Browsing by master's degree program "Magisterprogrammet i neurovetenskap"

The sensitivity of our conscious visual system comes remarkably close to the sensitivity limits imposed by the quantal nature of light. This exquisite sensitivity is made possible by the rod bipolar pathway (RBP), the most sensitive neural circuit studied to date, which allows us to consciously perceive light stimuli producing, in total, fewer than a dozen single-photon absorptions in rod photoreceptors. One of the central features of the RBP is the pooling of signals arising in thousands of rod photoreceptors scattered over the surface of the retina (spatial integration) into individual retinal ganglion cells (RGC), which subsequently encode visual scene as a train of action potentials and transfer these signals to the brain. However, the ultimate limits of sensitivity and the retinal circuitry underlying non-conscious vision at the absolute threshold of visual sensitivity are poorly understood. Here, we utilized the pupillary light reflex (PLR) as a functional readout of the non-conscious visual system to simultaneously measure and compare the threshold sensitivities of the conscious and non-conscious visual systems across different spatial scales in dark-adapted human observers. For this purpose, we designed, built, and calibrated an apparatus capable of producing precisely calibrated stimuli across five orders of magnitude in intensity, and four orders of magnitude in size. We find that the PLR and conscious vision express stimulus size-dependent differences in their threshold sensitivities, where when utilizing stimuli covering the whole visual field the PLR matches the sensitivity of conscious vision, by responding to stimuli producing, fewer than three photon absorptions spread over a pool of ten thousand rod photoreceptors, but when utilizing small stimuli the threshold sensitivity of the PLR falls short by an order of magnitude as compared to conscious visual system. Additionally, we find that the PLR produces a constant response to a constant number of photons (complete spatial summation), for stimulus sizes of up to 570µm in diameter. Thus, the PLR is capable of complete spatial summation over a retinal area 9-fold larger than conscious vision. Our results are consistent with RBP input into both visual systems, with each visual system providing a readout to the brain through separate RGCs.

Chronic stress has been linked to the pathogenesis of various disorders, such as generalized anxiety disorder, depression, and post-traumatic stress disorder (PTSD). Stress-induced hyperexcitability of the basolateral amygdala (BLA) has implications in anxiety-like behavior. Promising evidence points to the direction of GluK1 subunit containing kainate receptors (KARs) having a role in the modulation of GABAergic transmission in the lateral amygdala (LA). The aim of the present study was to investigate whether dysfunction of KARs contribute to stress-induced amygdala hyperexcitability and anxiogenesis in mice. Chronic restraint stress (CRS) is an animal model simulating chronic psychological stress. An in situ hybridization experiment was performed to investigate how CRS affects expression levels of GluK1 in the different neuronal populations in the LA. These data show that CRS leads to downregulation of GluK1 expression in the parvalbumin-positive (PV+) interneurons specifically. Patch clamp recordings of spontaneous inhibitory postsynaptic currents showed that CRS did not affect synaptic GABAergic transmission to the principal neurons in the LA. Lastly, conditional knock-out (cKO) mice that have the Grik1 gene knocked out selectively in the PV-expressing interneurons showed no change in anxiety-like behavior after CRS while their wild-type counterparts demonstrated an increase in anxiety-like behavior observable in the elevated plus maze test. Thus, ablation of GluK1 in PV+ interneurons affects the stress-induced anxiogenesis. Due to low number of animals, it cannot be confirmed yet whether the deletion leads to stress resilience or a phenotype where even regular handling is an aversive experience comparable to physical restraint. GluK1 KAR modulation of PV+ interneuron excitability and its susceptibility to stress-related alterations is only a recently discovered phenomenon, and even though this study provides some insight into the underlying mechanism, further research is needed. Systematic characterization of the mechanism could provide a novel tool for understanding and treating stress-related pathological anxiety, possibly helping patients suffering from anxiety disorders resistant to current treatments available.

