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

Diets of wild owls have been studied by pellets, when the nutritional value of owl prey remains unclear. Fatty acids are an essential part of animal nutrition, acting as energy reserve, building blocks of membranes and precursors for signaling molecules. I studied whether Finnish owl species have species-specific characteristics in the fatty acid profiles of adipose tissue due to different diets and feeding habits. I also compared fatty acid profiles between wild and captive owls to elucidate if the diet of captive owls resembles the diet in the wild. Fatty acids from visceral adipose tissue and a liver were transesterified to fatty acid methyl esters, which were identified and quantified by gas-chromatographs linked to either a mass spectrometry or flame ionisation detector. The fatty acid compositions were compared with Principal Component Analysis and statistical significance of the separations between species, origins and tissues were studied by Soft Independent Modelling of Class Analogies. Differences in the relative concentrations of individual fatty acids and their structural category sums were evaluated by the Kruskal-Wallis test followed by the Wilcoxon rank-sum test. Compared to wild owls, captive owl (snowy owl Bubo scandiacus and Eurasian eagle-owl Bubo bubo) adipose tissues had lower relative concentrations of many long-chain polyunsaturated fatty acids (PUFAs), such as arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid. The captive owls also had lower n-3 PUFA/n-6 PUFA ratio than the wild owls. I suggest that these differences arise from anthropogenic food given to the prey of the captive owls. In the future, the feeding and breeding of captive owls could be improved and potential health hazards prevented by giving the owls a diet rich in long-chain PUFAs, either by giving the prey more versatile diet or adding supplements to the owl’s diet. The adipose tissues of wild Eurasian eagle-owls contained higher total relative concentrations of monounsaturated fatty acids than the species belonging to the Strix genus. On the other hand, long-chain PUFAs were more prominent in the Strix owls. I suggest that these differences in the fatty acid composition are due to the disparities in feeding behaviour: the Eurasian eagle-owls eat carrions and large prey that cannot be swallowed whole, which may lead to consuming more the body outer parts of the prey. The fatty acid composition of the eagle-owls can also be affected by urban prey that have consumed anthropogenic food. In the future, the fatty acid composition of wild prey should be examined to be able to evaluate the prey composition of owls and its effect on the owl fatty acid profile with physiological consequences, the knowledge that could be used in the future decision-making that supports the conservation of owls.

Attention deficit hyperactivity disorder (ADHD) is a neuropsychiatric disorder characterized by inattention, hyperactivity and impulsivity. The symptoms appear in childhood and, if left untreated, can continue into adulthood affecting the quality of life. Currently, diagnosing a child's ADHD relies on subjective questionnaires filled out by a parent and an interview. The detection of changes in brain activity especially during everyday activities could bring important information that could help inform the diagnostics of ADHD. The changes in brain activity in persons diagnosed with ADHD during familiar, everyday events have been studied very little. However, ADHD brain imaging studies done during resting state and simple tasks have found changes in large-scale brain networks. These networks can be studied using functional connectivity approach, where the degree of synchronous activity in different brain regions is used to determine the connection strength between these regions. In this thesis, using functional magnetic resonance imaging, the differences in the functional connectivity of the whole brain between children diagnosed with ADHD (n=17) and controls (n=19) were investigated during a virtual reality task that simulates everyday life (EPELI), watching natural-like videos, and resting state. Connectivity matrices were generated with the NiLearn Toolbox program using Seitzman and colleagues' (2018) 300-area parcellation. The connectivity of the whole brain was examined using Network-Based Statistics. During movie watching the ADHD group showed increased connectivity compared to the control group in a network that included several areas of the motor cortex. This may indicate a role for these regions in the hyperactivity symptoms of ADHD. The same network also included the right superior temporal gyrus, which has previously been linked to impulsivity symptoms in individuals diagnosed with ADHD. During the virtual reality task and resting state, no differences in connectivity were observed between the groups. However, differences between the experimental situations were revealed in several networks when the connectivity was compared within the groups. Many of these networks were very extensive and included several subcortical and cerebellar structures in addition to cortical areas. Both the control and ADHD groups showed increased connectivity in the resting state compared to EPELI. This could possibly be due to the differences in the participants' actions during the task performance. To the best of the author's knowledge, this study is the first to examine the functional connectivity of brains diagnosed with ADHD during naturalistic stimuli. The clear differences between the controls and the ADHD group during movie watching are promising for future naturalistic brain studies. Based on the results, network models are effective in studying the functional connectivity of ADHD under different conditions. However, consideration for similarity of activities during the virtual task could have led to the detection of larger differences.

