive T cells (i.e., T cells able to recognize self, brain-derived antigens and promote an immune reaction), possibly underlies the pathogenesis of the disease. In order to test this hypothesis and to decipher the specific role of mLVs in the modulation of T cell-mediated neuro-immune response after TBI, we ablated the existing mLVs in adult male C57BL/6OlaJ mice (with the use of the AAV-mVEGFR3 1-4 Ig vector), induced TBI with controlled cortical impact, and examined the motor function of the mice and the activation of different T cell populations in the brain, as well as in the secondary lymphoid (spleen and lymph nodes – LNs) and non-lymphoid organs (meninges). Our data showed that the T cell-mediated adaptive neuro-immune response in TBI was unaffected by the depletion of mLVs. Our results, however, are preliminary, due to the limited sample size used in this study, which reduces the statistical power and restricts our ability to conclude for the effect of mLV depletion on TBI recovery.

Early life stress (ELS) has been associated with the development of psychiatric disorders such as anxiety and depression later in life. The central hypothesis is that these disorders are caused by a malfunctioning of the serotonin system and serotonin (5-HT) produced in the dorsal raphe nucleus (DRN). The DRN is anatomically connected to the medial prefrontal cortex (mPFC), especially to the infra- and prelimbic cortex, where 5-HT modulates behaviors such as impulsivity and cognitive flexibility. The DRN and mPFC mediate with low-frequency network oscillations, which are indicative of the state of the network and its funtional connectivity, as disturbances in these network oscillations have been connected to neuropsychiatric disorders. The aim of the thesis is to investigate whether and how ELS can influence the local field potential (LFP) activity of the mPFC and DRN and the functional connectivity of the DRN and mPFC. This is researched by characterizing and comparing the LFP activity recorded in the DRN, where 5-HTergic neurons are located, and in layer 5 of the infralimbic area of the mPFC. To accomplish these aims, a well-established animal model of early-life stress, the limited bedding and nesting model (LBN), was used. The model causes fragmented maternal care due to the stress of the dam, which in turn leads to the stress of the pups. Simultaneous multi-site recordings of LFP and multi-unit activity (MUA) within DRN and mPFC were performed in vivo during postnatal days (PND) 10-11 from control and LBN pups to characterize the network activity of these two brain areas and then investigate possible changes in their functional connectivity. The efficacy of the LBN model was determined by the observed decreased weight gain of LBN animals compared to controls. From the data, the LFP activity of the DRN and mPFC were characterized. The activity was characterized as power spectrum, wavelet spectrum, and MUA with DRN showing discontinuous activity with low signal-to-noise ratio and low frequency theta oscillations (4-12 Hz), while mPFC showed almost continuous activity with higher signal-to-noise ratio and developing gamma oscillations (20-50 Hz). The power of LFP signal of the areas was not found to be affected by ELS. To investigate if the coupling by synchrony between DRN and mPFC networks is altered by ELS, I analyzed wavelet coherence by computing coherence values between LFP signals in DRN and mPFC in a control and ELS for frequencies from 1 to 50Hz. The functional connectivity was affected by ELS. Statistically significant changes were observed in wavelet coherence in the lower frequencies of 1-2.8 Hz between the control and LBN treatment, suggesting impaired synchronization between DRN and mPFC at 1-2.8Hz frequency range immediately after ELS exposure at PND 10-11 mice. Caveats of the study were low signal-to-noise ratio of the recordings, the small group size of LBN animals (n=5) as well as the uneven sex distribution (male n=11, female n=3) which prevented the sex-based comparison of the effects of ELS. The thesis examines postnatal LFP brain activity in the DRN and mPFC and the functional connectivity between these brain areas. The results of the thesis show that ELS exposure is able to influence the functional connectivity of these two brain regions. The results support previous findings, which have found alterations in the functional connectivity of the neural networks underlying neuropsychiatric disorders in adulthood. The findings of this thesis suggest that ELS could affect the functional connectivity of a developing network and thus increase the risk of the development of neuropsychiatric disorders. Further studies are needed with larger group size, even gender balance, and better signal-to-noise ratio of recordings.

