Browsing by study line "Neurotiede"

The developing human hindbrain and its role in neuronal pathogenesis have been relatively difficult to study for ethical reasons. By using the dual-SMAD inhibition and WNT signalling induction, a new method to culture brain organoids to resemble the human hindbrain has recently been established. In this study the new method has been used to detect the developing hindbrain’s response to flaviviral infection. Model virus used in this study is the Zika virus (ZIKV) which is known to alter the development of central nervous system and cause microcephalia. Pathogenic activity of the virus is measured by detecting the morphology of the organoids during infection as well as screening the organoids activation against oxidative stress, in a form of KEAP1/Nrf2-ARE pathway activation. Three different clones of ZIKV, which differ from each other by one amino acid in their non-structural protein 1 (NS1) gene, were used in the infections as well as two different time points of development. Controversially to previous findings on ZIKV infections to brain organoids, our findings show that developing hindbrain-like organoids do not change in shape or size during ZIKV infection. There are no differences in the lack of morphological changes between one-month olds or two months old organoids or between the different ZIKV clone infected organoids. The activation of the KEAP1/Nrf2-ARE pathway was measured by screening the two final products of the pathway, Nqo1 and HO-1. By screening the mRNA levels of these two genes, it showed that different ZIKV clones affect the activation of the KEAP1/Nrf2-ARE pathway in different levels at different times of development. Also, the expression of the same gene can be altered by the age of the organoids. Additionally, the expression of the two genes were different from each other at given time points and in response to the different clones. These findings suggest that the different isoforms of NS1 of ZIKV may alter the developing hindbrain’s response to oxidative stress. Findings also show that the time of the infection can additionally play a critical role to the ZIKV infection. The altered response to oxidative stress may contribute to microcephaly: the oxidation homeostasis of the developing hindbrain is modified, and apoptotic cell death can take place.

Schizophrenia (SZ) is a neurodevelopmental psychiatric disorder with high heritability. Patients with SZ commonly suffer from sleep problems of different types, some of them with potential underlying abnormalities in sleep oscillations. These changes in sleep are usually accompanied by deficits in cognitive performance. However, the relationship between sleep, cognitive performance and genetic risk factors are not well known in SZ. In this study, patients were selected from a nation-wide SUPER -cohort. Sleep and circadian rhythm of patients with SZ (n = 26) and age-matched healthy controls (n = 11) were followed for a week with actigraphy and sleep diary, combined with word-pair -memory task and polysomnography at the end of the week. The results showed that patients spend more time in lighter sleep and awake during the night than controls. As expected, patients had impaired sleep spindle density compared to controls. Additionally, patient had worse overnight memory consolidation. However, sleep spindle density was not associated with memory performance. Lastly, polygenic risk score (PRS) for long sleep, but not PRS for SZ, predicted lower spindle density in patients, which could be indirect evidence for deviated neurophysiological processes of sleep behind the observed deviations in EEG oscillations among the patients. These results show that, as compared to controls, patients with SZ demonstrate abnormalities in their sleep, which can be seen both in macro- and microstructures of sleep. Further analyses of the interplay between sleep oscillations and genetic risk factors are likely needed to link sleep problems with overnight memory consolidation.

