Browsing by study line "Neurotiede"

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.

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.

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.

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.

The analysis of gaze behaviour is nowadays commonly employed to help with the diagnosis and exclusion of differential neurological conditions as well as to help researchers better understand cognition in the early stages of life. However, its application in the developmental evaluation and follow-up of children with early-onset epilepsy has not been profoundly studied yet. Therefore, the current study aimed to investigate the association between the gaze behaviour of infants with early-onset epilepsy and their future neurodevelopmental outcome. To study the association and its predictive ability, three models were created. Sixty-three infants with epileptic seizure onset before 12 months of age participated in the study with the voluntary consent of their parents. Infants’ gaze behaviour was recorded with Tobii Pro-X3-120 at two measure points. The results showed infants’ initial ability to fixate their gaze, changes in their gaze shift probability in the first 12 months of life, and structural aetiology to be significantly associated with the infants' developmental outcome at 24 months of age. Where the structural aetiology was significantly associated with poorer developmental outcome, good initial fixation ability and improvements in the infants’ gaze shift probability during their first year of life were significantly associated with more positive outcome. These findings suggest that gaze behaviour at an early age is an essential predictor of later development in infants with early-onset epilepsy. Hence, eye-tracking could provide means to evaluate the later neurocognitive outcome of infants with early-onset epilepsy at an early age.

Kainate receptors are known to regulate neuronal function in the brain (Li, H., & Rogawski, M. A. (1998), Braga, M. F. et al. (2004), Lerma & Marques (2013), Carta, M (2014)). In the amygdala, they have been shown to affect synaptic transmission and plasticity, as well as glutamate and γ-aminobutyric acid (GABA) release (Li, H. et al. (2001). Braga, M. F. et al. (2003), Braga, M. F. et al. (2009), Aroniadou-Anderjaska, V. et al. (2012), Negrete‐Díaz, J. V. et al. (2012)), however, their role during development of the amygdala circuitry is not known. In the present study, we wished to understand how GluK1 kainate receptors regulate synaptic population activity and plasticity in the developing amygdala by using extracellular field recordings in P15-18 Wistar Han rat pup brain slices. Since field excitatory postsynaptic potentials (fEPSPs) are not commonly measured from the amygdala, we first sought to pharmacologically characterize the basic properties of the extracellular signal, recorded from the basolateral amygdala in response to stimulation of the external capsulae (EC). Having confirmed the validity of the fEPSP as a measure of postsynaptic population response, we were able to show that blocking GluK1 with (S)-1-(2-Amino-2-carboxyethyl)-3-(2-carboxy-5-phenylthiophene-3-yl-methyl)-5-methylpyrimidine-2,4-dione (ACET), a selective GluK1 antagonist, had no effect on the fEPSP. Furthermore, activation of GluK1 with RS-2-amino-3-(3-hydroxy-5-tert-butylisoxazol-4-yl) propanoic acid (ATPA), a GluK1 agonist, reduced the amplitude of the fEPSP, without affecting its slope, suggesting an increase in inhibitory signaling within the network. Blocking GABAergic activity with GABAA- receptor antagonist picrotoxin significantly reduced the effects of ATPA. Additionally, the increase in inhibitory signaling due to the activation of GluK1 was confirmed with whole-cell voltage clamp, by measuring spontaneous inhibitory postsynaptic current (sIPSC) frequency. Activation of GluK1 heavily increased sIPSC frequency in the basolateral amygdala neurons. Finally, we were also able to show that activation of GluK1 with ATPA strongly attenuates LTP induction. These results show that GluK1 kainate receptors play a vital role in the modulation of synaptic transmission and plasticity in the developing amygdala.