Master's thesis project includes the backbone assignment of the human activity-regulated cytoskeleton-associated protein C-lobe (hArc, Uniprot ID: Q7LC44), 7-fluoroindole-based tryptophan-labeling method, and comparing that with the 100% double-labeled and 20%(13C) fractionally labeled samples. The project focuses on the effects of 7-fluoroindole-based fluorotryptophan-labeling. hArc C-lobe has only one tryptophan, which makes the analysis easier. Typically fluorotryptophan-labeling is a costly method – fluorotryptophan itself is very expensive and attaching the fluorine to the tryptophan while expressing is expensive and complicated. Fluoroindolebased labeling circles around the problem, as indole and serine are used in procaryotic systems for tryptophan biosynthesis – meaning that fluoroindole, which is cheap, could be used as an alternative for previous methods. Fluoro-labeled tryptophan is used in protein NMR; for example, in binding studies – fluorine-probes are sensitive, and binding of ligand or protein would move these peaks, indicating binding. This project aims to get an insight into the application of this labeling method. The goal is to see if one could utilize one sample with both (1H, 15N, 13C) labeling and 7-fluorotryptophan labeling for binding and structural studies. However, fluorine is very electronegative, affecting surrounding structures and possibly sequentially nearby amino acids. This possible effect will be observed and determined by comparing the 1H15N-chemical shifts between well-established labeling methods and fluoroindolebased labeling. To determine what amino acids in the protein are affected, if they are affected, will be determined by using the backbone assignment results and the results from the sample comparisons.

The degree of neurogenesis in the adult hippocampal dentate gyrus (DG) is the center of the discussion in the field of adult neurogenesis. Although there is an on-going controversy, accumulating evidence suggests that the neural stem cells (NSCs) in the adult human DG are very few. The question remains open as to why there are so few NSCs in the adult human DG when compared with the rodent DG. In order to address these questions, it seems necessary to understand the developmental process of the NSCs in the adult human DG. In this thesis, the neural stem and progenitor cells in the fetal human DG are characterized. In addition to these findings, a semi-automatic method for counting and categorizing cells in their expressions of immunochemistry markers is developed.

As the most common mental disorder, anxiety disorders present a major burden to healthcare worldwide and a challenging problem to overcome for the ones suffering from it. Recently, researchers have started to recognize that the relationship between sleep and anxiety disorders is bidirectional; disturbed sleep is a potential risk factor for the progression of anxiety and anxiety can lead to sleep disturbances. However, the neural mechanisms underlying anxiety and sleep problems are still poorly recognized. In this study, we used a chronic sleep fragmentation (SF) paradigm to investigate how disturbed sleep alters anxiety-like behavior in mice and what are the potential underlying neuronal mechanisms. This model was chosen because we wanted to focus on a common form of disturbed sleep in humans rather than total sleep deprivation. We measured anxiety-like behavior in the light-dark box and open field tests right after the 2-week SF period and again after a week of recovery. Additionally, we performed immunohistochemical analysis to study prolonged cell activity (transcription factor ∆FosB), parvalbumin (PV) interneurons and perineuronal net (PNN) structures in the medial prefrontal cortex (mPFC) of the mice. Changes in mPFC activity and related brain areas are associated to anxiety in humans and anxiety-like behavior in rodents alike. Similarly, changes in PV interneurons and PNNs, that regulates PV cell function, are associated to anxiety-like behavior. However, PV interneurons and PNNs have not been previously studied in a setting that combines sleep fragmentation and anxiety-like behavior. We found that chronic SF increases anxiety-like behavior in female mice and that this effect persists at least for a week. Conversely, we did not observe significant increase in anxiety-like behavior in male mice. Both female and male mice showed decrease in ∆FosB in the mPFC suggesting that SF treated mice had lower overall levels of cell activity. Similarly, we found that SF treated mice had decreased PV interneuron intensity in both sexes which could indicate changes in the cell activity. However, the pattern of changes in the IHC results was not identical in males and females. Based on the IHC results, we suggest that SF affects neuronal processes in both sexes but the disparity in them could explain the difference in the behavioral effect. This thesis shows that disturbed sleep can lead to increased anxiety-like behavior in rodent models and recognizes potential targets to study the mechanisms behind the phenomena.