Faculty: Faculty of Biological and Environmental Sciences Degree programme: Master’s Programme in Neuroscience Study track: Neuroscience Author: Emma-Helka Erkkilä Title: The brain physiology of stress and the effects of burnout on executive functions Level: Master’s thesis Month and year: 08/2022 Number of pages: 35 Keywords: executive functions, emotion, cognition, stress, burnout Supervisor or supervisors: Docent Kaisa Hartikainen and Lic.Med. Mia Pihlaja Where deposited: Helsinki University Library Additional information: Abstract: BACKGROUND- Burnout as a result of prolonged and excessive stress may impair higher order cognitive functions of the brain such as executive functions and their efficiency. This Master's thesis examines the effects of chronic stress on the brain, more specifically the effects of burnout on executive functions. The aim of this study was to specifically research the effects of burnout on executive and emotional functions and their interaction. The research was conducted at the Behavioral Neurology Research Unit, Tampere University Hospital as part of Sustainable Brain Health project funded by the European Social Fund. MATERIAL AND METHODS- 54 voluntary examinees of whom 51 were analyzed. The examinees were divided into two groups based on BBI-15 survey (27 suffering from burnout and 24 control subjects without burnout). The examinees performed a computer-based Executive reaction time (RT) test, during which a 64-channel electroencephalogram (EEG) was recorded. In additions all examinees received alternating transcutaneous vagus nerve stimulation (tVNS) and placebo stimulation. From the Executive RT test, we obtained objective measures reflecting the efficiency of executive functions (RT and total errors) and specific executive functions such as working memory, inhibition and attention. Additionally, the emotional stimulus included in the test enabled the assessment of the emotional functions and the interaction between emotional and executive functions. The EEG and tVNS results were not in the scope of this master’s thesis, and they will be reported later on. RESULTS- The results of this thesis are preliminary. Distinct positive correlation was observed between burnout assessment based on the BBI-15 survey and the results of the BRIEF-A self-report which measures the subjective experience of challenges in executive functions in daily life. There was no statistically significant (p<0.05) difference between the groups in RTs or errors made in the Executive function RT test. Instead, the groups differed on how the threatening emotional stimulus affected the accuracy of responses. Subjects suffering from burnout made less errors with a threatening emotional stimulus compared to a neutral stimulus and vice versa the control subjects made more errors with the threatening emotional stimulus compared to neutral. This difference was statistically significant (p=0,025). DISCUSSION- Challenges experienced in everyday executive functions were linked with burnout. However, RTs and errors in the Executive reaction time test did not correlate with the severity of the burnout nor were the self-evaluated problems in executive functions depicted in the test performance. Instead, the subjects suffering from burnout differed from the control group in how the threatening stimulus affected the accuracy of responses in the test. It is possible that the subjects suffering from burnout benefit from the increase in arousal caused by the threatening emotional stimulus which was shown as improved accuracy of responses when there was a threatening stimulus, whereas the control group's accuracy of responses was disrupted by the threatening stimulus. We speculate that if the control group’s baseline level of arousal was optimal then the threatening emotional stimulus may have increased arousal to suboptimal level causing decrease in performance. Subjectively experienced challenges in executive functions and objective changes in the interaction between emotions and the executive functions were observed in the study. In conclusion, burnout causes changes in executive functions.

Spinal cord injury (SCI) in human patients is the most expensive clinical condition worldwide, restricting individuals’ ability to manage with daily-life activities independently. With very limited available treatment possibilities, the understanding and validating of regenerative mechanisms and treatment options in animal models is crucial for their translation to clinical practice. The majority of SCIs in human patients are contusive in the cervical level of the spinal cord. However, thoracic injury rodent model is more commonly studied, with only recent studies working with cervical contusion injury model. Chondroitin sulphate proteoglycans (CSPGs), and especially their CS chains, are thought to be the major inhibitory structures for neurite regeneration after SCI. However, current research has led to a new idea that the inhibitory effect of CS chains can be reversed to regeneration enhancing by heparin-binding growth-associated molecule (HB-GAM). This endogenously secreted molecule is highly up-regulated in the central nervous system (CNS) during postnatal development, but in the adult CNS the expression is down-regulated. This suggests that postnatal-level concentrations might be needed for inducing neurite regeneration in adult CNS. In this study, HB-GAM treatment was tested on both cervical hemicontusion and hemisection injury models. Here we show that repeated intrathecal injections of HB-GAM were sufficient to increase grey matter myelin optical density in mouse hemicontusion injury model, and partly induce functional recovery in hemisection model. Obtained anatomical evidence suggests that enhanced myelination is potentially involved in the repair mechanism of HB-GAM. The connection between HB-GAM treatment and functional recovery, and also other mechanisms of HB-GAM-induced regeneration need further exploration. In broader perspective, the results are promising for translation of a novel treatment approach to clinical use.

