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

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.

Faculty: Faculty of Biological and Environmental Sciences Degree programme: Master’s Programme in Neuroscience Study track: Neuroscience track Author: Ariel Olivia Vasques Ojeda Title: The effects of sleep disruption on sleep architecture and microglial morphology Level: Master’s thesis Month and year: May 2024 Number of pages: 50 pages Keywords: Sleep disruption, microglia, frontal cortex, adolescents, older mice, EEG, microglial morphology, hippocampus Supervisor or supervisors: Birgitte Rahbek Kornum, Christine Egebjerg Jensen Where deposited: University of Helsinki library Additional information: Abstract: Although sleep is an essential biological need for all beings, we have yet to understand why exactly it is a crucial aspect of our lives. The loss of sleep is seen as a natural occurrence that increases as we begin to age. The consequences of sleep deprivation are not yet fully understood but have been associated with a range of detrimental effects on comorbid conditions, including reduced quality of life, cognitive impairments, immune suppression, and various other adverse outcomes. The role of microglia in response to sleep deprivation is a discussion that is also yet to be understood, but that can be a pivotal point for future understanding. This master's thesis investigates the impact of sleep deprivation on sleep architecture in aged mice and microglial activation in adolescents. The study aims to understand how sleep disruption affects these age groups, focusing on microglial morphology and overall sleep patterns. Using EEG/EMG recordings, sleep disruption was induced by introducing novel objects for four hours daily at ZT 2-6 over seven days. The study found that older mice experienced a shift in their sleep patterns, with significant changes in NREM and REM sleep occurring during the dark phase, highlighting the influence of the circadian rhythm. In adolescent mice, sleep disruption led to increased morphological changes, suggesting a reduction in microglial activity or an intermediary state of activation. The results underscore the importance of sleep in maintaining neural homeostasis and highlight age-dependent differences in the response to sleep loss. The study discusses the implications of these findings for understanding the neurobiological mechanisms underlying sleep and its disruption, particularly in relation to microglial function and brain health. 

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.

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.

Kainate receptors (KARs) act as prominent regulators of neuronal excitability, network activity as well as neurotransmitter release in the developing brain. In the neonatal hippocampus the GluK1 subunit containing KARs take part in regulating the activity of the CA3 interneurons and hence the maintenance of early synchronous network oscillations, which are thought to be vital for developing connections. In the interneurons of the CA3 subfield this regulatory activity is likely performed through a noncanonical, G-protein mediated inhibition of a Ca2+ sensitive medium-duration afterhyperpolarizing current (ImAHP). As in various central neurons the ImAHP has been shown to be regulated by voltage-gated calcium channels (VGCCs) and as the activity of the voltage-gated calcium channels has been in turn shown to be modulated by G-protein coupled signaling of GluK1 KARs, we went on to investigate whether a direct link between GluK1 KARs and VGCCs could be detected in the CA3 stratum radiatum interneurons of neonatal hippocampus. Here we show that the pharmacological inhibition of GluK1 KARs does not affect the amplitude of Ca2+ influx through VGCCs in the CA3 stratum radiatum interneurons of acute hippocampal slices from neonatal mice. As G-protein mediated signaling has been shown to induce alterations in the voltage-dependence of the VGCC-mediated currents, we similarly investigated the effects of GluK1 inhibition on the current-voltage relationship of Ca2+ currents in CA3 interneurons during the first postnatal week as well as during the second postnatal week, since GluK1 subunit is known to undergo developmental changes in its expression during this time. No significant effect was however detected in either of the age groups. Although in our experiments the GluK1-KAR inhibition seemed to induce no statistically significant changes in the Ca2+ current amplitudes or in the voltage-dependence of VGCC-mediated currents in the CA3 interneurons, further, more specific studies should be encouraged to investigate the phenomenon in specific interneuron subtypes and in distinct calcium channel families.

