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Browsing by study line "Neuroscience"

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  • Rappe, Anna (2021)
    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.
  • Ouabbou, Sophie (2019)
    Tiivistelmä – Referat – Abstract Mental disorders are among the leading causes of global disease burden and years lived with disability. Their pathogenesis is poorly understood and there are enormous challenges in the development of biomarkers to aid in diagnosis and more effective therapeutic options. It has been documented that the microbiota-gut-brain axis shows alterations in mental disorders such as anxiety, depression, autism spectrum disorders, bipolar disorder and schizophrenia. Here we study the gut microbiota of individuals with axis I mental disorders and their unaffected siblings by 16S RNA gene amplicon sequencing. In the Central Valley of Costa Rica, a total of 37 participants were recruited and diagnosed using a Best Estimate Diagnosis protocol. For each of the individuals diagnosed with a mental disorder a healthy sibling was selected after matching by age and gender. A total of 13 pairs of 26 siblings, affected and unaffected, was used for the analysis. In a subsequent analysis, individuals were also divided into the three categories of “unaffected” (UA), “affected without psychosis” (AA) and “affected with psychosis” (AP). They underwent clinical assessments about their habits and diet and about resilience (Connor-Davidson Resilience Scale), current status (SADS-C) and disability (WHODAS 2.0). Their fecal samples were collected freshly and stored at -80°C. DNA was extracted, libraries constructed by PCR and subjected for Illumina MiSeq 300 paired-end 16S RNA amplicon sequencing for analysis of the gut microbiota. The sequencing data were analyzed using the R packages mare and vegan for gut microbiota composition, diversity and richness, taking into account the identified confounders. All participants were of Hispanic ethnicity, residents of the San José Greater Metropolitan Area, adults and 69% of them were women. Affected individuals had major depression, bipolar affective disorder, psychosis non-otherwise specified or schizoaffective disorder. Based on beta-diversity analysis as a measure of the community-level microbiota variation, it was found that the use of levothyroxine (R2=0.08, p=0.005) and of irbesartan (R2=0.068 ,p=0.001) had a significant impact on the microbiota composition and hence the use of these drugs was included as confounder in further analyses. Several statistically significant differences in the relative abundance of intestinal bacteria were identified: Differences were found in the relative abundance of bacterial families Peptostreptococcaceae, Ruminococcaceae, Porphyromonadaceae, and in bacterial genera Pseudomonas, Barnesiella, Odoribacter, Paludibacter, Lactococcus, Clostridium, Acidaminococcus and Haemophilus. Our results indicate that affected individuals have more pro-inflammatory Proteobacteria (Pseudomonas) and less bacteria associated to healthy phenotype, such as Barnesiella and Ruminococcaceae, the former being dose-dependently depleted in AP and AA compared to UA. Furthermore, we documented decreased bacterial richness among affected participants while no significant differences were detected in alpha diversity. Our study identified significant differences in the microbiota of individuals affected by mental illness when comparing to their healthy siblings. The results may have important implications for the holistic understanding of mental health and its diagnosis and therapeutics. Larger studies to confirm these findings would be justified.
  • Tienhaara, Samu (2021)
    In visual detection, thresholds for light increments are higher than thresholds for light decrements. This asymmetry has been often ascribed to the differential processing of ON and OFF pathways in the retina, as ON and OFF retinal ganglion cells have been found to respond to increments and decrements, respectively. In this study, the performance of human participants in detecting spatially restricted (diameter 1.17 degrees of visual angle) and unrestricted increments and decrements was measured using a two-interval forced choice task. Background light intensities ranged from darkness through scotopic to low photopic levels. The detection threshold asymmetry found in earlier experiments was replicated with local stimuli. In contrast, however, the asymmetry between increment and decrement detection thresholds disappeared with fullfield stimuli. An ideal observer model was constructed to evaluate the role of two factors, Poisson variations and dark noise, in determining detection thresholds. Based on the model, these factors are insufficient to account for the increment-decrement asymmetry.