Recent studies have associated ER stress with various types of hearing loss, such as drug- and noise-induced, age-related, and hereditary hearing loss. However, the research has mostly focused on auditory sensory cell (hair cell) death, and it is not well understood if other molecular mechanisms can drive ER stress-dependent hearing loss. We used Manfflox/flox;Pax2-Cre conditional knockout (cKO) mice under the C57BL/6J (B6) mouse strain to study the effects of genetically-induced chronic ER stress on hearing function. In these mice, the gene coding for mesencephalic astrocyte-derived neurotrophic factor (Manf) has been silenced specifically in the cochlea. Manf is thought to act as an ER homeostasis regulator, and it has shown cytoprotective properties in different disease models both in vitro and in vivo. However, Manf’s mode of action is still poorly understood and even less is known about its function in the inner ear. Previously, cKO mice were found to upregulate ER stress markers in the cochlear hair cells. These mice develop progressive high-frequency hearing loss characterized by high-frequency outer hair cell (OHC) death. However, they have elevated hearing thresholds already at postnatal day 22 (P22) before any OHC death takes place and have elevated hearing thresholds in hearing frequencies where OHCs are retained. Therefore, there has to be another pathological mechanism besides OHC death accounting for the elevations in their hearing thresholds. Hence, we wanted to study the effect of ER stress on the outer hair cell hair bundle structure. The hair bundle is located at the apical pole of the hair cells, and it consists of filamentous actin (F-actin)-filled stereocilia. In mechanotransduction (MET), sound stimuli-induced motions of cochlear fluids cause stereocilia to deflect towards the tallest stereocilia row, allowing for depolarization of hair cells and transformation of mechanical force into electrical signal. Therefore, hair bundle is an essential structure for the hearing function. We used scanning electron microscopy (SEM) and fluorescent microscopy to study OHC hair bundles of cKO mice. We saw disorganization of the bundle structure already at P22. It progressed with age and advanced to strong stereocilia fusion by P56. At this age, all of the high-frequency OHCs of cKO mice displayed stereocilia fusion. We used cochlear whole mounts and immunostainings to study the protein composition of OHC stereocilia of Manf-deficient mice. The base of the stereocilia, termed as the tapering region, contains proteins that link the plasma membrane of stereocilia to their F-actin core, ensuring the cohesion of individual stereocilia. Mutations in these proteins have been associated with stereocilia fusion and hair bundle disorganization. At P56, we saw that stereocilia tapering region proteins radixin (RDX) and myosin 6 (Myo6) were mislocalized from the tapering region towards the apical tips of stereocilia in the high-frequency OHCs of cKO mice. Additionally, we saw that PTPRQ – a tapering region protein that is under normal conditions expressed only in the IHCs of mature cochlea – was upregulated in OHCs of cKO mice, yielding an expression pattern similar to RDX and Myo6. In addition, we used the F-actin probe phalloidin to quantitatively compare F-actin densities in the cuticular plates of cKO and WT mice. Cuticular plate is a structure responsible for attaching stereocilia to hair cell body. It consists of a dense F-actin network and prior studies have associated defects in the cuticular plate composition with hearing loss and stereocilia bundle abnormalities. We found a significant decrease in phalloidin staining intensity in the cuticular plates of high-frequency OHCs of cKO mice, indicating that their cuticular plate F-actin rigidity had been reduced. Together our data shows that Manf deficiency promotes diverse impairments in the OHC hair bundles, consequently inducing hearing loss. To conclude, our study presents novel insights into the complexity of ER stress-induced cochlear pathology. We show that ER stress impairs MET by inducing structural changes in the OHC hair bundle. It appears to be the major reason for hearing loss in the cKO mice, rather than hair cell death. In the future, the impact of Manf deficiency to the inner ear should be further studied. For example, younger and aged cKO mice could be studied to better characterize the progression of Manf deficiency-induced cochlear pathology and hearing loss. Similarly, Manf’s effect on hearing should be studied in other ER stress models to determine its role in the hearing function.

