Browsing by study line "Neurotiede"

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.

Meningeal lymphatic vessels (mLVs), a recent functionally characterized structure in the meninges, contribute to the clearance of macromolecules, immune cells and metabolic waste from the central nervous system to peripheral lymph nodes. Having been identified as a route of clearance, there is a focus on understanding their role in neurological disease pathology. Here we consider their function in the pathology of traumatic brain injury (TBI) particularly in blood solute clearance, lesion progression and neuroinflammatory response. We use a transgenic model of mLV developmental dysfunction, K14-VEGFR3-Ig, to analyze the progression and severity of a controlled cortical impact (CCI) injury. We show that in mice lacking mLVs there are a higher percentage of microglia cells in an activated state in the hippocampus whereas the progression of hematoma and lesion size does not differ from wild type. Our results suggest that at two months post injury, meningeal lymphatics could be functionally important in modulating microglia activation, which is associated with chronic inflammation.

Sleep is one of the most vital functions of newborns and infants, and it is essential for neuronal network development. Therefore, long-term sleep disturbances have been associated with growth delays and behavioral disorders. Commonly reported infant sleep disturbances, such as night awakenings and difficulties falling asleep, cause distress to parents. Yet, the development of infant sleep in the home environment has not been fully elucidated due to lack of objective measurement parameters. In the current study, we assessed the feasibility of a motion sensor, attached to wearable pants, and ECG textile electrodes to monitor sleep-related respiration and heart rate of newborns and infants. First, we compared signals recorded by the motion sensor’s measurement channels to the standard respiratory piezo effort belt’s signal during daytime EEG recordings. According to our results, the motion sensor’s gyroscope proved to measure respiratory rate most accurately, while the ECG signal transmitted by the sensor was reliable in interpretable sections. We then provided wearable garments and smartphones to families with infants to assess overnight home-use. Our results indicate that different sleep states could likely be identified based on respiration fluctuation visible in the gyroscope’s signals. Moreover, the wearable system was considered practical and easy to use by the parents. Future studies should focus on validating the sensor with clinically approved measures, in order to train the algorithms to automatically identify different sleep-wake states. By doing so, the wearable sensor could provide information on natural infant sleep structure development over long time periods. Additionally, clinical validation of the sensor may result in the development of a companion diagnostic tool for infant cardiorespiratory and movement disorders.

Efficient processing of auditory information begins to emerge early in human ontogeny and establishes foundations for learning language from speech exposure. Here we show that repeated exposure to spoken words causes in neonatal brain attenuated neural responses that are linked to language skills at the age of 24 months. In the study, 75 newborn infants were exposed to repeated presentation of two spoken disyllabic pseudowords. During the word exposure, event-related potentials to presented pseudowords were measured with electroencephalography. The study provides three kinds of findings regarding neonatal brain dynamics and repetitive word exposure. Firstly, the results show that continuous exposure to spoken pseudowords modulates neonatal brain activity and can lead to attenuation of neural responses. This neural suppression likely reflects neonates’ early capacity to recognize spoken words and form neural representations of the stimuli through repetition. Secondly, the attenuated neural responses were bound to the presentation of the first syllable and did not occur after presentation of the second syllable. Thirdly, occurrence of neonatal neural suppression was associated with better expressive language skills later, at the age of 2 years. Altogether, the results provide preliminary evidence that neonatal brain responses to word repetition can be utilized to indicate efficiency of learning language from speech exposure and later state of language development.

The sense of hearing is dependent on the sensory cells of the cochlea: inner and outer hair cells. The critical functional structure of these cells is the stereocilia bundle, the mechanotransduction (MET) organelle. The outer hair cells (OHCs) are highly vulnerable to environmental assaults, the effects of aging, and gene mutations. This vulnerability is thought to be mediated by challenges in maintaining intracellular calcium homeostasis. Endoplasmic reticulum (ER) stress is a form of cellular stress that was previously shown to cause hair cell impairment. A possibility is that this impairment is mediated by perturbations in calcium homeostasis. In this thesis, the aim was to find out how the OHC calcium homeostasis is affected by specific ER stress-inducing mutations and age in mouse models exhibiting progressive hearing loss. I studied OHC calcium homeostasis in Manf conditional knock-out (cKO) mice under C57BL/6 (B6) strain in which ER homeostasis-promoting protein MANF (Mesencephalic Astrocyte-derived Neurotrophic Factor) is depleted in cochlear hair cells. Previous studies have shown that these mice develop progressive hearing loss that includes OHC loss and prominent stereocilia pathology, stereocilia fusion. By itself, the B6 mouse strain is a model of age-related hearing loss due to a Cdh23 missense mutation that is known to cause progressive hearing loss and, according to earlier evidence, may be a source of ER stress. I studied B6 mice at 6–9 months of age and Manf cKO mice at 2 months of age to comparatively examine changes to OHC calcium homeostasis that may correlate with the changes in the stereocilia bundle morphology and with hearing loss. I studied hearing function by auditory brainstem recordings in vivo. I estimated the functionality of MET channels in OHCs by FM1-43 uptake. I used immunofluorescence microscopy to study subcellular expression of key calcium-buffering and calcium-extrusion proteins in OHCs. I used a novel super-resolution imaging technique, expansion microscopy (ExM), to study stereocilia bundle morphology. OHCs of Manf cKO mice showed changes in calcium homeostasis in all the studied aspects: (1) FM1-43 uptake through MET channels was reduced, (2) the expression of the calcium extrusion pump PMCA2 and its obligate partner, the cell adhesion protein neuroplastin, was decreased, (3) and the expression of calcium-buffering protein oncomodulin was increased. All this data indicated OHC calcium dyshomeostasis. These molecular changes were consistent with the hair bundle pathology—stereocilia fusion—revealed by phalloidin staining of the actin-rich core of the stereocilia followed by ExM imaging. OHCs of 6–9-month-old B6 mice exhibited reduced FM1-43 uptake, yet not to the extent seen in cKO mice, and there were no changes in PMCA2 and neuroplastin expression and no prominent stereocilia fusion. Together, I show in this study that OHC hair bundle dysmorphology is linked with changes in calcium homeostasis in the mouse model of ER stress-induced hearing loss. This is consistent with the fact that calcium dyshomeostasis is an integral part of cellular ER stress. An intriguing, yet unanswered question is whether these changes in stereocilia bundle physiology could actually be the trigger for the death of these sensory cells.