Browsing by master's degree program "Neurotieteiden maisteriohjelma"
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(2019)Introduction and aims. Multiple different neurobiological alterations have been hypothesized to underlie Major Depression Disorder (MDD), but no unifying theory exists to explain the mechanisms of the disorder. The aberrant brain dynamics in MDD can be seen in the alterations of long-range temporal correlations (LRTCs), which have been proposed to be an indication of criticality in healthy brain. Alterations in LRTCs have been suggested to reflect deficiencies in excitation-inhibition (E/I) balance, neuromodulation or connectivity patterns, which have also been proposed to be the underlying mechanisms of MDD. There has been controversy whether the pathology is related to attenuated or increased LRTCs, and the sources of altered brain dynamics have not yet been localized. The aim of this study was to find in which frequency bands and where in the brain the neuronal LRTCs are altered in MDD on source level. In addition to analyzing the correlations between neuronal LRTCs and depression severity in parcel level, we studied correlations in functional networks to get a better understanding of the system level alterations in MDD. We also studied whether behavioral LRTCs correlate with depression severity or with behavioral performance. Methods. We investigated the long-range temporal correlations in a cohort of 19 depressed subjects by using magnetoencephalography (MEG) for recording brain activity during resting state and response inhibition task and performed DFA analysis on the amplitude envelopes of cortical oscillations. The depression severity was measured with BDI-21 questionnaire. Results and conclusions. We found the LRTCs to be positively correlated with depression severity in the alpha frequency band (8–12Hz) predominantly in the limbic system that underlies emotional control. This result was supported by the parcel level analysis in which correlations between alpha band LRTCs and depression severity were observed in the orbitofrontal cortex and temporal pole, indicating that the hyper-activation of limbic system could explain the negative bias characteristic to depression. Positive correlations were also found in frontoparietal, ventral, and dorsal attentional networks that support cognitive control. Alpha band LRTCs correlated also with behavioral LRTCs during both resting state and task conditions. However, we observed more wide-spread correlations between alpha range LRTCs and depression severity than between neuronal LRTCs and behavioral LRTCs. Behavioral LRTCs correlated with depression severity, but not with behavioral performance. These results indicate that depression is characterized by vast alterations in the brain dynamics and imply that the wide range of different symptoms in MDD could be explained by alterations in the excitation/inhibition balance in the limbic system and cognitive networks.
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(2019)Parvalbumin (PV) interneurons are GABAergic inhibitory neurons that shape neuronal network activity and plasticity. They are involved in both developmental and adult plasticity and have recently been divided into subpopulations that differ in birthdate, intrinsic properties and are involved in different types of learning; while late born PV neurons, expressing low levels of PV, are required for the acquisition of new information, early born PV neurons, expressing high levels of PV, are involved in the consolidation of the information. PV cells can be enwrapped with perineuronal nets (PNNs), an extracellular matrix structure that stabilizes synapses and indicates a mature state of the cell. The development of PNNs correlates with the closure of critical period of plasticity in development, and the enzymatic removal in adulthood can reopen those periods. Similarly, antidepressants like fluoxetine have been proven to reopen critical periods of plasticity in adulthood (iPlasticity) and decrease PNN structures in PV cells. However, whether the effect of fluoxetine is restricted to a subpopulation of PV interneurons is unknown. In addition, no previous studies have yet investigated the maturity state of the PV subpopulation by analyzing its PNN structures. In this thesis we aimed to elucidate differences in the maturity state of the subpopulations and the fluoxetine effect in those. To do that, we treated a cohort of adult mice with a chronic fluoxetine treatment previously reported to be capable of the reopening of critical periods. Following, we performed an immunohistochemistry analysis to detect PV and PNN levels in the CA3b hippocampal area. In addition, our mice line expressed TdTomato (TdT) in PV cells which allowed a more sensitive detection of PV neurons. After imaging the slices with a confocal microscope, we analyzed the PV and PNN intensity both by manual counting and with a semi-automatic macro script in ImageJ software that we developed and validated. The PV intensity of control mice was used to divide the cells in two groups; low PV and high PV expressing cells. PNNs in those subpopulations in both the control and fluoxetine treated group were analyzed and statistically compared. The low PV subpopulation showed a significantly low PNN intensity compared to the high PV subpopulation, indication a plastic or immature low PV subpopulation and a mature or consolidated high PV subpopulation. Interestingly, fluoxetine selectively decreased the PNN structures in the high PV subpopulation, by bringing the PNN intensity to comparable levels found in the low PV network. No effect of fluoxetine in the low PV network was detected. Fluoxetine induced a change towards a plastic state in the network believed to be involved in memory consolidation by decreasing its PNNs structures. This discovery gives new insights on the understanding of antidepressant plastic actions, suggesting that a chance for strong memories to change could be facilitated with the drug, and explain the antidepressant’s effects when combined with psychotherapy. However, supplementary experiments to compare and define PV subpopulations and a confirmation of the selective effect of fluoxetine are needed to confirm the preliminary hypothesis suggested by our data.