Background: Despite the well-established link between diabetes and dementia risk, the impact of prediabetes and diabetes on the prodromal dementia phase remains controversial. In this study, we investigated whether prediabetes and diabetes increase the risk of cognitive impairment–no dementia (CIND) and accelerate its progression to dementia, as well as the possible underlying mechanisms. Methods: In the Swedish National Study on Aging and Care-Kungsholmen (SNAC-K), one cohort of cognitively-intact individuals (n=1,837) and one cohort of individuals with CIND (n=671) aged ≥60 years were followed for up to 15 years. At baseline and each follow-up (every 3 or 6 years), a neuropsychological test battery was administered, and the domains of episodic memory, processing speed, executive function, visuospatial abilities, and language were derived. CIND was defined as having no dementia and cognitive performance ≤1.5 SDs below age group-specific means in at least one cognitive domain. Dementia was diagnosed according to DSM-IV criteria. Diabetes (controlled and poorly-controlled) was diagnosed by physicians through medical assessment, clinical records, and glycated hemoglobin (HbA1c) ≥6.5%. Prediabetes was identified as HbA1c 5.7-6.4% in diabetes-free participants. Clinicians diagnosed heart disease and collected blood samples used to measure C-reactive protein (CRP). Data were analyzed with Cox regression models adjusted for possible confounders. Results: At baseline, in the cognitively-intact cohort, 133 (7%) participants had diabetes and 615 (34%) had prediabetes. During follow-up (mean 9.2 ± 3.0 years [range=2.2-15.5 years]), 544 (30%) individuals in the cognitively-intact cohort developed CIND. Poorly-controlled diabetes (HbA1c ≥7.5%) was associated with 2-times higher risk of CIND (HR 2.0, 95% CI:1.11-3.48) than diabetes-free participants. In the CIND cohort, 84 (13%) had diabetes and 238 (36%) prediabetes. During follow-up (mean 7.7 ± 4.0 years [range=0.2-15.2 years]), 132 (20%) individuals progressed to dementia. Poorly-controlled diabetes was associated with 3-times higher risk of dementia progression (HR 3.3, 95% CI: 1.29-8.33). Furthermore, comorbid heart disease and diabetes was associated with 2.5-times higher risk of progression to dementia (HR 2.5, 95% CI: 1.17-5.47), particularly if the diabetes was poorly-controlled (HR 5.8, 95% CI: 1.72-19.3). Similarly, having elevated CRP levels and diabetes was associated with increased risk of progression to dementia (HR 4.1, 95% CI: 1.15-14.2), especially in participants with poorly-controlled diabetes (HR 13.6, 95% CI: 1.89-98). No associations between prediabetes and CIND were detected in either cohort. Conclusions: Diabetes, especially if poorly-controlled, increases the risk of cognitive impairment and accelerates its progression to dementia. The diabetes-associated progression from CIND to dementia is further exacerbated by the presence of heart disease and elevated levels of systemic inflammation.

Kainate receptors (KARs) act as prominent regulators of neuronal excitability, network activity as well as neurotransmitter release in the developing brain. In the neonatal hippocampus the GluK1 subunit containing KARs take part in regulating the activity of the CA3 interneurons and hence the maintenance of early synchronous network oscillations, which are thought to be vital for developing connections. In the interneurons of the CA3 subfield this regulatory activity is likely performed through a noncanonical, G-protein mediated inhibition of a Ca2+ sensitive medium-duration afterhyperpolarizing current (ImAHP). As in various central neurons the ImAHP has been shown to be regulated by voltage-gated calcium channels (VGCCs) and as the activity of the voltage-gated calcium channels has been in turn shown to be modulated by G-protein coupled signaling of GluK1 KARs, we went on to investigate whether a direct link between GluK1 KARs and VGCCs could be detected in the CA3 stratum radiatum interneurons of neonatal hippocampus. Here we show that the pharmacological inhibition of GluK1 KARs does not affect the amplitude of Ca2+ influx through VGCCs in the CA3 stratum radiatum interneurons of acute hippocampal slices from neonatal mice. As G-protein mediated signaling has been shown to induce alterations in the voltage-dependence of the VGCC-mediated currents, we similarly investigated the effects of GluK1 inhibition on the current-voltage relationship of Ca2+ currents in CA3 interneurons during the first postnatal week as well as during the second postnatal week, since GluK1 subunit is known to undergo developmental changes in its expression during this time. No significant effect was however detected in either of the age groups. Although in our experiments the GluK1-KAR inhibition seemed to induce no statistically significant changes in the Ca2+ current amplitudes or in the voltage-dependence of VGCC-mediated currents in the CA3 interneurons, further, more specific studies should be encouraged to investigate the phenomenon in specific interneuron subtypes and in distinct calcium channel families.

Beta frequency (15-25 Hz) oscillations in the extracellular field potential recorded by cortical EEG and depth electrodes have been connected to stopping. Especially short increases in beta power, so called beta bursts, occur more frequently close to stopping an ongoing movement or when cancelling a planned action. However, there are discrepancies about the causal role of these beta bursts on stopping. Although some studies indicate causality, in others the bursts occur too late for being causal or their number does not increase prior to stopping. One explanation to the disagreement may lie in the behavioral task commonly used to study the neural correlates of action inhibition, the stop signal task. In this task the movement is cancelled before it starts, and actual stopping is thus hidden from the experimenter. Instead, an estimated stop signal reaction time is mathematically modelled. It is likely that this reaction time varies trial by trial, which causes inaccuracy in the results. We were able to define an exact stopping time using head fixed rats running on a treadmill. This enabled us to align brain activity precisely with stopping. With this task, we showed that the number of transient beta bursts increases just prior to stopping. Moreover, the increase correlates with the velocity. These results indicate that beta bursts are causal to stopping. Beta bursts have been noted to be disturbed in Parkinson’s disease and our results may open new doors for early diagnoses or treatments.