  • Blom, Sonja (2022)
    Pain is a subjective feeling often difficult to interpret or study and thus, pain of those unable to communicate their pain is difficult to recognize. According to the new definition of pain by IASP (Raja et al 2020), verbal description is only one of the many behaviours that can be used to express pain, and the inability to communicate pain does not negate the possibility of experiencing it. This addition to the definition points out that non-human animals, too, even if they cannot express it in words, are capable of both experiencing and communicating pain. Can we as humans interpret a state of pain in an animal in a trustworthy way – and in a manner that would be respectful and non-invasive to the animal? Infrared thermography (IRT) is a technology based on using infrared radiation instead of normal light to form images. These images can be used to quantify the surface temperature of an object with high resolution. The intensity of the radiation emitted by the object being imaged depends on the surface temperature and for this reason thermal imaging enables detecting and measuring changes of surface temperature. Pain and stress might manifest physiologically as activation of the autonomic nervous system, which in turn might result in changes in surface temperatures of the body. These changes might be detectable with a thermal camera. If we could establish a link between certain intricate temperature changes of the head area to certain type of activation of the sympathetic nervous system resulting from pain, thermal imaging could have the potential to detect this. In this study I investigated if there were detectable temperature changes in animal patients before and after a standard examination conducted to each patient admitted to the Wildlife Hospital of Helsinki Zoo, where my data was gathered. Another question was whether the patients that had pain differed in their temperature changes as compared to other patients. The question at the heart of my research was whether there would be a change in peripheral facial temperatures of patients before and after the examination. Another question was whether thermal patterns would be different for pain- and non-pain patients. I found that for some parameters, the temperature differences between pain- and non-pain patients were indeed different, for example the crown temperature of birds seemed to change with examination for patients without pain but not for patients with pain. A more prominent finding was that temperatures decrease across many parameters after an examination as compared to prior to it, across all or many patient groups. My research does not univocally show that thermal imaging could be used to detect pain; rather it affirms the thought that the measurement of changes in peripheral temperatures could be a potential window to non-invasively detect some changes of activation of the sympathetic nervous system in animals.
  • Pasculli, Maria Samuela (2024)
    The S209F variant of the Abelson Interactor family member 3 (ABI3) gene has emerged as a risk factor for late-onset Alzheimer’s Disease (LOAD). The ABI3 protein is functionally related to the WAVE Regulatory Complex (WRC) participating in the control of cytoskeletal processes favoring either filopodia for chemotaxis or pseudopodia for phagocytosis. The S209F coding variant is thought to impair phosphorylation of the ABI3 protein leading to dysfunctional association with WRC. In the brain, the ABI3 gene is mainly expressed by microglia, macrophages representing the resident immune cells of the brain. Despite some research about the variant based on rodent models and reporting sometimes contrasting results, the role of the ABI3 S209F variant in AD remains poorly understood. Here, human-induced pluripotent stem cells (h-iPSCs) reprogrammed from fibroblasts of controls and variant carriers are sequenced to ensure retention of the original phenotype upon reprogramming. H-iPSCs are differentiated into microglia (iPSC-derived microglia, iMGL) following an established protocol. Morphological changes and microglia-specific gene expression partially show that iMGL between days 31 and 38 of differentiation in vitro can be considered mature. To assess the functional properties of microglia, cytokines/chemokines production, cathepsin gene expression, lysosomal activity, and Apolipoprotein E (ApoE) protein levels are measured. It is found that S209F microglia downregulate CCL5/RANTES and upregulate cathepsins B and L (CTSB and CTSL) upon LPS+IFNg stimulation which may lead to motility, migratory and endo-lysosomal dysfunctions. Lysosomal activity is found to positively correlate with CD163, but not with either CTSB or CTSL expression. ApoE protein levels show an upregulation trend in S209F microglia which may indicate modifications in lipid metabolism. Metabolic assessment based on mitochondrial respiration and glycolysis does not show any difference between S209F and control microglia, but ABI3 knock-out (KO) shows glycolysis dysfunctions. Overall, this study offers some hints into the mechanisms that make the ABI3 S209F variant a risk factor for AD pointing at the need to investigate microglia motility and migration focusing on pathologically relevant protein aggregates and their clearance and with particular attention to phagocytosis and endo-lysosomal pathway.