4-Methylmethcathinone (Mephedrone) is one of the the most prevalent synthetic cathinones that bears close structural similarity to amphetamines. Like other stimulants, mephedrone is often used with alcohol (ethanol). In animal studies ethanol has been observed to potentiate the neurotoxicity of amphetamine-type stimulants, and same has been observed when mephedrone and alcohol is combined. The long-term effects of mephedrone have still remained largely elusive. The aim of this thesis is to study the effects of mephedrone, methamphetamine, and ethanol on dendritic spine density and morphology in the hippocampus, nucleus accumbens and caudate putamen, and compare the spine densities with changes in brain activation observed in manganese-enhanced magnetic resonance imaging (MEMRI). Dendritic spines are small membranous protrusions on dendrites that act as the post-synaptic sites for most of the excitatory synapses. Amphetamine and methamphetamine have been shown to affect the density and morphology of the spines. The goal of this thesis was to investigate the long-term effect of binge-like (two times a day, four consecutive days) stimulant treatment on dendritic spines using Golgi-stained rat brain sections. The brains of 48 male Wistar rats were imaged using AxioImager Z2 microscope and the number and the size of the spines was analyzed using Reconstruct software. In this thesis no effect on dendritic spines was observed in the hippocampus and nucleus accumbens in animals treated with mephedrone, methamphetamine, ethanol or combination of them. In the caudate putamen significant increase in the total density of dendritic spines and in the density of filopodia-like spines was observed in mephedrone-treated animals. Other treatments showed no observable effect. These results were conflicting with previous studies where amphetamine-type stimulants have been shown to increase the spine density in the nucleus accumbens and the hippocampus and increase the density of branched spines. In the caudate putamen methamphetamine has been observed to decrease the spine density. There was no correlation between spine densities and brain activation observed in MEMRI. To my best knowledge this is the first time when the effect of mephedrone on dendritic spines has been studied. It is possible that the treatment regimen used here was not strong enough to produce marked long-term changes on dendritic spines. It is also possible, that mephedrone is not as neurotoxic as other amphetamine-type stimulants, which may explain why the effects remained limited and conflicting. More research is still required to establish the long-term structural effects of mephedrone.

Neurotrophin, Brain-derived neurotrophic factor (BDNF) and its cognate receptor Tropomyosin receptor kinase B (TrkB), have been concomitantly linked with neuronal plasticity as well as antidepressant mechanism of action. Adult hippocampal neurogenesis involves proliferation and survival of new-born neurons and has been related to antidepressant mechanisms and cognitive improvement. Environmental enrichment (EE) enhances adult hippocampal neurogenesis (AHN) and induces anxiolytic-like effects. This study postulates that EE-living conditions could restore the abnormal serotonergic modulation on AHN of our transgenic mice. In this study, a transgenic mouse line wherein TrkB receptor is compromised from serotonergic neurons and AHN found to be impaired was used. To assess the behavioural effects and the changes in learning and memory tasks produced by 10-weeks of EE, a behavioural battery test was performed. Our results suggested anxiolytic-like effects from EE in the transgenic mice. Likewise, cognitive improvements were also observed in both control and transgenic mice promoted by EE. Moreover, hyperactivity observed in transgenic mice in standard conditions could be rescued, and no phenotypical differences were observed between control and transgenic mice subjected to EE. To further study the effects of EE on AHN, cellular proliferation and survival were studied through the incorporation of BrdU. The results indicate that the abnormal serotonergic regulation of AHN was rescued upon EE-living conditions. Moreover, molecular methods used to measure the alteration of gene expression revealed significant upregulation of genes related to neuronal plasticity and epigenetic modifications. Altogether, these results suggest EE promotes the neuronal plasticity, rescues the impaired regulation of AHN and modulates the genetic expression of the transgenic mice. Findings from this study could provide new insights regarding novel targets that could modulate adult brain plasticity.

