Browsing by study line "Neuroscience"

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.

Schizophrenia, a mental disorder affecting over 1% of the world’s population, has a 41-65% chance of being acquired in monozygotic twins, and shows a complex heritable pattern. Research has shown the involvement of various neuronal and glial cell types in the disorder’s progression. Recent studies are focusing on cortical interneurons, as clinical features of schizophrenia such as working memory deficits emerge due to the abnormal activity of these cells . The advent of induced pluripotent stem cell (iPSC) technology has made it easier to study schizophrenia disease mechanisms, with studies revealing differences in morphological and physiological properties of cortical interneurons in patients with schizophrenia. In this thesis , the aim was to optimize iPSC-interneuron differentiation protocol and live-cell imaging method suitable for disease modelling. Interneurons were differentiated from iPSCs with overexpression of inducible transcription factor, Achaete-scute homolog 1 (ASCL1). The iPSCs were derived from twin pairs discordant for schizophrenia and from healthy controls. Expression of interneuron-specific markers was verified using RT-qPCR and validated at the protein level by an immunocytochemistry (ICC) assay in the control cell lines first. Additionally, to estimate the formation of neurites and differences in neurite length and branching, the differentiated interneurons from the controls were subjected to live-cell imaging by IncuCyte S3 live-cell imaging system. Imaging parameters such as cell body cluster filter was optimized to visualize the neurites. To study interneuron involvement in schizophrenia, iPSCs from one twin pair discordant for schizophrenia were successfully differentiated. Interneurons strongly expressed Gamma-aminobutyric acid (GABA) neurotransmitter related neuronal markers: glutamate decarboxylase 67 (GAD67) and GABA at protein level. The neurons were identified as somatostatin (SST) subtype GABAergic neurons by their mRNA and protein expression. While it was possible to observe differences in gene expression, there were no clear differences in the morphology of the differentiated cells as well as the localization of markers in comparison to the healthy controls. Further studies should focus on having a protracted time for differentiation where more mature interneurons can be produced by establishing co-cultures with excitatory neurons. This will help replicate the in vivo cortical machinery which in turn will aid in better understanding of disease mechanisms.

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.

Gliomas are the most common malignant brain tumours. The most aggressive and lethal type of glioma is glioblastoma. It has a dismal prognosis, and, despite aggressive treatment, the average patient survival is 1-2 years. Although glioblastoma has a heavy impact on individuals and their families, as well as on healthcare systems, our current lack of mechanistic knowledge hinders the development of improved treatments and diagnostics. Recent studies showed that glutaminolysis, a metabolic pathway utilizing glutamine to produce α-ketoglutarate, is promoted in tumour cells, suggesting a significant role of α-ketoglutarate concentration in tumour progression. Therefore, I hypothesise that reduction of α-ketoglutarate concentration in glioblastoma might suppress glioblastoma aggressiveness. To address this hypothesis, I focus on another metabolic pathway controlling α-ketoglutarate concentration, namely the GABA metabolism. Here, I show that the expression of ABAT and GAD1, which encode rate-limiting enzymes of the GABA metabolism, is associated with the lower-grade of glioma and a better prognosis for patients. Interestingly the expression of ABAT and GAD1 negatively correlates with the expression of CD109, a glioma stemness marker. Furthermore, suppression of glioblastoma stemness by CD109 silencing induces ABAT and GAD1 expression. Taken together these results suggest that the upregulation of the GABA metabolism reduces glioblastoma stemness and proliferation. In future, I am planning to examine the effect of ABAT and GAD1 overexpression and knockdown on glioblastoma stemness and proliferation, as well as the underlying molecular mechanisms to understand how the GABA metabolism suppresses the glioblastoma progression.

Temporal lobe epilepsy (TLE), a condition defined by unprovoked and recurrent seizures originating from the temporal lobe, is among the most ubiquitous of the various forms of epilepsy. Despite being chronic and highly prevalent, the available treatment options concerning the same remains a critical issue. Since the current therapeutic condition of epilepsy requires more development, renewed focus studying its molecular mechanisms and therapies is imminent. One of the longstanding theories trying to decode the molecular perturbations in TLE has been deficits in GABAergic inhibition resulting in abnormal neuronal activation. K+ - Cl- co-transporter (KCC2) activity is vital for maintaining a hyperpolarizing GABA response. The past decades have intimately and causally linked the prognosis of the seizures observed in TLE with deficits in KCC2 functioning. However, the precise mechanisms relevant to the disruption of KCC2 activity are still blurry. Here we show how KCC2 de-stabilization/localization in the neuronal bilayer is a characteristic of epileptic animal tissue. With the help of co-immunoprecipitation assays, western blot, and mass spectrometry, we found that in normal healthy brain tissue, GM1 ganglioside present in the membrane has specific and direct interactions with the KCC2 cotransporter. However, in the pilocarpine model of TLE, the interaction of this complex was significantly disturbed, primarily in the hippocampus and to some extent in the cortex. Our results act as an extension to previous research which stated that the structural association of the KCC2 clusters with neuronal lipid rafts is crucial for the functionality of the KCC2 cotransporter. Having learned about the unique nature of the pathophysiology of TLE, it is imminent to note that additional research in the direction of studying its biochemical pathways is required. The findings of this experimental study support the claim that KCC2 and GM1 as a complex are closely associated in the epileptic conditions and hence, this research paves the way to further explore the role of KCC2 and GM1 as a consequential complex in the pathophysiology of TLE.

