Browsing by master's degree program "Master's Programme in Neuroscience"

During the brain development, GABAergic neurons, also referred as interneurons, migrate tangentially from the subpallium to the pallium. After intracortical dispersion, the interneurons start radial migration towards their final location in the cortex. Although the radial migration of interneurons is extensively studied, mechanisms guiding the migration remain relatively unknown. Here we studied how manipulation of cortical activity affects the radial migration and allocation of the cortical GABAergic neurons in the developing mouse brain. For this purpose, we utilized whisker trimming induced sensory deprivation in GAD67-GFP mice at postnatal days 2-5 (P2-P5) followed by cell counting in brain slices derived from P5 and P10-aged mice. In addition, we performed live-imaging of migrating neurons in organotypic cultures derived from P2 SST-TdTomato and 5HT3aR-GFP mice and cultured for 1 day in vitro. These two mouse lines roughly represent early- and late-born subpopulations of the GABAergic neurons. Live-imaging was accompanied by activity manipulations using different drugs and the Designer Receptors Exclusively Activated by Designer Drugs (DREADD) technology. Analysis of the interneurons’ allocation on the barrel cortex after the unilateral sensory deprivation revealed misallocation of GAD67+ neurons on deep cortical layers of the contralateral hemisphere of the ablation group at P5. Analysis of the tracks from the live-imaged migrating interneurons revealed altered saltatory movement behaviour of 5HT3aR+ interneurons when clozapine-N-oxide (CNO) was used to activate the electroporated GFP-GCaMP3-mCherry-hM3Dq neurons located on L2/3 of the cortex. Moreover, we observed reduced motility of migrating interneurons in the organotypic cultures treated with a KCC2 inhibitor that alters GABAA-receptor mediated transmission. Altogether, our results suggest that activity is important in promoting the radial migration of late-born interneurons during the first days of the postnatal development.

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.

Nitric oxide (NO) is an important signalling molecule in the brain. NO regulates the function of many proteins by e.g. interacting with tyrosine and cysteine residues, thus inducing post-translational modifications. In animal models, inhibition of NO production triggers behavioural effects similarly to those induced by antidepressant drugs. Receptor tropomyosin-related kinase B (TRKB) has been identified as a key player mediating antidepressant drug (AD) induced effects, and it’s a potential target for NO since it displays multiple potential sites for nitration. Preliminary results from our group indicate that TRKB nitration impairs its signalling, and AD uncouple many proteins from TRKB and reorganizes TRKB protein complex. We examined the effect of selective nitric oxide synthase (NOS) inhibitor N⍵-propyl-L-arginine (NPA) in mice submitted to the contextual fear conditioning and found out that inhibiting NO production with NPA has an antidepressant-like effect on mice. We also found out that AD fluoxetine prevents nitration of TRKB receptors in vivo and antidepressant drugs fluoxetine, phenelzine and imipramine disrupt the interactions of TRKB, NOS1 and NOS1 adaptor protein (CAPON) in co-immunoprecipitation assay. To understand the nature of TRKB and NOS1 interaction, we thus examined the protein domains in NOS1 and TRKB using Uniprot database, and we were unable to identify sites that could interact directly. Literature search for NOS1 adapting proteins followed by Uniprot data mining indicated CAPON as a potential candidate to mediate NOS1: TRKB interaction. Our data shows for the first time that antidepressant drugs disrupt TRKB:CAPON:NOS1 interaction, thus protecting TRKB from NOS1-induced nitration. ADs might induce their behavioural effects by preventing NO-induced impair in TRKB signalling