Histamine and hypocretin/orexin are neuromodulators important for regulation of alertness and wakefulness. These systems project to major areas of the brain, are highly conserved among vertebrates and they significantly innervate each other. Different studies have indicated an interaction between the histaminergic and orexin systems, however the role of histamine in this interaction is still not well-established. The goal of this study was to examine possible changes in orexin neurons development and larvae behaviour, after genetic loss of histamine decarboxylase (hdc), the histamine-synthesizing enzyme. Using whole-mount in-situ hybridization and immunofluorescence staining we observed a significant reduction in the expression of the hcrt mRNA and the orexin A peptide in 6 dpf hdcKO zebrafish larvae. However, KO of hdc had no effect on startle response, dark flash response and sleeping behaviour of 6 dpf larvae. To further investigate the regulatory role of the histaminergic system, we employed treatment of hdcWT and KO larvae with ciproxifan, a histamine H3 receptor inverse agonist. Ciproxifan treatment increased darkness habituation in 7 dpf hdcWT and KO larvae but reduced the intensity of the dark flash response only on hdcWT larvae. Furthermore, ciproxifan treatment differentially affected the expression of the orexin A peptide in 7 dpf hdcWT and KO larvae but had no effect on the expression levels of the hcrt mRNA. Collectively, these findings suggest the significance of histaminergic signaling for normal development of orexin neurons and the implication of histamine in the execution of the dark flash response. Lastly, this study indicates the complex role of the histamine H3 receptor and the requirement of further studies for better characterization of its function.

This thesis investigates whether the antidepressant-like and plasticity-promoting effects of LSD depend on TrkB expression among parvalbumin-positive interneurons (PV+ INs). Given the pivotal role of PV+ INs in facilitating critical-period-like plasticity, both during development and under the influence of conventional antidepressants, we hypothesized they may be involved in mediating psychedelic-induced neuroplasticity. Our findings challenge prevailing hypotheses, suggesting that LSD's plasticity-promoting effects may not rely on PV+ INs. We found that LSD did not increase the spine density of PV+ INs among wildtype mice, nor affected the expression of parvalbumin in PV+ INs or the perineuronal nets (PNNs) that enwrap them. Unexpectedly, we found that LSD did have subtle effects on PV+ IN spine density and the expression of parvalbumin in a mouse model with reduced TrkB expression among PV+ INs, suggesting a possible kind of compensatory mechanism at play. Our results reveal the multifaceted nature of LSD's actions on plasticity, shedding light on its therapeutic potential and prompting further exploration into its underlying mechanisms.

Mesencephalic astrocyte-derived neurotrophic factor (MANF) is an evolutionarily conserved, secreted protein. Structurally, MANF has a saposin-like and SAP-like domain that is commonly found in proteins in the lysosome and nucleus, respectively. In addition to it, it has a RTDL motif which is a KDEL-like sequence representing the ER retention signal. Previous studies have revealed that MANF has cytoprotective effects in several disease models, making it a putative therapeutic molecule. However, its exact function and molecular mechanism are yet to be elucidated. Therefore, knowing the exact function of MANF is crucial for its effective use as a therapeutic. Subcellular localisation is an effective way to determine a protein’s exact function as it provides an enclosed environment and surface for possible chemical interaction in which a protein can execute its specialised function(s). Due to the lack of studies on the subcellular localisation of endogenous MANF at a basal state and preliminary unpublished results from the group on MANF’s ability to regulate catalase mRNA expression, the aim of this study was to investigate MANF’s subcellular localisation through the development and validation of biochemical methods. Methods such as immunofluorescence, immunoelectron microscopy and biochemical fractionation were explored and validated for studying subcellular localisation. Here, the limitations of immunoelectron microscopy and biochemical fractionation are described, as the protocols used need further optimisation. Using immunofluorescence microscopy, MANF was detected particularly in the perinuclear region and ER. The findings are suggestive of MANF localisation but not conclusive due to the inadequate physiological condition of the cells; indicating the need for further optimisation of the methodology to study MANF’s localisation.