Adolescent ill-being has in recent years become a prominent health concern globally. Ill-being during adolescence can have negative consequences for future health and wellbeing, as important patterns of health are formed during this time. This highlights the importance of early identification of risk factors and overarching patterns of mental and physical ill-being and arguments for early intervention during adolescence. The aim of this study was to identify risk factors and co-occurrence of subjective ill-being symptoms in the form of depressive symptoms and subjective health complaints. This study also examined whether the subjective ill-being of students was reflected in cortisol patterns in a naturalistic setting. This since stress has been identified as a key etiological factor in ill-being, through the damaging effect of prolonged exposure to elevated cortisol levels. By applying a novel measure of school atmosphere, the study also aimed to examine the potential protective role of the social atmosphere in school on subjective ill-being and cortisol levels. A total of 329 students from eleven Finnish-Swedish upper secondary schools participated in the cross-sectional study by answering a questionnaire. The salivary cortisol samples were collected from a subsample of the participants, with of a total of 209 participant that met the salivary sampling criteria applied in the study. The methodological framework for the statistical analysis of the study consisted of independent samples t-test, ANOVA, Pearson’s correlation, and multiple linear regression. The results showed a higher prevalence of ill-being in girls and second year students. A significant co-occurrence was found for the subjective ill-being measures of depressive symptom and subjective health concerns. The subjective ill-being was however not reflected in the daily cortisol patterns of students in a naturalistic setting. Furthermore, a positive school atmosphere was significantly negatively associated with subjective ill-being of student in the form of depressive symptoms and subjective health complaints. When controlling for covariates, the subjective meaning of school experienced by the students was identified as a significant protective factor against symptoms of ill-being. These findings identify students in need of additional support and highlights the need of applying an overarching view on student ill-being in future adolescent research. Since no associations was found between daily cortisol patterns and subjective ill-being this study contributes to the understanding of HPA axis in early disorder onset. This study also highlights the importance of subjective meaning in a school context and posits increasing the subjective meaning as a prominent strategy to decrease ill-being among Finnish-Swedish upper secondary school students. Further studies are however needed to assess the causality and to examine these relationships further.

Aging is the progressive accumulation of cellular dysfunction, stress and inflammation. The mitochondrial network plays a central role in the maintenance of cellular homeostasis, with a growing body of evidence assigning dysfunctional regulation of this network as cause or effect of age-related diseases including metabolic disorders, neuropathies, various forms of cancer and neurodegenerative diseases. Neuronal sensitivity to changes in energy supply and metabolic homeostasis make neurons especially susceptible to alterations in the mitochondrial network. Mitophagy, a specified form of autophagy, is the selective degradation and quality control mechanism of mitochondria by engulfment and fusion with acidic endolysosomal compartments of the cell. Mitophagy has been extensively characterised in cultured cells and short-lived model organisms. However, our understanding of physiological mitophagy during mammalian aging is unknown. This study utilizes mito-QC mitophagy reporter mice that enable in vivo detection and monitoring of mitochondrial turnover due to the distinct physicochemical properties of the tandem GFP-mCherry reporter. Using cohort groups of young and aged reporter mice, age-dependent alterations of mitophagy were quantified in the cerebellum and the outer nuclear layer (ONL) of the retina. Specific autophagy and mitophagy markers were used to assess the longitudinal alterations in the mitophagic landscape. Images of fixed brain tissue sections were attained by high-speed spinning disc confocal microscopy for the quantitative and histological analysis. This study characterises the longitudinal alterations of mitophagy in distinct regions of the central nervous system (CNS) of mitophagy reporter mice, demonstrating tissue-specific alterations in mitochondrial turnover throughout physiological time. Åldrande kan definieras som den successiva ackumuleringen av cellulär dysfunktion, stress och inflammation. I upprätthållandet av cellens funktioner och homeostas har det mitokondriella nätverket en central roll. Omfattande forskning visar att åldersrelaterade sjukdomar såsom neuropati, ämnesomsättningssjukdomar, olika cancerformer samt neurodegenerativa sjukdomar föranleds av mitokondriell dysfunktion. Neuroner är beroende av oavbruten energitillförsel och upprätthållen metabolisk homeostas, vilket gör dem speciellt mottagliga för förändringar i det mitokondriella nätverket. Mitofagi är en selektiv form av autofagi som degenererar och kvalitetskontrollerar mitokondrier genom att leverera dem till lysosomer där de bryts ned av hydrolytiska enzymer. Den aktuella kunskapen inom regleringen av och mekanismerna bakom mitofagi baserar sig på gedigen forskning av kortlivade organismer och cellkulturer. Däremot är vår kunskap inom åldrandets inverkan på mitofagi i däggdjur begränsad. I denna studie används musmodellen mito-QC vars rapportörgen består av ett binärt GFP-mCherry-komplex som besitter olika fysikaliska och kemikaliska egenskaper, vilket möjliggör upptäckt och analys av mitofagi in vivo. En kvantitativ jämförelse av mitofagi i unga och åldrande möss genomfördes i vävnadssnitt av cerebellum och av det yttre nukleära lagret av retinan. Specifika autofagi- och mitofagimarkörer användes för att utvärdera de longitudinella förändringarna i mitokondriell degenerering. Bilder för kvantitativ och histologisk analys erhölls med höghastighets spinning-disk-konfokalmikroskop. Denna forskning karaktäriserar de longitudinella förändringarna av mitofagi i definierade regioner av det centrala nervsystemet i musmodellen mito-QC och presenterar vävnadsspecifika förändringar i degenereringen av mitokondrier under åldrandets framskridande.