Cranial windows are commonly used in neuroscience for direct brain access, but they require skull removal which can lead to neuroinflammation, potentially affecting experiment outcomes. As the nature of the window varies from experiment to experiment along with parallel treatments, it is of particular interest to map the quality and extent of the resulting brain inflammation on a case-by-case basis to have a better understanding of inflammation-related confounding factors in future experiments. As the focus of our future experiments is on finding electrophysiological biomarkers of depression employing state-of-the-art in vivo electrophysiological tools through a cranial window, we would like to characterise the extent of inflammation linked to the cranial window of interest in a mouse model of depression. To achieve this, we implanted mice with a soft cranial window and afterwards, treated them with corticosterone for a month. The animals were sacrificed and astrocytic glial fibrillary acidic protein (GFAP) and microglial ionized calcium-binding adapter molecule 1 (IBA) were labelled on coronal brain sections and imaged using a confocal microscope. We quantified the percentage of GFAP+ and IBA+ pixels on brain slices as measures of neuroinflammation using an automatic global thresholding-based approach and a semi-automatised machine-learning-based approach of the QUINT workflow. Our thresholding-based analysis revealed a significant elevation in GFAP-positive pixels, indicative of astrogliosis, in mice subjected to both cranial window implantation and corticosterone treatment compared to controls. However, no significant changes in microglial reactivity were observed under similar conditions. Importantly, it appears that the cranial window alone did not evoke long-lasting brain inflammation and corticosterone only slightly affected astrocytic reactivity. And despite results using the QUINT workflow presented some confounds, our results provide important considerations for future experiments employing a combination of soft cranial window and chronic corticosterone treatment, but more research is needed to enhance the generalisability of our findings.

The mammalian circadian clock is critical to physiological homeostasis and oversees many important processes like sleep and nociception. Chronic pain, particularly, neuropathic pain (NP) is defined as pain caused by nerve injury and is commonly studied in rodents using the spared nerve injury (SNI) model. The SNI model strongly mimics NP pathology by causing hypersensitive pain responses. As pain and the circadian rhythm are actively linked (as proven by diurnal fluctuations in pain sensitivities in individuals with NP), these responses can disrupt overall well-being and increase an individual’s predisposition to diseases like cancer and depression. Therefore, avenues to discern the workings of the circadian clock have been explored, and some include the use of small-molecule modulators like TH301, that regulate specific core circadian genes. In this study, we explored the possible role of TH301 in normalizing disrupted circadian rhythms in a rodent model of NP, while also examining its potential as an analgesic agent.

Research conducted on neural oscillations have paved the way to unravel the complexities of the brain dynamics underlying behavior and cognition. Neuronal oscillations characterize neuronal activity and processing at all spatial scales from neuronal microcircuits to large-scale brain dynamics and hence link cellular and molecular mechanisms to circuit dynamics underlying behavior. Large-scale oscillations and their inter-areal synchronization can be identified from in vivo electrophysiological data from animal models as well as from human magneto- and electroencephalography (M/EEG) data. Large-scale oscillation dynamics identified from human M/EEG data has been critical for resolving whole-brain oscillation dynamics view but is hindered by the indirectness of the measures. In contrast, rodent in vivo electrophysiology has been conventionally used to resolve oscillation dynamics locally in brain microcircuits. Although these measurements yield critical information of the mechanisms behind local oscillation dynamics, they are difficult to link with whole-brain dynamics view obtained from human M/EEG data. The newly established setup at the Neuroscience Center aims overcome these limitations and allows the measurements directly from the brain of awake head-fixed mice with over 1000 channel measuring simultaneously from both cortical and subcortical structures. This Master’s thesis project objective was to obtain proof-of-concept data to characterize oscillation dynamics during resting-state (RS) from awake behaving mice and to investigate whether these dynamics could be modulated by the manipulating E/I balance. More specifically, the current project aimed to investigate the oscillatory profile of the default-mode network (DMN) activity while manipulating the E/I balance with pharmacological mediums. Electrophysiological data was collected from RS activity from awake mice with two µECoG grids comprising together 512 channels and two laminar Neuropixel probes with each consisting 348 channels. The areas of interest were targeted to capture the DMN activity, covering anterior cingulate cortex (ACC), secondary motor cortex (M2), retrosplenial areas, visual cortical layers, pre- and infralimbic areas, hippocampal areas such as CA1 and dentate gyrus as well as lateral and posterior thalamic areas. The network activity was modulated with pharmacological mediums (sedative, stimulant, control) administered in low acute doses to see their effects on the oscillatory profile. Data from four mice were included into this Master’s thesis work and each mouse was recorded first for 30-minute daily baseline, following a 30-minute pharmacological measurement. This Master’s thesis included the data obtained from the µECoG data to the data analysis focusing on the large-scale cortical activity of the DMN. Power spectral density analysis showed a prominent alpha peak, also seen in humans, across condition with a mild decrease in volume in the stimulant condition. Synchronization was assessed with imaginary part of the phase locking value (iPLV), and the results showed increased synchronization in the stimulant condition and decreased in sedative condition in comparison to the control condition. The amplitude correlation coefficient showed also expected results in both pharmacological conditions, namely higher correlation in stimulant and lower in sedative. This project was able to obtain valuable information of the newly established in vivo electrophysiology setup and the results were in line with our expectations. This promising outcome solidifies the translational potential of the setup and its ability to serve as a translational counterpart in numerous research designs in health and disease.

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.