Tutkimuksellinen opetus ja tutkimuksellisuus oppilaiden suorittamissa tehtävissä ovat tärkeä osa biologian opetusta. Tutkimuksellisuus voi sisältää kokonaisen tutkimuksen, tai vain osia siitä. Tutkimuksellisten työtapojen hallinta on osa opetussuunnitelman mukaista oppimisen arviointia peruskoulussa ja lukiossa. Lukion opetussuunnitelman ihmisen biologian kurssin sisällöissä mainitaan myös pienimuotoisen tutkimuksen toteuttaminen osana oppimisen tavoitteita sekä arviointikehystä. Tutkimuksellinen lähestymistapa tukee ja korostaa oppilaan itseohjautuvuutta ja aktiivista, oma-aloitteista oppimisprosessia ja sen kautta opetusta voidaan myös eriyttää erilaisten oppijoiden tarpeita tukevalla tavalla. Tutkimuksellisten tehtävien ja työtapojen on todettu lisäävän motivaatiota ja parantavan oppimistuloksia. Ihmisen biologian oppimateriaaleissa tutkimuksellisia tehtäviä on todettu olevan lukumääräisesti vähemmän, kuin muissa biologian aihepiireissä. Tässä tutkimuksessa tarkastelluissa oppimateriaaleissa tutkimuksellisia tehtäviä on vähän suhteessa tehtävien kokonaismäärään, osassa materiaaleista ei välttämättä ollenkaan. Tutkimuksellisuuden taso ja eri tasojen edustus vaihtelee jonkin verran oppikirjojen välillä. Tutkimuksellisten tehtävien osuus eri aihekokonaisuuksissa vaihtelee niin aihekokonaisuuksien, kuin myös oppikirjojen välillä, mutta samat aihepiirit jäävät toistuvasti ilman tutkimuksellisia tehtäviä, kun taas muutama aihepiiri erottuu joka oppikirjassa tutkimuksellisten tehtävien runsaammalla lukumäärällä. Osa tehtävistä sisältää tutkimuksellisia piirteitä olematta kuitenkaan varsinaisesti tutkimuksellisia tehtäviä.

Schizophrenia (SCZ) is a chronic neuropsychiatric disorder believed to arise from the intricate interplay between genetic predisposition and environmental factors. Though the aetiology of SCZ is unknown many findings support an excessive synaptic pruning hypothesis. Maternal immune activation (MIA), encompassing prenatal infection and systemic inflammation, constitutes a significant environmental risk factor implicated in SCZ onset (Patterson, 2009; Brown, 2012). MIA induces persistent alterations in the microglia of offspring termed microglial priming, characterized by heightened reactivity to inflammatory stimuli (Choudhury and Lennox, 2021). Notably, studies have reported increased sensitivity to activation, elevated expression of inflammatory markers, and an increase in the total number of microglia (Perry and Holmes, 2014; Choudhury and Lennox, 2021). Primed microglia may contribute to excessive synaptic pruning, thereby compromising neuronal connectivity and potentially leading to the onset of SCZ. This thesis investigated the impact of microglia on neurons and explored the microglial tendency for hyperactivation in the context of SCZ predisposition. It utilized induced pluripotent stem cell (iPSC) technology to create a rat astrocyte/unaffected control human iPSC-derived neuron/induced microglia-like cell (iMGL) tri-culture model. Uniquely, iMGLs were differentiated from a library of monozygotic twin lines discordant for SCZ, and unaffected controls. This allows for exploration of the differences between iMGLs from unaffected twins with a genetic predisposition for SCZ, affected twins with clinical manifestation of SCZ, and unaffected controls without a known genetic predisposition for SCZ. The tri-culture system was subjected to lipopolysaccharide (LPS) and polyinosinic:polycytidylic acid (poly(I:C)) treatments to activate iMGLs, and differences in cytokine release, synapse pruning, and neuronal activity were assessed. The principal outcomes of our investigation revealed enhanced cytokine release from SCZ-derived iMGLs when exposed to inflammatory stimuli, alongside increased network connectivity among samples containing genetically predisposed iMGLs. While most of the results did not reach significance, they suggest a potential link between SCZ pathophysiology and hyperactive microglia. Future research will focus on enlarging the study cohort, establishing tri-culture models featuring neurons and iMGLs derived from the iPSCs of the same patient, conducting CBA analysis to confirm the elevated cytokines finding, and scrutinizing iMGL morphology.