  • Tervi, Anniina (2020)
    The diversity of different neuronal types lays the foundation for different functions in the brain. The development of different subpopulations and special features of neurons in the central nervous system are still partly unknown. Finding answers to these developmental issues could help in the process of characterisation of cell types and mapping of neuronal networks between the brainstem nuclei in the brain. Previous studies have shown that a ventrolateral neuroepithelial domain in the anterior hindbrain, rV2, produces excitatory (glutamatergic) and inhibitory (GABAergic) neurons, which are related to monoaminergic nuclei in the brainstem (Lahti et al., 2016). In this master’s thesis project, the development of a subpopulation of neurons expressing Gsc2 transcription factor in the interpeduncular nucleus was studied. This project was based on single-cell RNA sequencing results conducted in E13.5 mice. Predicted by single-cell RNA sequencing results, Gsc2 expressing cells are GABAergic interneurons and originate from the rV2 domain of the rhombomere 1 region in the hindbrain. Co-expression pattern with another transcription factor Sall3 with Gsc2 during development was also addressed in the study. Furthermore, the role of Notch signalling in the binary cell fate decision between GABAergic and the glutamatergic fate of rV2 neurons was investigated. Validation of single-cell RNA sequencing results was performed using in situ hybridisation and immunohistochemistry methods with mice embryos at the age of E12.5 and E15.5. This study verified previously shown origin of Gsc2 expressing cells to the rhombomere 1 region and in addition, showed that Gsc2 expressing cells are GABAergic. Co-expression pattern of Gsc2 with Sall3 neither in the rV2 domain nor in the interpeduncular nucleus was seen in our results. In the rV2 domain, the depletion of Notch signalling decreased the expression of differentiating GABAergic neurons. This indicates that Notch has a role in GABAergic neurotransmitter identity during the development of brainstem neurons in mice. Based on our results, Gsc2 could be used as a lineage marker for GABAergic interneurons originating from the rhombomere 1 region and as a marker for a subpopulation of the interpeduncular nucleus. Furthermore, results from the role of Notch signalling could help in discovering the mechanisms related to the determination of neurotransmitter identity in rV2 neurons. Further investigations, in different developmental time points and with additional markers, are needed to verify these results.
  • Wong, Carlton (2019)
    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.
  • Anastasiadou, Maria (2019)
    Tiivistelmä – Referat – Abstract Genetic variations within the MYO16 gene indicate a common predisposition to severe psychiatric, neurocognitive and neurodevelopmental disorders (NDD), as well as bipolar disorders (BD) and schizophrenia spectrum disorders (SSD). Myosin XVI’s ability to regulate actin and its involvement in cytoskeleton remodeling highlights the protein’s significance in neuronal circuitry development and signaling. Mutations in actin regulator protein-encoding genes like MYO16 have been found to shift cytoskeletal dynamics, as well as cause irregular dendritic spine and excitation/inhibition (E/I) synapse phenotypes. Interestingly, altered actin dynamics and E/I synapse dysregulation are two commonly detected molecular deficits associated with neuropathologies, namely autism spectrum disorders (ASD), SSD, and intellectual disability (ID). Therefore, synaptic E/I profiles are good candidates for investigating the neuropathologies they accompany, and also for revealing potential functional abnormalities. Hence, we determined that quantifying the levels of inhibitory synaptic proteins VGAT and gephyrin is the most suitable approach to investigate inhibitory synapse profiles and their relation to pathologies. Specifically, we investigated how microRNA (miRNA)-mediated myosin XVI protein knockdown (KD) affects pre- and postsynaptic inhibitory synapse density in rat primary hippocampal neurons. We achieved this by analyzing the density of VGAT and gephyrin puncta, signifying pre- and postsynaptic inhibitory synapses, respectively, and also by measuring their diameter to determine differences in inhibitory synapse size. Moreover, we quantified and assessed inhibitory synapse density and size differences between groups by comparing Myo16 KD-plasmid expressing hippocampal neurons to scrambled control cells. Common for both Myo16 KD plasmids was the active suppression of myosin XVI by 33%. However, Myo16 KD plasmids did not affect inhibitory synapse density and size to the same degree. Specifically, there was a significant reduction of inhibitory synapse density in the Myo16 KD3-plasmid expressing neurons, yet, no changes were observed in Myo16 KD5-plasmid expressing neurons. Finally, pre- and postsynaptic inhibitory synapse size differences were not significant between groups for either Myo16 KD plasmid when compared to scrambled control. Aberrant actin cytoskeleton remodeling, as well as altered E/I synapse ratios may lead to hyper/hypo-transmissive neuronal states or cause E/I imbalance, suggesting a complex relationship between actin regulator genes and inhibitory synapses. Our understanding behind their interplay is fairly limited, thus, gaining insight into the mechanisms associated with altered E/I balance remains the primary aim.