Sensory systems display a topographical organization, and in the murine somatosensory system there is oneto-one correspondence between individual whiskers and individual cortical columns called barrels. Functional connectivity in the whisker-to-barrel system is formed prenatally and refined after birth, guided by both spontaneous and whisker-evoked activity. GABAergic connectivity emerges already prenatally and includes transient circuits, but the exact role of GABAergic signalling in early development is elusive. The neuronal, major chloride extruder, potassium-chloride cotransporter (KCC2) is heavily upregulated in the cortex during the first two postnatal weeks resulting in the emergence of hyperpolarizing inhibition. However, in cortical interneurons (INs) KCC2 expression can be detected already at the time of birth. The role of this early interneuronal KCC2 expression is unclear. The aim of this thesis was to study the role of KCC2 in the network activity of cortical INs during the perinatal period. Transgenic mice with conditional inactivation of Kcc2 gene, and expression of the calcium indicator GCaMP6f in GAD2+ neurons (INs) were used to image cortical Ca2+ activity. Transcranial widefield Ca2+ imaging in awake head-fixed mice was performed at the day of birth (P0) and showed that spontaneous, but not evoked, activity was significantly reduced in the knock-out animals. Moreover, immunostaining for the activity-induced transcription factor Egr1 showed that thalamic network activity was significantly decreased in the knock-out and heterozygous animals, suggesting involvement of subcortical areas in the decreased cortical activity. Additional experiments are needed to elucidate the role of other mechanisms contributing to the observed change in activity.

In this master’s thesis project, I studied the association of lipid molecules phosphatidylinositol 4-phosphate (PI4P) and phosphatidylinositol 3-phosphate (PI3P) with autophagy in neurons. One of the aims of the study is to determine the level of basal autophagy in primary hippocampal neurons and to come up with a protocol for autophagosome observation without forcing radical changes in cell culture conditions. Other mammalian cells have extremely weak basal autophagy, but they increase it significantly in response to starvation, for example. However, neurons are extremely sensitive to any changes in their surroundings. They change their morphology, behaviour and biochemical properties, and often they simply do not survive. Therefore, the goal is a protocol for successful autophagy observation with minimal external influence. Despite the debate around basal autophagy in neurons, I observed high levels of basal autophagy in neuronal cells incubated in media without supplements. Also, my observations revealed that the inhibition of the last step of autophagosome processing with Bafilomycin A1, was enough to cause the massive accumulation of large autophagosomes. Results demonstrated that primary hippocampal neurons exhibit high levels of basal autophagy, suggesting that on the contrary to other mammalian cells neurons might not have enough potential to increase autophagy when it is induced pharmacologically or by stressful conditions. This would explain why autophagy induction is often claimed to be ineffective for neuronal cultures. The main goal is to observe and compare PI4P presence on autophagosomes in normal conditions and when autophagosome:lysosome fusion is inhibited with Bafilomycin A1. The side goal is to observe PI3P presence on autophagosomes as well. I transfected primary hippocampal neurons with fluorescent probes for PI4P or PI3P as well as for autophagosome-related protein LC3. Localization data was collected with live-cell imaging on a confocal microscope. As expected, PI3P was not detected on autophagosomes located in soma. It is involved in the initial vesicle biogenesis in distal axons but not in later events taking place closer to the cell body. PI4P showed high degree of colocalization with LC3, indicating PI4P presence on autophagosomes, but only when the fusion was presumably inhibited by Bafilomycin A1. These results suggest that PI4P appears on autophagosomes either as a result of compensatory pathway, where autophagosomes fuse with late endosomes instead of lysosomes; or as a molecule normally involved in autophagosome:lysosome fusion. Literature supports the latter explanation, but it cannot be confirmed without further research. These results give an insight into PI4P role in neuronal autophagy and might be relevant for the future research of autophagy disruption and aggregate accumulation in neuronal diseases as a consequence of abnormal lipid signalling, lipid metabolism and transport.