Morphological features are considered as markers of microglial functionality, and they show regional heterogeneity in the brain. Recently the sleep-wake cycle was shown to affect microglial morphology in mice and correlate with cortical sleep slow wave activity (SWA). Microglial sizes and ramification increased during the dark period and decreased during the light period in cerebral areas associated with SWA, suggesting that neuronal activation could be affecting microglial morphology through SWA. I studied microglia in the hindbrain areas with and without functional connection to SWA to further investigate the association between SWA and alterations in morphology, and to investigate if there are differences in microglial morphology and their diurnal alterations in brain regions other than those commonly investigated. I examined three hindbrain areas (cerebellar cortex (CC), deep cerebellar nucleus (DCN) and medial vestibular nucleus (MVN)) and somatosensory cortex (SC) of mice (n=15) at two timepoints: 6 hours after the light onset (high SWA) and offset (low SWA). My aims were to answer if there are morphological differences in microglia between 1) the four brain areas at both timepoints and 2) between the two timepoints in each brain area. My hypotheses were that CC and DCN which have functional connections to cortical SWA, would show similar diurnal morphology alterations as demonstrated in the cerebral areas, and MVN that has no known cortical SWA connection, would lack significant alterations. As microglia are heterogenous throughout brain, I expected microglia to differ between different brain areas, especially the hindbrain and the SC. I found that microglial morphologies significantly differed between the hindbrain and the cortex, while the hindbrain areas were more similar in morphology. Moreover, the brain areas demonstrated diurnal morphology alterations of microglia with varying extent: CC and DCN microglial morphology did not correlate with SWA as clearly as SC did, and interestingly, morphological features of MVN microglia showed a pattern opposite to other areas, microglia being larger during the light period than the dark period. These results highlight the importance of the diurnal time to microglial morphology and the heterogeneity of microglia between different brain regions.