  • Pihl, Enni-Eveliina (2023)
    Microglia, the resident macrophage-like glial cells of the central nervous system (CNS), form the first line of defense against pathogens in the brain, and regulate both innate and adaptive immunity. Any abnormalities in their microenvironment lead to microglial activation, characterized by alterations in their gene expression, morphology, and functional behavior. Once activated, microglia respond to CNS injury and inflammation by, e.g., migrating to the site of damage, releasing pro-inflammatory cytokines, as well as phagocyting cell debris and pathogens. Prolonged activation of microglia expressing pro-inflammatory phenotypes can lead to exacerbated CNS damage. Hence, limiting CNS inflammation by stimulating microglial polarization towards their pro-resolving phenotypes would be of great clinical relevance. The research of our laboratory focuses on CNS injury and repair, as well as finding novel therapies for ischemic stroke. Specialized pro-resolving mediators (SPMs) derived from essential fatty acids have been proposed to offer a potential therapeutic approach for ischemic stroke via promoting resolution of post-stroke inflammation. Previous studies have revealed the ability of SPMs to induce a transformation of macrophages, the immune cells strongly resembling microglia, towards their anti-inflammatory phenotypes. The aim of this study was therefore to assess whether SPMs have similar effects on BV2 microglia, specifically on their lipopolysaccharide (LPS)-induced production of pro-inflammatory cytokines, tumor necrosis factor α (TNF-α) and interleukin 6 (IL-6). In addition to assessing the cytokine levels, our aim was to determine the optimal conditions for studying the effects of SPMs on microglial migration. In the present study, the levels of TNF-α and IL-6 were determined by specific ELISAs, and the transwell assay was used to measure microglial migration. Resolvins E1 (RvE1) and D1 (RvD1), as well as protectin D1 (PD1) and 15-epimer of lipoxin A4 (15-epi-LXA4) were all associated with decreased levels of TNF-α and IL-6, with RvE1 having the most potential as a resolving agent. In addition, we observed that serum starvation notably decreases the release of IL-6 and affects microglial migration. Overall, our results support the idea that SPMs could provide a novel therapeutic strategy for stroke therapy as they contribute to the resolution of CNS inflammation.
  • Hein, Emil (2022)
    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.
  • Jalonen, Sonja (2023)
    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.
  • Seiffert, Nina (2021)
    An increasing number of people are diagnosed with depression. One possible reason for the development of depression is faulty wiring and information processing in certain neural networks (network hypothesis) in the central nervous system. It has been shown that antidepressant drugs (ADs) can induce a juvenile-like plasticity state in the brain (iPlasticity) comparable to the plastic state of critical periods during development. iPlasticity enables the rewiring of neuronal networks in combination with environmental stimuli. At the molecular level, the binding of brain-derived neurotrophic factor (BDNF) to its high-affinity receptor tropomyosin kinase receptor B (TRKB) leads to TRKB dimerization and activation, triggering a downstream signalling cascade promoting brain plasticity. Activation of the TRKB signalling cascade is triggered by neuronal activity as well as AD treatment. Recent findings demonstrate that classical as well as rapid-onset ADs bind directly to the transmembrane domain of TRKB, leading to increased translocation of intracellularly stored TRKB to the plasma membrane and enhanced BDNF binding. Cholesterol, a sterol lipid known to regulate TRKB signalling, has been found to ensure optimal