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(2019)Caged photolysable compounds have served to be pivotal to neuroscientific investigations; allowing the cognizing of molecular kinetics and properties of neuronal micro-machinery such as neurotransmitter receptors. Precision in terms of temporal and spatial resolution of neurotransmitter release endowed by photolysis has multitudinal applicabilities in the realm of GABAA receptors (GABAARs), their neuronal niche and effects on neuronal and network activity. Caged compounds, in their caged form, may display certain unideal traits such as undesired interactions with the system and antagonistic activity on the target receptor. This study aims to reevaluate the GABAAR antagonistic actions of caged Rubi-GABA, which was found to antagonize these receptors at significantly lower concentrations than those reported in the literature. Furthermore, this study electrophysiologically characterizes the possible antagonistic properties of a novel quinoline-derived UV-photolysable caged GABA compound, 8 DMAQ GABA, whose activity, in its caged form appears to have a much more favorable antagonism profile compared to the widely used RuBi-GABA. To assess the antagonistic effects of these compounds on GABAAR-mediated miniature inhibitory postsynaptic currents (mIPSCs) patch-clamp recordings were carried out in the whole-cell voltage clamp configuration on cortical layer 2/3 cortical pyramidal neurons in acute neocortical slices prepared from 16-18 day-old rat rats. The results of this study indicate a revised antagonism profile for caged Rubi-GABA, with marked GABAAR toxicity in the low micromolar range. The study also scrutinizes the photo-kinetic properties of both caged GABA compounds and reveals that the rate of GABA release from 8-DMAQ is slower than from RuBi-GABA.
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(2018)Background and objectives: Autism spectrum disorders (ASD) are developmental neuropsychiatric disorders in which core symptoms are problems in communication and interaction as well as restrictive and repetitive behaviour and interests. ASD is 2-5 times more common in males than in females. In recent years, researchers have found, that there are differences between females and males in ASD symptoms, neuropsychological characteristics, comorbid problems, neurobiology and etiology. The purpose of this systematic review is to give a comprehensive picture about the role of female sex/gender in ASD. To establish this, the review covers symptoms of autism, neuropsychology, neurobiology, comorbidity, neurogenetics and neuroendocrinology. Research questions were the following: 1) Is there evidence of sex/gender differences in ASD symptoms and comorbidity disorders? 2) Are there sex/gender differences to be found in ASD etiology? 3) What kind of support different explanations about sex/gender bias have gotten in various research areas? The purpose of the study is also to integrate the existing theories into one model that takes account to different aspects of sex/gender differences in ASD. Methods: The protocol of this systematic review follows "The Preferred Reporting Items for Systematic Reviews and Meta-Analyses" (PRISMA) when applicable. Eligibly criteria and search terms were selected in a way that would offer the widest range of articles covering the subjects of this study. Literature search was conducted using the Medline and PsychINFO as search engines. The final sample consisted of a total of 129 articles. Data was extracted on all relevant variables of the study, that were the number of participants, age of participants, specific diagnoses, methods and results. Results: Sex/gender differences in ASD were found in all areas that were included in this systematic review. Females with high function ASD (HFASD) were found to have less problems in social communication and interaction and less repetitive and restricted behavior and interests than males with HFASD. In addition, HFASD were found to have better language skills than males with HFASD. However, females with ASD were found to have more sensory processing problems, mental health problems and epilepsy than males with ASD. Females with ASD were also found to have lower full-scale intelligence quotient than males with ASD. In the context of etiology, it has been found that there are sex/gender differences in neuroanatomy, susceptibility genes and hormone levels. Conclusions: Results from this systematic review suggest that females with HFASD are underdiagnosed. This results from etiological sex/gender differences that cause partially different clinical presentation of ASD between females and males. ASD research has also concentrated mostly on males with ASD while ignoring females with ASD. Underdiagnosing can have many unfavorable consequences for females with HFASD since if they do not have a diagnosis, they do not get support. In the future, it is crucial to pay attention to females with ASD in the clinical work and scientific research.