During recent years obesity has been under extra scrutiny due to its globally rising prevalence, multifaceted effects on the human body and common occurrence of comorbidities. It is estimated that one third of the Finnish population over age 40 will be obese by 2028 (THL, 2022). Consequently, development of mitigation strategies has become a high priority in today’s societies leading to a rising need for new treatments. Several studies have shown how pathological adipogenesis has deleterious effect on brain functionality. The neuropathology of obesity could be explained by increased blood-brain barrier (BBB) leakage, oxidative stress, neuroinflammation and glial activation. Pathologically activated astrocytes (astrogliosis) exhibit phenotypical and functional differences compared to healthy astrocytes, typically exhibiting enlarged cell bodies and swollen cytoskeleton. Astrogliosis has been mainly studied in the context of CNS diseases. Recent studies also shed light on the role of astrocytes in the progression of peripheral diseases including cancer metastasis or inflammation (Ma et al., 2023). However, the active astrocytic profile in obesity is relatively underexplored. In this study we report astroglial phenotypic shifts induced by high-fat diet (HFD) feeding and weight loss (WL). No significant change in GFAP expression was seen between mice that were given an HFD for different durations and their corresponding controls. However, we noted a non-significant trend for increased GFAP expression in response to shorter timepoints (5 days of diet change). This suggested an early astrocytic response to diet, which later normalizes over time. We reported healthy morphologies in astrocytes from chow group exhibiting typical simple thin cytoskeleton with long cell protrusions. Astrocytes in HFD-conditions exhibited reactive phenotypes evidenced by swollen cytoskeletal structures and high GFAP immunofluorescence, extensive lipid droplet (LD) accumulation and upregulated metabolic activity. These observations indicated stressful conditions caused by the diet. Astrocytes in WLconditions exhibited varying phenotypes displaying both reactive and healthy characteristics, slight increase of metabolic activity and lipid accumulation. In addition, we reported different immunofluorescence profiles between glial differentiation promoting marker Meteorin and ER stress regulated cytoprotective marker MANF between the experimental groups. These results show that HFD-induced obesity and consecutive weight loss induce a reactive-like phenotypic shift on astrocytes involving both morphological and functional changes.

This thesis presents a comprehensive exploration of cerebral palsy, acknowledged as the predominant childhood disability. Traditionally viewed through a narrow lens as primarily a motor disorder, recent investigations have broadened this perception significantly. Beyond motor impairments, cerebral palsy manifests an array of comorbidities spanning sensory, emotional, social, and cognitive domains, reshaping our comprehension of its profound impact on individuals' lives. Challenging the static characterization long associated with cerebral palsy, contemporary research has unveiled a compelling dimension - persistent neuroinflammation. Contradicting the notion of a stable condition, these findings suggest potential progressive aspects. The revelation of persistent neuroinflammation prompts a fundamental reconsideration of cerebral palsy's nature. Should its etiological significance be established, it could revolutionise our understanding, suggesting a dynamic condition evolving over time. Conducted through a rigorous search across Pubmed and MEDLINE databases, this thesis stems from an exhaustive exploration of 900 articles. The literature review was conducted in accordance with the PRISM framework. The literature review provides a comprehensive foundation covering the historical context,pathophysiology, and neuropathology of cerebral palsy. Furthermore, it delves deeply into the aforementioned non-classical perspectives, shedding light on the multifaceted nature of this neurological condition. By synthesising classical and contemporary viewpoints, this study endeavours to broaden the discourse surrounding cerebral palsy, fostering a more inclusive and nuanced comprehension of its complexities. This thesis seeks to bridge the gap between traditional views of cerebral palsy as solely a motor disorder and the evolving understanding of its diverse manifestations across various domains. By integrating insights from multiple disciplines and challenging existing paradigms, it aims to contribute to a more holistic framework for conceptualising cerebral palsy. This integrated perspective aims to enhance not only our theoretical understanding but also the practical implications for interventions and support strategies tailored to the multifaceted needs of individuals living with cerebral palsy.

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.

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.

In visual detection, thresholds for light increments are higher than thresholds for light decrements. This asymmetry has been often ascribed to the differential processing of ON and OFF pathways in the retina, as ON and OFF retinal ganglion cells have been found to respond to increments and decrements, respectively. In this study, the performance of human participants in detecting spatially restricted (diameter 1.17 degrees of visual angle) and unrestricted increments and decrements was measured using a two-interval forced choice task. Background light intensities ranged from darkness through scotopic to low photopic levels. The detection threshold asymmetry found in earlier experiments was replicated with local stimuli. In contrast, however, the asymmetry between increment and decrement detection thresholds disappeared with fullfield stimuli. An ideal observer model was constructed to evaluate the role of two factors, Poisson variations and dark noise, in determining detection thresholds. Based on the model, these factors are insufficient to account for the increment-decrement asymmetry.

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.

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.