TRKB-BDNF signalling by changing the TRKB dimers’ relative orientation when altering the membrane thickness. A point mutation of TRKB tyrosine 433 to phenylalanine (TRKB.Y433F) has been found to hinder TRKB dimerization. Molecular dynamic simulations reveal that other membrane lipids are likely to participate in AD binding to TRKB. The aim of this thesis was to investigate whether lipid and drug compound treatments affect TRKB dimerization in Neuro2A cells expressing TRKB. Furthermore, we assessed whether the Y433F mutation modulates TRKB dimerization in such treatments. Protein fragment complementation assay (PCA) was used as in vitro protein-protein interaction assay to quantify dimerization of overexpressed TRKB carrying two split luciferase reporter proteins. Additionally, to avoid variability caused by transient transfection and be able to test large compound libraries, the establishment of a stably TRKB-expressing N2A cell line was initiated. The results show that lipid compounds, such as Allopregnanolone, as well as ADs, such as Imipramine and (2R,6R)-Hydroxynorketamine, increased TRKB dimerization in vitro in a dose-dependent manner within 40 minutes. The increase was more pronounced in the TRKB WT-expressing cells. This indicates that the compounds tested here may be directly interacting with TRKB, facilitating dimerization. Moreover, data seem to confirm previous research on the less effective TRKB.Y433F mutation. While stable expression of TRKB carrying one of the luciferase reporter proteins was successfully achieved in a monoclonal cell line, the amount of protein expressed seems to require further optimization before utilising it for PCA. In conclusion, lipid and AD treatments can induce an increase in TRKB dimerization in a dose-dependent fashion. Further investigations are needed to determine where the compounds bind and by which mechanisms they exert their effects on TRKB. Furthermore, the work on the stable cell line will be completed to avoid variability of transient transfection in the future.
  • Kuutti, Mirjami (2022)
    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.
  • Törrönen, Essi (2020)
    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.
  • Moog, Maia (2022)
    Catastrophic childhood epilepsies are characterized by persistent seizures and are frequently associated with cognitive and developmental impairments. Many, approximately 30%, of these epilepsies are rare genetic disorders that do not have effective therapeutic options. The bench to drug process is lengthy and expensive, and thus it is critical to find more affordable drug screening options. Zebrafish are an ideal model organism for screening studies as they share considerable (70%) genetic similarities with humans and are cheap to maintain with efficient breeding capabilities. In the present study, 37 zebrafish lines were screened for epileptic brain activity to identify high priority genes for future pharmacology studies. Each zebrafish line, generated by CRISPR-Cas9 represents a single gene loss of function mutation associated with 3 epilepsy based on genome wide association studies. Larval zebrafish were screened for spontaneous seizure activity using electrophysiological assays. The following 8 genes were significantly associated with spontaneous seizure activity in zebrafish: EEF1A, ARX, GRIN1, GABRB3, PNPO, STRADA, SCN1A, and STXBP1. There is now an open-source database for all 37 zebrafish lines, which contains sequencing information, survival curves, behavioral profiles, and electrophysiological data. The findings reveal novel target genes for future drug development and discovery. Future work is needed to explore whether zebrafish also model co-morbidities commonly seen in human patients with epilepsy.
  • Pazos Boubeta, Yago (2019)
    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.