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(2020)Charcot-Marie-Tooth disease (CMT) is a collective name for inherited neuropathies affecting the peripheral nerves. CMT affects 1:2500 children and adults worldwide. The disease is genetically highly heterogeneous, and the pathogenic mechanisms are largely unknown. Thus far, there is no cure known for the Charcot-Marie-Tooth disease. Therefore, the study of the genetic factors involved in the disease and the understanding of the underlying molecular mechanisms will benefit the development of strategies to prevent or treat these diseases. In this thesis, a new candidate gene for CMT was investigated in patient fibroblasts. The novel gene variant was originally found at University of Helsinki in a pair of Finnish brothers with CMT; and in later examinations, in their affected family members. The gene encodes an ER calcium channel receptor that is responsible for Ca2+ release from the endoplasmic reticulum (ER) and plays an important role in the regulation of various cellular processes. In this thesis, I studied the effect of the variant in patient fibroblasts by Western blotting, quantitative reverse transcriptase PCR (RT-qPCR) and calcium imaging. I also knocked down the gene using siRNA in healthy fibroblasts to investigate if the loss of the receptor has a similar effect on calcium signaling as the patient variant. My results showed that siRNA treatment significantly decreased the targeted protein levels and delayed the ATP-evoked Ca2+ release from ER without profound effect on the amplitude of the release. Similar effects of the studied mutation were observed in one patient cell line, but not in the other. Patient cell line, which did not have alterations in the levels of the protein and Ca2+ release, had elevated levels of mRNA of the affected gene. The results suggest that the gene variant does not impair the total volume of the ATP-evoked Ca2+ release from ER. The possible effect of the studied mutation may be related to the decreased levels of the mutated protein, which at the functional level may affect the timing of total Ca2+ release from ER. However, the functional effect of the variant could not be confirmed with the fibroblast cells; further experiments are needed to clearly confirm the variant’s effect on calcium signaling.
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(2020)The progressive myoclonus epilepsy of Unverricht-Lundborg type (EPM1) is a neurodegenerative disease caused by loss-of-function mutations in the cystatin B gene (CSTB) with juvenile onset, stimulus sensitive action-activated myoclonus, generalized tonic-clonic seizures and ataxia. The cystatin B (CSTB) protein inhibits cysteine proteases, such as cathepsin L, which has been reported to cleave histone H3 N-terminal tails in mouse embryonic stem cell differentiation. We have shown previously that histone H3 cleavage is an irreversible epigenetic chromatin modification, which occurs in cystatin B-deficient (Cstb-/-) mice derived neural progenitor cells during differentiation. In this study, first, we used the wild-type E13.5 mice brain derived neural cells in culture to determine the effect of extrinsic signaling factors to our earlier developed ex vivo neurosphere cell model. We also confirmed that the histone H3 cleavage positive progenitor cells are primarily neuronal cells. Then, we used phenotype rescue of Cstb-/- neural progenitor cells and showed that CSTB is a negative regulator of histone H3 cleavage. In wt mouse neurosphere cryosections, we showed that cathepsin B and L are not expressed in the nucleus of neural cells before differentiation.