The insular cortex has been implicated in the neurocircuitry underlying alcohol addiction. The role of the insular cortex and its projections in regulating ethanol intake in AA (Alko-Alcohol) rats has been studied using chemogenetic tools. Chemogenetic activation of the anterior agranular insula (aAI) in AA rats through excitatory DREADDs expressed in the aAI has been found to decrease ethanol consumption. The aAI projects to the central nucleus of the amygdala (CeA), another brain region involved in the development of addiction, particularly in the withdrawal/negative affect stage. In the current study, we sought to further investigate the role of the aAI and the CeA in regulating voluntary ethanol consumption in AA rats. First, we characterized the efferent projections of the aAI in AA rats by chemogenetically activating the aAI with DREADDs and then measuring c-Fos expression in various regions of interest throughout the brain. Next, we investigated the role of the aAI --> CeA projection in ethanol intake by chemogenetically activating or inhibiting the aAI --> CeA projection using the dual viral Cre-dependent DREADD approach. We examined the effects of this manipulation on voluntary ethanol consumption in AA rats in a two-bottle choice paradigm. Finally, we examined the roles of CeA D1Rs (dopamine receptors) and 5-HT2ARs (serotonin receptors) in regulating ethanol intake by examining the effects of pharmacological agonism or antagonism of these receptors on voluntary ethanol consumption in AA rats. Our results from the first experiment reveal significant activation of brain regions including the posterior agranular insula, the mediodorsal nucleus of the thalamus, and the posterior piriform cortex following chemogenetic activation of the aAI. The projections from the aAI to these regions are potentially important in the aAI circuitry in AA rats and are therefore of interest in future studies on the role of aAI circuitry in ethanol intake. In the second experiment, we found no significant effects of aAI --> CeA projection activation or inhibition on ethanol consumption in AA rats, indicating that this projection may not be a key component in regulating ethanol intake in these rats. Finally, we found no significant effects of pharmacological D1R antagonism, 5-HT2AR antagonism, or 5-HT2AR agonism in the CeA on ethanol intake in AA rats, although there was a non-significant trend towards a dose-dependent decrease in ethanol consumption with increasing dose of the D1R antagonist. Our results reveal new neural projections that should be investigated in future research on the role of the aAI in regulating ethanol intake. Studies on the neurobiology underlying alcoholism may reveal new pharmacological or anatomical targets for treatments of alcoholism in humans.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) is a master regulator of both steroidogenesis and gonadal development. Disruptions of NR5A1 can result in differences in sexual development (DSD). With proven interspecies differences in NR5A1 functioning and human material not being available, human stem cells are one of the most achievable, ethical, and accurate models to study the earliest developmental stages of foetal life. However, in currently existing human stem cell-derived gonadal models the expression of NR5A1 has been insufficient without artificial induction due to the lack of knowledge of its distinct biological mechanisms, endogenous ligands, and co-factors. A functional reporter cell line would enable high throughput microscope screening of differentiation protocols with expressed NR5A1. The aim of this thesis was to generate a functional monoclonal human embryonic stem cell (hESC) reporter line for the gene NR5A1 with Alt-R CRISPR-Cas9 ribonucleoprotein (RNP) complex. Firstly, an efficient guide RNA was determined for NR5A1 by T7 assay, and a homology-directed repair (HDR) donor plasmid was designed based on it. Secondly, monoclonal hESC lines were generated with the Alt-R CRISPR-Cas9 RNP complex knock-in method and HDR donor plasmid via electroporation and single-cell sorting. Finally, monoclonal hESC reporter lines were screened with Touchdown PCR and a functionality analysis based on fluorescence and mRNA expression was performed. Two monoclonal hESC reporter lines H9-NR5A1-eGFP cl. 1 and dual-inducible H9-NR5A1-DDdCas9VP192-eGFP cl. 28 were established by using Alt-R CRISPR-Cas9 RNP complex. However, a functional validation performed on H9-NR5A1-DDdCas9VP192-eGFP cl. 28 cells showed the cell line to be non-functional upon NR5A1 upregulation regardless of the expressed eGFP mRNA detected with RT-qPCR.