Poor quality of sleep and the following health problems affecting daily life are in many cases caused by cognitive and physiological arousal resulted from a stressful event. Such stress detrimental to sleep may originate from psychosocial factors such as feelings of shame and social rejection. Our goal was to elucidate the impact of acute psychosocial stress occurring before bedtime on sleep macrostructure and the early night non-rapid eye movement sleep (NREMS). In addition, virtual reality solutions are emerging as options to simulate social threats in laboratory environments. We studied whether a virtual reality variation of a public speaking scenario was sufficient in producing a physiological stress response evident in heart rate variability (HRV) parameters. We compared two experimental groups of healthy young adults (n=34), which differed in the scenario completed within the virtual reality. The stress condition involved a public speaking simulation in front of an attentive virtual audience whereas the control condition involved listening to a neutral presentation in the same but empty virtual seminar room. The participants’ physiological responses were measured with a HRV monitor for 38 hours and the quality of sleep during the laboratory night following stress induction with electroencephalography (EEG). The examined early sleep period was divided into two separate cycles of NREMS, whose results were juxtaposed. For analysing frequency band activity during sleep, we processed the data from EEG with Fourier transformation to yield power spectral density values i.e. frequency activity values. Comparing the two conditions, we observed a distinct effect of stress both during the virtual public speaking scenario and in the subsequent early sleep in the participants from the stress group. We found a significant increase in heart rate and rising fluctuations in the LF/HF (HRV power spectrum high frequency/low frequency) ratio around the stress task period contrasting the results of the control condition, reflecting increased sympathetic tone in the stress group. In the following night, the percentage of stage N3 sleep significantly increased at the cost of N2 sleep during the first NREMS cycle in the stress condition, but this effect resolved in the second NREMS cycle where group differences were absent. As a key finding, the stress group exhibited higher beta frequency activity in proportion to delta activity throughout both cycles and sleep stages. This effect was significantly magnified in N3 sleep where the delta/beta activity ratio decreased in the stress group from cycle 1 to 2, indicating worsening quality of sleep as the night progressed. We reflected our results through a homeostatic point of view, where the increased high frequency beta activity at sleep onset and early sleep in the stress group might explain their increased N3 sleep duration in the first NREMS cycle. A stronger affinity for the important N3 sleep may be a sleep protective mechanism to counter the stress induced abnormally high frequency EEG activity at sleep onset and early sleep to ensure the restorative benefits of slow-wave activity.

In recent years, psychedelics have shown promise in the treatment of conditions like depression and addiction. The therapeutic effects of psychedelics have been linked to their ability to increase plasticity in the brain, an effect that has also been seen for antidepressants. Brain-derived neurotrophic factor (BDNF) is a member of the neurotrophin family, which has an important role in the development of the nervous system, as well as promotion of neuronal survival and differentiation during adulthood. BDNF, through its receptor TrkB, has been implicated in antidepressant action, and BDNF-TrkB signalling is involved in many aspects of plasticity. Recently, antidepressants have been reported to bind directly to TrkB, and through this binding mediate their plasticity-enhancing, as well as behavioural effects. Psychedelics have been shown to increase structural and functional plasticity, but the mechanisms behind these effects are not fully understood. For example, the serotonergic receptor 5-HT2A is known to be behind the acute hallucinogenic effects of psychedelics, but its role in plasticity is still debated. The aim of this study was to investigate the mechanisms of LSD-induced plasticity. The dimerization of TrkB was examined after LSD treatment in the protein-fragment complementation assay (PCA). Phosphorylation of TrkB signalling markers mTOR and ERK, which have known effects on plasticity, was assessed in Western blot, and the total expression of BDNF was examined with the enzyme-linked immunosorbent assay (ELISA). The timeline of the effects was investigated, and the involvement of 5-HT2A in TrkB dimerization and the phosphorylation of ERK was assessed by combining LSD treatment with the 5-HT2A antagonist M100907. Dimerization was also assessed in a TrkB mutant (Y433F) that has previously been shown to disrupt antidepressant effects on plasticity. These experiments showed that LSD treatment increased TrkB dimerization as well as phosphorylation of mTOR and ERK. The Y433F mutation interfered with LSD-induced TrkB dimerization, but the effects of LSD on TrkB dimerization or ERK phosphorylation were not blocked by M100907. Together, these data suggest that 5-HT2A is not involved in LSD-induced promotion of TrkB dimerization or ERK phosphorylation. The increases in phosphorylation and dimerization were found to be most robust after a 1 h LSD treatment, however an increase in BDNF expression was seen in cortical neuron cultures only after a 24 h treatment with LSD. The results reported in this study support the view that 5-HT2A might not be needed for the plasticity-inducing effects of psychedelics. If this is true, the development of treatments that target plasticity without hallucinatory effects could be possible. Overall, this research provides insight into the mechanisms of LSD-induced plasticity and offers new and interesting directions for future research in the field.