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(2019)Tiivistelmä – Referat – Abstract Mental disorders are among the leading causes of global disease burden and years lived with disability. Their pathogenesis is poorly understood and there are enormous challenges in the development of biomarkers to aid in diagnosis and more effective therapeutic options. It has been documented that the microbiota-gut-brain axis shows alterations in mental disorders such as anxiety, depression, autism spectrum disorders, bipolar disorder and schizophrenia. Here we study the gut microbiota of individuals with axis I mental disorders and their unaffected siblings by 16S RNA gene amplicon sequencing. In the Central Valley of Costa Rica, a total of 37 participants were recruited and diagnosed using a Best Estimate Diagnosis protocol. For each of the individuals diagnosed with a mental disorder a healthy sibling was selected after matching by age and gender. A total of 13 pairs of 26 siblings, affected and unaffected, was used for the analysis. In a subsequent analysis, individuals were also divided into the three categories of “unaffected” (UA), “affected without psychosis” (AA) and “affected with psychosis” (AP). They underwent clinical assessments about their habits and diet and about resilience (Connor-Davidson Resilience Scale), current status (SADS-C) and disability (WHODAS 2.0). Their fecal samples were collected freshly and stored at -80°C. DNA was extracted, libraries constructed by PCR and subjected for Illumina MiSeq 300 paired-end 16S RNA amplicon sequencing for analysis of the gut microbiota. The sequencing data were analyzed using the R packages mare and vegan for gut microbiota composition, diversity and richness, taking into account the identified confounders. All participants were of Hispanic ethnicity, residents of the San José Greater Metropolitan Area, adults and 69% of them were women. Affected individuals had major depression, bipolar affective disorder, psychosis non-otherwise specified or schizoaffective disorder. Based on beta-diversity analysis as a measure of the community-level microbiota variation, it was found that the use of levothyroxine (R2=0.08, p=0.005) and of irbesartan (R2=0.068 ,p=0.001) had a significant impact on the microbiota composition and hence the use of these drugs was included as confounder in further analyses. Several statistically significant differences in the relative abundance of intestinal bacteria were identified: Differences were found in the relative abundance of bacterial families Peptostreptococcaceae, Ruminococcaceae, Porphyromonadaceae, and in bacterial genera Pseudomonas, Barnesiella, Odoribacter, Paludibacter, Lactococcus, Clostridium, Acidaminococcus and Haemophilus. Our results indicate that affected individuals have more pro-inflammatory Proteobacteria (Pseudomonas) and less bacteria associated to healthy phenotype, such as Barnesiella and Ruminococcaceae, the former being dose-dependently depleted in AP and AA compared to UA. Furthermore, we documented decreased bacterial richness among affected participants while no significant differences were detected in alpha diversity. Our study identified significant differences in the microbiota of individuals affected by mental illness when comparing to their healthy siblings. The results may have important implications for the holistic understanding of mental health and its diagnosis and therapeutics. Larger studies to confirm these findings would be justified.