The vagus nerve is the longest nerve of the autonomic nervous system. It innervates, among other organs, the stomach, the lungs and the heart, and it reaches several areas of the brain, including the locus coeruleus and the amygdala. The invasive stimulation of this nerve (vagus nerve stimulation, or VNS) is a currently used method for the treatment of refractory epilepsy and pharmaco-resistant depression (Englot et al. 2011; O’Reardon et al., 2006), but the impact that this technique might have on the brain physiology and functions is still under investigation. Various studies (Frangos et al., 2015; Yakunina et al., 2016; Hansen, 2019) have shown that VNS increases noradrenaline production in the brain, a neurotransmitter that is involved in several cognitive processes, such as sleep and mood control. Furthermore, in a study on patients with epilepsy, by Sun et al. in 2017, VNS appeared to have a clear effect on working memory and emotion-attention interaction. Nevertheless, VNS presents all the risks and potential complications that characterize invasive procedures requiring surgery. Therefore, research is now focusing on safer, non-invasive alternatives, such as transcutaneous vagus nerve stimulation (tVNS). This technique allows to stimulate the nerve through its sensory fibres, located in the cymba and tragus of the ear. The scope of the present study was to see whether tVNS would have the same effects on cognitive and affective functions as VNS. The sample for this single blind placebo-controlled study was composed of 30 healthy subjects between 18 and 45 years old. Exclusion criteria included a history of psychiatric, neurological or cardiovascular diseases. All subjects were asked to complete a computer-based task, the Executive Reaction Times-Test. Throughout the test the subjects alternately received an active or a placebo stimulation, and their brain activity was recorded for the whole duration of the test using a 64-channel EEG cap. The Executive-Reaction Times-Test was chosen for this study because it allows to test multiple executive functions simultaneously. The subjects were presented with a series of stimuli on a screen and were asked to react as fast and accurately as possible to “Go” signals, and to refrain from responding when “NoGo” signals appeared. The test started with a triangle pointing either up- or downwards, followed by a brief pause and a traffic light image. The traffic light showed either a red or a green light and included an emotional distractor in the form of a spider or a flower. The red and green lights were alternately used as “Go” or “NoGo” signals, and the rule changed at each test block. In order to complete the task, subjects needed to keep the image of the triangle in their working memory, stay focused on the stimuli and be ready to react or be able to inhibit any responses, thus several main executive functions are being tested: inhibitory control, working memory, attention and emotion-attention interaction. Active stimulation was delivered through clip electrodes that were attached to the tragus of the left ear, whereas placebo stimulation was delivered through clip electrodes that were attached to the left ear lobe. The subjects were not aware of the difference between the two locations. Only the data of 18 subjects was used for the results analysis, because of technical difficulties with the EEG data (some recordings were too noisy, some presented flat channels). The behavioural data was divided into reaction times and errors, which were separately analysed. The EEG data was used to extract the amplitudes of the ERP peaks N2 and P3. The former is a negative peak visible at 200-350ms; the latter is a positive peak visible at 300-500ms. Previous studies have shown the peaks to be associated with response conflict and inhibition (Falkenstein et al., 1999; Donkers et al., 2004; Smith et al., 2013). The behavioural data analysis did not show any significant effect of stimulation on reaction times or error amounts. The ERP analysis, instead, returned interesting results. We observed a main effect of stimulation (p=0.04) in “NoGo” conditions. There was a significant reduction in the N2P3 amplitude and the N2 amplitude in “NoGo” conditions, with active stimulation compared to placebo. These results seem to suggest that with tVNS, fewer cognitive resources are allocated to resolve the inhibitory task, without worsening the subjects’ performance. The lack of significance in the behavioural results might have been due to a ceiling effect, with the Executive Reaction Times-test being too easy for our sample. Overall, the number of errors was too low to conduct a reliable statistical analysis. Nevertheless, the effects we observed on brain physiology would suggest that further research is needed to explore the actual impact of tVNS on cognitive and affective functions.