Diabetes mellitus is an incurable disease caused by dysfunctional insulin signaling. The brown adipose tissue (BAT) serves as a hotspot for both lipid and glucose consumption and is thus an attractive target for treating metabolic diseases. Newly surfacing evidence suggest that the endothelial cells (ECs) lining the inner layer of vessels might regulate the morphology and function of adipose tissues. Several studies, including our own, suggest that the vessel density is negatively affected by metabolic diseases. As the BAT is an important organ for systemic lipid and glucose metabolism, and as the effects of metabolic diseases on BAT vessels are not adequately explored, I wanted to investigate how the BAT vasculature changes upon early time points of type 1 (T1D) and 2 (T2D) diabetes in this thesis work. To this end, I used mouse models with chemically induced T1D and genetic T2D and characterized these models with immunohistochemical analyses and immunoassays. To explore the transcriptomic landscapes of ECs and adipose stem cells (ASCs), I analyzed scRNAseq data of BAT stromal vascular fractions (SVF), focusing on changes in gene expression and EC-ASC interactions at a transcriptomic level. Also, by using a publicly available single-cell RNA sequencing (scRNAseq) dataset, I compared BAT SVF gene expression to complement the data resulting from our experiments. The results from this work reveal differential angiogenic responses in the T1D and T2D mouse models and open new avenues of research into how these different pathways are activated and how we can take advantage of these differences to treat diseases. All in all, this work will support the efforts in developing better options for future diabetes prevention, diagnosis, and care.

Normal sex differentiation depends largely on the healthy development of the bipotential gonad, which is identical in both sexes during early stages of embryonic development. Sex differentiation towards the female phenotype is initiated by the expression of pro-ovarian genes, among which Forkhead Box L2 (FOXL2) is an important regulator. Moreover, FOXL2 was found to be one of the genes most widely implicated in female disorders of sex development (DSD). However, there is a lack of understanding regarding its precise role during ovarian differentiation and development. In order to study the gene during early gonadal development, human embryonic stem cells (hESCs) were used as a model. An inducible FOXL2 activation line was generated in vitro, by applying the CRISPR/Cas9 technique in combination with the tetON and destabilized DHFR systems. The cells were also subjected to gonadal differentiation, based on a previously established protocol. The results showed that the establishment of the activation line was successful, and expression of FOXL2 could only be observed in cells that were treated with trimethoprim and doxycycline. Similar findings were observed in the differentiated activator cells, as again only the induced cells expressed FOXL2. On the other hand, both induced and non-induced differentiated cells showed expression of bipotential gonadal marker genes LHX9, EMX2, GATA4 and WT1. However, in the induced cells a lower relative expression of these markers could be observed. Therefore it seems that relative expression of bipotential gonadal markers was affected by FOXL2 activation. The expression of female gonadal marker genes RSPO1, FSHR, WNT4, AMH and FST was not influenced by FOXL2 activation during gonadal differentiation, as most of the markers showed similar levels of expression in both induced and non-induced cells. Therefore further research needs to be conducted to determine optimal time point of FOXL2 activation during differentiation. Nevertheless, an in vitro model could be generated, which could help in the future to further study the role of FOXL2 in gonadal differentiation, and to better understand pathological mechanisms underlying female DSDs.

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.

Educational technology is advancing rapidly, with VR (virtual reality) emerging as a promising branch of XR (extended reality) technology for educational purposes. Utilizing head-mounted displays (HMD), immersive VR experiences immerse users in a virtual environment, limiting their awareness of the physical world. VR proves valuable in education by complementing traditional teaching methods, offering experiences impossible in the physical realm. Studies indicate enhanced affective factors, understanding, motivation, and memorization among students. In biology education, VR serves as a visual aid, helping students grasp complex biological concepts difficult to visualize from a two-dimensional textbook. It also shows potential in supplementing hands-on activities like laboratory work and anatomical dissections, experiences outside classrooms, and sustainability education. However, challenges persist in VR's educational application, including uncertainty about learning outcomes, health concerns, high costs, and a general lack of expertise in VR design and pedagogical implementation. Educational VR design has thus far lacked a foundation in pedagogy and learning theories. This thesis aims to address this gap by reflecting on the development of a pedagogically meaningful VR experience within sustainability education. Collaborating with the Global Campus project of the University of Helsinki, the thesis introduces a VR experience integrated into the immersive virtual sustainability learning experience, Serendip. The design process involved literature research, user and expert interviews, and consideration of learning theories such as constructivist learning, experiential learning, flow theory, gamification, CTML, SDL, and CLT. Specific aspects of VR design, like immersion levels and prior knowledge of users, were also considered. The thesis's significance lies in pioneering pedagogy-based design for educational VR, particularly addressing complex, abstract, and multidisciplinary subjects. It emphasizes the need for collaboration among pedagogy, content, and VR animation experts in future educational VR design. This work serves as a potential template and inspiration for further research in the field, aiming to refine the integration of pedagogical principles into VR experiences for education.

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.

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.

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.