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(2020)The diversity of different neuronal types lays the foundation for different functions in the brain. The development of different subpopulations and special features of neurons in the central nervous system are still partly unknown. Finding answers to these developmental issues could help in the process of characterisation of cell types and mapping of neuronal networks between the brainstem nuclei in the brain. Previous studies have shown that a ventrolateral neuroepithelial domain in the anterior hindbrain, rV2, produces excitatory (glutamatergic) and inhibitory (GABAergic) neurons, which are related to monoaminergic nuclei in the brainstem (Lahti et al., 2016). In this master’s thesis project, the development of a subpopulation of neurons expressing Gsc2 transcription factor in the interpeduncular nucleus was studied. This project was based on single-cell RNA sequencing results conducted in E13.5 mice. Predicted by single-cell RNA sequencing results, Gsc2 expressing cells are GABAergic interneurons and originate from the rV2 domain of the rhombomere 1 region in the hindbrain. Co-expression pattern with another transcription factor Sall3 with Gsc2 during development was also addressed in the study. Furthermore, the role of Notch signalling in the binary cell fate decision between GABAergic and the glutamatergic fate of rV2 neurons was investigated. Validation of single-cell RNA sequencing results was performed using in situ hybridisation and immunohistochemistry methods with mice embryos at the age of E12.5 and E15.5. This study verified previously shown origin of Gsc2 expressing cells to the rhombomere 1 region and in addition, showed that Gsc2 expressing cells are GABAergic. Co-expression pattern of Gsc2 with Sall3 neither in the rV2 domain nor in the interpeduncular nucleus was seen in our results. In the rV2 domain, the depletion of Notch signalling decreased the expression of differentiating GABAergic neurons. This indicates that Notch has a role in GABAergic neurotransmitter identity during the development of brainstem neurons in mice. Based on our results, Gsc2 could be used as a lineage marker for GABAergic interneurons originating from the rhombomere 1 region and as a marker for a subpopulation of the interpeduncular nucleus. Furthermore, results from the role of Notch signalling could help in discovering the mechanisms related to the determination of neurotransmitter identity in rV2 neurons. Further investigations, in different developmental time points and with additional markers, are needed to verify these results.
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(2019)Meningeal lymphatics vessels (mLVs), the recently characterized lymphatics in the central nervous system (CNS), provide a link between the adaptive immune system and the CNS. mLVs could be important for the activation of T cell-mediated adaptive immune response, by draining antigens from the brain to the deep cervical lymph nodes, where they are presented to T cells. In traumatic brain injury (TBI), we hypothesized that the activation of self-reactive T cells (i.e., T cells able to recognize self, brain-derived antigens and promote an immune reaction), possibly underlies the pathogenesis of the disease. In order to test this hypothesis and to decipher the specific role of mLVs in the modulation of T cell-mediated neuro-immune response after TBI, we ablated the existing mLVs in adult male C57BL/6OlaJ mice (with the use of the AAV-mVEGFR3 1-4 Ig vector), induced TBI with controlled cortical impact, and examined the motor function of the mice and the activation of different T cell populations in the brain, as well as in the secondary lymphoid (spleen and lymph nodes – LNs) and non-lymphoid organs (meninges). Our data showed that the T cell-mediated adaptive neuro-immune response in TBI was unaffected by the depletion of mLVs. Our results, however, are preliminary, due to the limited sample size used in this study, which reduces the statistical power and restricts our ability to conclude for the effect of mLV depletion on TBI recovery.
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(2019)Tiivistelmä – Referat – Abstract Genetic variations within the MYO16 gene indicate a common predisposition to severe psychiatric, neurocognitive and neurodevelopmental disorders (NDD), as well as bipolar disorders (BD) and schizophrenia spectrum disorders (SSD). Myosin XVI’s ability to regulate actin and its involvement in cytoskeleton remodeling highlights the protein’s significance in neuronal circuitry development and signaling. Mutations in actin regulator protein-encoding genes like MYO16 have been found to shift cytoskeletal dynamics, as well as cause irregular dendritic spine and excitation/inhibition (E/I) synapse phenotypes. Interestingly, altered actin dynamics and E/I synapse dysregulation are two commonly detected molecular deficits associated with neuropathologies, namely autism spectrum disorders (ASD), SSD, and intellectual disability (ID). Therefore, synaptic E/I profiles are good candidates for investigating the neuropathologies they accompany, and also for revealing potential functional abnormalities. Hence, we determined that quantifying the levels of inhibitory synaptic proteins VGAT and gephyrin is the most suitable approach to investigate inhibitory synapse profiles and their relation to pathologies. Specifically, we investigated how microRNA (miRNA)-mediated myosin XVI protein knockdown (KD) affects pre- and postsynaptic inhibitory synapse density in rat primary hippocampal neurons. We achieved this by analyzing the density of VGAT and gephyrin puncta, signifying pre- and postsynaptic inhibitory synapses, respectively, and also by measuring their diameter to determine differences in inhibitory synapse size. Moreover, we quantified and assessed inhibitory synapse density and size differences between groups by comparing Myo16 KD-plasmid expressing hippocampal neurons to scrambled control cells. Common for both Myo16 KD plasmids was the active suppression of myosin XVI by 33%. However, Myo16 KD plasmids did not affect inhibitory synapse density and size to the same degree. Specifically, there was a significant reduction of inhibitory synapse density in the Myo16 KD3-plasmid expressing neurons, yet, no changes were observed in Myo16 KD5-plasmid expressing neurons. Finally, pre- and postsynaptic inhibitory synapse size differences were not significant between groups for either Myo16 KD plasmid when compared to scrambled control. Aberrant actin cytoskeleton remodeling, as well as altered E/I synapse ratios may lead to hyper/hypo-transmissive neuronal states or cause E/I imbalance, suggesting a complex relationship between actin regulator genes and inhibitory synapses. Our understanding behind their interplay is fairly limited, thus, gaining insight into the mechanisms associated with altered E/I balance remains the primary aim.