Intersectins (ITSNs) are important scaffold and adaptor proteins that play an important role in various cellular processes such as endocytosis. Although we know a lot about their function, there is little information on the regulation of these proteins. On the other hand, microRNAs have been shown to have an extensive function in regulating numerous genes in animals and their dysfunction is credited for down regulation of many proteins. In this study, I demonstrate that microRNAs are potential regulators of ITSNs in HEK293 cells and human neuronal cell cultures. In this study, I cloned 3’UTRs of different isoforms of intersectins (ITSNs) and microRNAs to the expression vectors to express them in cells. I then transfected HEK293T or neuronal stem cell line (HEL47.2) with the constructed vectors and used various methods to analyse the effect of microRNAs on the expression of ITSNs. The main methods I used were dual-luciferase assay, reverse transcription quantitative PCR and western blotting, human neuronal stem cell culturing and lentiviral transduction. My results demonstrate that there were two microRNAs that stood out from other and had a significant downregulation of ITSNs mRNA levels in HEK293T cells. Those were miR-124 and miR-19. However, in the human neuronal cell line I did not observe a significant alteration of the ITSNs transcript level. Additionally, I suggest that the given microRNAs regulate protein levels by promoting the decay of the ITSN transcripts. However, more studies are needed to show a stronger causative effect of microRNAs on ITSNs. Subsequent studies should also look at how multiple microRNAs can influence gene expression cooperatively.

Goal-directed behavior is reliant on the ability to choose correct actions to perform given the context of the situation while minimizing the interfering effect of goal-irrelevant stimuli. The ability to suppress inappropriate responses is called response inhibition. It can be seen as a higher order cognitive function which is one of the cornerstones for adaptive behavior in ever changing environment. Neural oscillations have been previously used to study at the neuronal processes underlying cognitive processes such as response inhibition. Neural oscillations are rhythmic fluctuation in the excitability of a neuron or a group of neurons. These temporal windows of excitability are thought to underlie efficient communication by changing the efficacy of the synaptic transmissions between neurons/group of neurons. Although, a lot has been uncovered about the different oscillations and their possible role in response inhibition, very little is known how the spectral content (power of a frequency) adapts across as the animal is learning to suppress their responses to new novel stimuli. This kind of learning associated spectral content adaptations has been observed previously in humans during motor learning for example. In the current study we aimed to look how spectral content adapts as the animals learn to suppress their responses to novel stimuli. We used head fixed rats on a treadmill that were trained to perform Go/NoGo task. Each rat performed 1-4 learning scenarios during which the “rules” for Go/NoGo task changes in an attentional set-shifting paradigm. We measured EEG from most of the rat’s cortex. EEG was measured from the point where the rat was first introduced to these novel stimuli until the rat had learned the new stimulus-response contingences. This EEG was divided into learning stages and the power spectrum was calculated for each of them. We observed power peaks centered around 1Hz, 2Hz, 4Hz, 8Hz and 11Hz across learning stages. However further analyses comparing average power across learning stages showed that these changes were not statistically significant. Thus, we did not observe gradual changes in power while rats were learning to suppress their responses to novel stimuli.

Introduction: Oxidative stress occurs in cells when reactive oxygen species are generated as a by-product of oxygen metabolism and start to accumulate excessively. While extensive oxidative stress is highly detrimental to the cells, trophic factors help them survive. Trophic factor MANF has interested especially Parkinson’s disease researchers, but recent findings suggest that MANF plays a role in many diseases, also ones with an early childhood-onset. For this reason, it is important to investigate MANF function in different cell types. We have studied how MANF-knockout human embryonic stem cells react to oxidative stress compared to wild-type human embryonic stem cells, by exposing the cells to hydrogen peroxide and ethanol. Results: MANF-knockout human embryonic stem cells were more sensitive to oxidative stress than wild-type cells, but the variation between measurements was remarkable and the differences were statistically insignificant. We found that a transcription factor of our interest localized in the cell nuclei of MANF-knockout cells upon oxidative stress exposure. Such a nuclear translocation did not occur in wild-type cells. Moreover, we found that high concentrations (>2%) of ethanol reduced the viability of cells in only four hours. Discussion: Our findings suggest that MANF-knockout human embryonic stem cells react to oxidative stress differently than wild-type cells. Additional studies are necessary to clarify whether MANF-knockout human embryonic stem cells are indeed more sensitive to oxidative stress than wild-type cells. In the future, it would be interesting to inspect whether MANF protects human embryonic stem cells when the cells are exposed to physiologically relevant ethanol concentrations for longer periods of time.