  • elDandashi, Rahaf (2021)
    Epigenetics is the study of changes in gene function without affecting the DNA sequence. Epigenetics studies the effects of the environment and behavior on the genome. Researchers have been able to detect several epigenetic modifications such as –DNA methylation, histone acetylation, and microRNA-associated gene silencing. Changes in the epigenome are essential for proper cell function and normal development and can also be induced by environmental factors. Stress is defined as a biological response to physiological and psychological demands which can affect cellular homeostasis. Factors such as prenatal life stress can affect gene function without directly altering the DNA nucleotide sequence. Elevated levels of stress can immobilize with the ability to impair cognitive function. There is evidence that suggests the involvement of epigenetic regulation in disorders such as addiction, depression, schizophrenia, and cognitive dysfunction. Therefore, this systematic review discusses recent findings of the role of epigenetics in prenatal exposure to stress. To achieve this, the thesis will cover different subtopics from genetics, neurobiology, and diseases, neuroscience, biological psychiatry, life sciences, medicine, behavioral brain research, biochemistry & molecular biology, as well as neuroendocrinology. Research questions are 1) Is there an association between epigenetics and prenatal stress? 2) What kind of mechanisms have been found? 3) What kind of techniques have been used in the identification of potential epigenetic mechanisms? What genes are associated with these epigenetic changes?. This study followed the "The Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) guideline checklist. Eligibility criteria and search terms where be selected and documented to offer the widest range of articles covering the subjects of this study. A literature search was done using PubMed/Medline, Google scholar, and gray literature. The last sample comprised 59 articles. Data were extracted so that the participants, intervention, comparisons, and outcomes were included. The literature search conducted in this systematic review identified a few findings. First is that the majority of animal and human studies found a significant or moderate association between epigenetics and prenatal stress. Second, DNA methylation is the most studied epigenetic mechanism in maternal exposure to stress Third, genome-wide studies were more common in human studies than in animals and the most widely used method used is Infinium HumanMethylation450 Bead Chip. However, the common methods used in human and animal studies are most likely because of the small sample size and causation cannot be determined. Finally, NR3C1 and FKBP5 genes were the most studied in human studies where they showed the strongest association between prenatal stress and epigenetic modifications. While in animal studies, the most studied genes were Bdnf and Dnmt1 as they showed a significant methylation level after maternal prenatal stress exposure. In conclusion, maternal prenatal stress could trigger epigenetic alterations in neonates in both animals and humans. This holistic review detailed and evaluated locus-specific and studies exploring current knowledge about associations between maternal prenatal stress and epigenetic changes.
  • Suonpää, Pinja (2022)
    Each year many new-borns are at risk for long-term developmental deficits due to adverse perinatal events. Early gross motor abilities have been shown to link with cognitive development and studying infant motor behaviour may provide means to assess global neurodevelopment. This thesis aims to explore a potential association between early gross motor abilities recorded at infancy with a multi-sensor wearable jumpsuit MAIJU and later neurocognitive development assessed at two years of age. The study sample (N=26) consisted of healthy full-term infants and those with prematurity or perinatal asphyxia. Spontaneous motor activity was recorded at home with the jumpsuit. Machine learning methods were used to quantitate the time infants spent in different postures and estimate the maturity of their motor abilities, which were compared to cognitive development at two years of age with correlational- and regression analyses. There was a positive trend between early motor abilities and later cognitive development. Specifically, standing posture explained the association, such that infants who spent more time standing had better cognitive abilities at two years of age. Standing may support cognitive development by increasing opportunities for visual and manual exploration and learning. Shared neuronal circuitries for motor and cognitive functions and faster neuronal maturation may also underlie the association. The current study supports the creation of future studies with larger sample sizes to establish the potential for the use of postural and movement information obtained from wearable jumpsuit MAIJU to assess the variability of neurocognitive development of at risk and typical infants with potential goal to identify future cognitive deficits at early stage.
  • Järvi, Vilja (2019)
    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.
  • Lewis, Serena (2021)
    Histamine receptors are known to be expressed throughout the peripheral nervous system and are involved in regulating the gut and immune system. The gut-brain axis, which consists of bidirectional signaling between the central nervous system and gastrointestinal tract, links gut functions to emotional and cognitive controls in the brain. Many animal models are known to express histamine receptors in their gut and brain tissue which can be altered by a compromised gut-brain axis like stress. Histamine receptors also play an important role in many gastric and intestinal disorders. However, the precise expression pattern of histamine receptors in zebrafish gut tissue is unknown, as is whether their expression levels also change with stress. Here, I show that zebrafish gut contains several histamine receptors, but their role involving stress within the gut remains unknown. I found that histamine receptors hrh1 and hrh3 as well as the enzyme that synthesizes histamine, histidine decarboxylase (hdc), are expressed in zebrafish gut and brain in wildtype and hdc knockout adult zebrafish using in situ hybridization. Stress induction on wildtype male zebrafish through chronic social defeat and analysis of histamine receptor and hdc mRNA levels using quantitative real time PCR showed no differences in subordinate, dominate, or control fish. However, it did provide quantitative data that hrh1, hrh2, and hdc mRNA expresses in the adult gut. My results demonstrate the first data to suggest histamine receptors are expressed in zebrafish gut, and that even though stress can alter the gut-brain axis, it may not do so through the regulation of these receptors.