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(2019)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.
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(2019)The insular cortex has been implicated in the neurocircuitry underlying alcohol addiction. The role of the insular cortex and its projections in regulating ethanol intake in AA (Alko-Alcohol) rats has been studied using chemogenetic tools. Chemogenetic activation of the anterior agranular insula (aAI) in AA rats through excitatory DREADDs expressed in the aAI has been found to decrease ethanol consumption. The aAI projects to the central nucleus of the amygdala (CeA), another brain region involved in the development of addiction, particularly in the withdrawal/negative affect stage. In the current study, we sought to further investigate the role of the aAI and the CeA in regulating voluntary ethanol consumption in AA rats. First, we characterized the efferent projections of the aAI in AA rats by chemogenetically activating the aAI with DREADDs and then measuring c-Fos expression in various regions of interest throughout the brain. Next, we investigated the role of the aAI --> CeA projection in ethanol intake by chemogenetically activating or inhibiting the aAI --> CeA projection using the dual viral Cre-dependent DREADD approach. We examined the effects of this manipulation on voluntary ethanol consumption in AA rats in a two-bottle choice paradigm. Finally, we examined the roles of CeA D1Rs (dopamine receptors) and 5-HT2ARs (serotonin receptors) in regulating ethanol intake by examining the effects of pharmacological agonism or antagonism of these receptors on voluntary ethanol consumption in AA rats. Our results from the first experiment reveal significant activation of brain regions including the posterior agranular insula, the mediodorsal nucleus of the thalamus, and the posterior piriform cortex following chemogenetic activation of the aAI. The projections from the aAI to these regions are potentially important in the aAI circuitry in AA rats and are therefore of interest in future studies on the role of aAI circuitry in ethanol intake. In the second experiment, we found no significant effects of aAI --> CeA projection activation or inhibition on ethanol consumption in AA rats, indicating that this projection may not be a key component in regulating ethanol intake in these rats. Finally, we found no significant effects of pharmacological D1R antagonism, 5-HT2AR antagonism, or 5-HT2AR agonism in the CeA on ethanol intake in AA rats, although there was a non-significant trend towards a dose-dependent decrease in ethanol consumption with increasing dose of the D1R antagonist. Our results reveal new neural projections that should be investigated in future research on the role of the aAI in regulating ethanol intake. Studies on the neurobiology underlying alcoholism may reveal new pharmacological or anatomical targets for treatments of alcoholism in humans.
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(2019)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.
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(2019)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.
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(2019)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.