Dermatologia on visuaalisia taitoja vaativa lääketieteen osa-alue, joka tutkii iholla, päänahassa, hiuksissa ja kynsissä esiintyviä sairauksia ja syöpiä. Ihotautien diagnosointi vaatii visuaalista asiantuntijuutta, joka tarkoittaa kykyä tehdä havaintoja ja tulkita niitä tarkoituksenmukaisesti. Asiantuntijuus syntyy havainto-oppimisesta, joka perustuu kokemuksen kautta kehittyneeseen kykyyn erotella ja luokitella tietoa. Se tarkoittaa kykyä erottaa olennainen epäolennaisesta, yhdistellä tietoa ja niiden välisiä suhteita sekä käsitellä poimittua tietoa nopeammin ja tehokkaammin. Visuaalista tarkkuutta vaativilla aloilla silmänliikkeiden tutkiminen voi paljastaa lääketieteellisten taitojen taustalla vaikuttavia ajatusprosesseja sekä oppimiseen liittyviä mekanismeja. Silmänliikkeiden ja digitaalisten havainto-oppimismoduulien yhdistäminen voi tarjota hyödyllistä tietoa visuaalisen asiantuntijuuden kehittymisestä ja auttaa suunnittelemaan toimivampia opetusmenetelmiä. Digitaalisista oppimismoduuleista saatu tutkimustieto on tarjonnut viitteitä siitä, että ne vaikuttavat positiivisesti visuaalisiin taitoihin. Tässä tutkimuksessa tutkittiin Helsingin yliopiston lääketieteen perustutkinto-opiskelijoiden visuaalisen asiantuntijuuden kehittymistä ihotautikurssin ja havainto-oppimismoduulien avulla. Opiskelijat (N = 63) tekivät ihotautikurssin yhteydessä kahta erilaista digitaalista havainto-oppimismoduulia, jotka sisälsivät kliinisiä ihotautikuvia. Niiden avulla tutkittavien tuli tunnistaa kuvissa esiintyvät ihosairaudet niistä näkyvien piirteiden perusteella. Suoriutumista mitattiin onnistumisprosentin, vastausajan ja varmuuden perusteella. Pienempään osaotokseen osallistui 13 opiskelijaa ja 7 ihotauteihin erikoistunutta lääkäriä. Suoriutumisen lisäksi tallennettiin heidän silmänliikkeensä (fiksaatioiden määrä, kesto ja sijainti), jotta nähtäisiin, mihin heidän katseensa kiinnittyy moduulien tekemisen aikana. Lopuksi opiskelijoiden ja asiantuntijoiden tuloksia verrattiin keskenään. Tulokset osoittivat, että opiskelijat suoriutuivat kohtalaisen hyvin moduuleissa kurssin alussa, mutta erot asiantuntijoihin olivat suuria. Visuaalisessa prosessoinnissa (fiksaatioiden määrä, kesto ja sijainti) eroja oli erityisesti fiksaatioiden määrässä: opiskelijoilla oli selvästi enemmän fiksaatioita kuin asiantuntijoilla. Kurssin lopussa opiskelijoiden ja asiantuntijoiden väliset erot suoriutumisessa pienenivät merkittävästi ja visuaalisessa prosessoinnissa eroja ei ollut enää lainkaan. Tutkimus osoitti, että opiskelijoiden visuaalinen asiantuntijuus kehittyi kurssin aikana ja havainto-oppimismoduulit olivat hyödyllinen opetusväline dermatologian opetuksessa. Toimivien opetusmenetelmien kehittäminen on tärkeää, jotta ne tukisivat opiskelijoiden oppimista ja visuaalisen asiantuntijuuden kehittymistä. Opiskelijoiden diagnoositarkkuuden ja kliinisen päättelykyvyn tehostuminen voisivat vähentää aikaisemmissa tutkimuksissa havaittuja eroja eksperttien ja noviisien välillä.