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(2019)Semantics is a study of meaning in language and basis for language comprehension. How these phenomena are processed in the brain is still unclear especially in naturalistic context. In this study, naturalistic language comprehension, and how semantic processing in a narrative context is reflected in brain activity were investigated. Subjects were measured with functional magnetic resonance imaging (fMRI) while listening to a narrative. The semantic content of the narrative was modelled computationally with word2vec and compared to voxel-wise blood-oxygen-level dependent (BOLD) brain signal time courses using ridge regression. This approach provides a novel way to extract more detailed information from the brain data based on semantic content of the stimulus. Inter-subject correlation (ISC) of voxel-wise BOLD signals alone showed both hemispheres taking part in language comprehension. Areas involved in this task overlapped with networks of mentalisation, memory and attention suggesting comprehension requiring other modalities of cognition for its function. Ridge regression suggested cerebellum, superior, middle and medial frontal, inferior and medial parietal and visual cortices bilaterally and temporal cortex on right hemisphere having a role in semantic processing of the narrative. As similar results have been found in previous research on semantics, word2vec appears to model semantics sufficiently and is an applicable tool in brain research. This study suggests contextual language recruiting brain areas in both hemispheres and semantic processing showing as distributed activity on the cortex. This activity is likely dependent on the content of language, but further studies are required to distinguish how strongly brain activity is affected by different semantic contents.
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(2019)Understanding the link between the gut microbiota, diet and the enteric nervous system is of significant importance in the prevention of gastrointestinal disorders. The aim of the study was to answer two questions: Firstly, is butyrate able to stimulate the luminal release of serotonin? Secondly, in which parts of the gastrointestinal tract does this possibly occur? These questions are of interest, due to the importance of the serotonergic signalling in the enteric nervous system. We created a luminal perfusion system to investigate the effect of butyrate in the gastrointestinal tract of male Wistar rats (500-550g). We isolated the stomach and 4 cm long segments of the duodenum, jejunum and colon. To our knowledge this form of physiological ex vivo studies investigating the entire gastrointestinal tract have not been done previously. The isolated stomach and the isolated intestinal segments were luminally perfused with 100 mM butyrate for 10 min respectively 45 min. The tissues were homogenized after the luminal perfusion. Serotonin and its main metabolite 5-hydroxyindoleacetic acid (5-HIAA) were assayed using commercial ELISA kits. Our results showed that butyrate significantly stimulates the release of 5-HIAA in the stomach, duodenum, jejunum and colon. Butyrate seems also to have a positive trend-effect on the release of serotonin itself in the stomach, duodenum, jejunum and colon. Although, there is a future potential for preventing gastrointestinal disorders with the help of diet and gut microbiota, the possible clinical significance of our results should be considered carefully.
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(2020)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.
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(2020)Alzheimer’s disease is the most common form of dementia and one of the highest causes of death worldwide. Recent discovery of lymphatic vessels from the dura mater, the outermost meningeal layer covering the central nervous system, has led to reassessment of the role of lymphatic vessels in neuropathological diseases. The meningeal lymphatic vessels drain macromolecules from the cerebrospinal fluid into the deep cervical lymph nodes and their proper function could be crucial for preventing amyloid-beta aggregation into the brain parenchyma. The function of the meningeal lymphatic vessels is still partly unknown. They have been hypothesized to function as an immune cell hub for the brain and dysfunction of the meningeal lymphatic vessels could lead to immune cell changes in the brain parenchyma. In my thesis, the role of the lymphatic vessels in Alzheimer’s disease was investigated by inducing atrophy of the meningeal lymphatic vessels with VEGF-C depletion in an APdE9 mouse model of Alzheimer’s disease. Single cell sequencing was used to identify the cell types present in the dura mater and in the deep cervical lymph nodes of an Alzheimer’s disease mouse model with and without atrophy of the meningeal lymphatic vessels. The amyloid-beta accumulation was immunohistochemically assessed from the brain and the cognitive decline was studied with behavioral tests. The results showed that atrophy of the meningeal lymphatic vessels did not increase the amount of amyloid-beta in the brain or affect the cognitive decline. The single cell sequencing from the meninges provided a more comprehensive cell atlas than has been published before. It was also found that the atrophy of the meningeal lymphatic vessels was associated with changes in the number of immune cells in the dura mater. The biggest changes were in the number of neutrophils and B-cells, which increased. Further studies are needed to evaluate the role of the meningeal lymphatic vessels in Alzheimer’s disease progression, as the results in this thesis were opposite to the results published before.
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(2019)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.
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