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Item Maternal immune activation and sex modulate hippocampal microglial properties in developing mice and result in behavioral changes in adult mice(2024) Loewen, Sophia; Tremblay , Marie-ÈveMaternal immune activation (MIA) is an inflammatory response during pregnancy that can result in an increased likelihood for the exposed offspring to develop neurodevelopmental disorders. MIA can be reliably induced in mouse models using polyinosinic:polycytidylic acid (poly I:C) at embryonic day 9.5, when microglia are colonizing the brain. Microglia, the resident immune cells of the brain, play critical roles during development though these roles vary throughout development, especially in the context of MIA. Furthermore, there is regional variability to microglia, including following MIA. We chose to focus on the polymorphic layer (PO) of the dentate gyrus and the cornu ammonis 1 (CA1) region (specifically the stratum radiatum (SR) and stratum lacunosum-moleculare (SLM)) of the ventral hippocampus as we know these regions are heavily involved in emotion and stress regulation—things that can be altered/involved in neurodevelopmental disorders. In these regions, we investigated microglial changes in density by staining against ionized calcium-binding adapter molecule 1 (Iba1; a typical microglial marker) and C-type lectin domain family 7 member A CLEC7a (pattern recognition receptor expressed on the surface of myeloid cells, including altered microglial states) in postnatal day 15 male and female mice. We show that CLEC7a-positive (+) cells have a tendency to increase in density in the CA1 SR region of the ventral hippocampus in MIA-exposed male P15 mice. We also highlight sex differences in development, namely that females have a lower density of Iba1+ cells compared to males at P15. We also show from our behavioral testing that females display slightly more anxiety-like behavior in adulthood. This work highlights the importance of continuing to investigate sex differences in microglia during development, both in the context of MIA and in health, as it may influence altered behavior in adulthood and may represent a window of potential therapeutic intervention.Item Reelin, stress, and inflammation: Implications for treatment of major depression and diagnosis of Alzheimer’s disease(2025) Reive, Brady S.; Caruncho, Hector; Kalynchuk, LisaReelin has been recognized for playing a role in various neuropsychiatric disorders including major depression and Alzheimer’s disease (AD). It was demonstrated that Reelin signaling is implicated in various molecular pathways that are affected in these disorders, including but not limited to trafficking of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) required for long-term potentiation and synaptic plasticity, regulation of amyloid beta and the phosphorylation of tau. Both major depression and AD are marked by reduced Reelin expression and alterations to Reelin cleavage. More recently, it has been shown that Reelin can be administered to animals exposed to chronic stress to relieve depression-like behaviour and spatial memory deficits associated with chronic stress exposure. While it has long been recognized that inflammatory processes contribute to both major depression and AD pathogenesis, it was only recently shown that Reelin expression is altered with inflammatory challenges and in several inflammatory conditions, including COVID-19. The observation that Reelin possesses antidepressant-like properties and inflammatory functions suggests Reelin dysregulation could contribute to inflammatory changes observed in depression and Alzheimer’s. Although altered Reelin expression has been shown in disorders of inflammation, depression and AD, it is currently unknown whether administration of recombinant Reelin modifies inflammatory processes, or in other words, whether there exists a bidirectional relationship between Reelin expression and inflammatory processes. With this research, evidence is provided showing exogenous Reelin administration can modulate inflammatory processes in chronic stress and non-stressed conditions. Additionally, it has been shown that a blood-based biomarker for depression is modulated by Reelin signaling. Early findings related to this biomarker, membrane protein clustering of the serotonin transporter protein on peripherally circulating lymphocytes, show this biomarker can reliably differentiate depressed from non-depressed controls. Additionally, within depressed populations this biomarker can identify those that will respond to treatment and those that are likely to be resistant to antidepressant treatment effects. However, large-scale validation studies for this biomarker have yet to be conducted, in part due to the massive manual labour requirements for analyzing membrane protein clustering. This led to the development of an automated method for analyzing membrane protein clustering, which is outlined in Chapter 5 of this thesis. Finally, while it is known that Reelin can modify membrane protein clustering and more neuropsychiatric conditions are marked by dysregulation of Reelin signaling than just major depression, there has yet to be an assessment of membrane protein clustering in many of these additional conditions (ex. epilepsy, multiple sclerosis, AD, etc.). This led to the evaluation of membrane protein clustering in a small sample of individuals diagnosed with mild cognitive impairment or AD. Results show relationships exist between membrane protein clustering characteristics and performance on tests of cognitive function as well as depressive symptoms. The results of membrane protein clustering in mild cognitive impairment and AD warrants further evaluation of membrane protein clustering in disorders marked by dysregulation of Reelin signaling.Item Elucidating the mechanisms underlying fetal alcohol spectrum disorders and therapeutics: The effects of postnatal choline supplementation and exercise intervention on cellular proliferation in the neurogenic niche of the hippocampus(2024) Ramnaraign, Fiona O.; Christie, Brian R.Alcohol exposure to a developing fetus can produce a wide variety of deleterious and transient effects; these effects present as a collection of symptoms and in clinical populations, are referred to as Fetal Alcohol Spectrum Disorders (FASDs). With prevalence rates of around 7 in 1000 cases, globally, FASDs are among the leading causes of neurodevelopmental damage. The amount and timing of alcohol use are factors that play into the severity and type of damage seen in patients with an FASD. Symptom types can range from craniofacial abnormalities to central nervous system (CNS) dysfunction. The third trimester of fetal development contains a growth spurt for the hippocampus, a region of the brain responsible for learning and memory processes and during this time, it is particularly susceptible to teratogenic insult. Additionally, the hippocampus contains one of the few neurogenic niches in the peri-adolescent brain, within the subgranular zone (SGZ) of the dentate gyrus (DG). As a result, it is expected that alcohol exposure during this period will disrupt neurogenic processes, but whether this effect is transient remains unclear. In recent years, professionals have moved towards novel approaches for the treatment of FASDs involving choline and aerobic exercise as effective treatment options; research is ongoing. Choline is an essential nutrient that plays a vital role in many neural and bodily processes including cell membrane synthesis, methylation, and neurotransmission. Aerobic exercise involves sustained, rhythmic physical activity and improves cardiovascular endurance through an increase in heart rate and oxygen flow to muscles. Previous work has demonstrated choline as an effective treatment to counter the aberrant effects of prenatal alcohol exposure (PAE) on the developing brain, particularly in hippocampal-dependent behaviours and similarly, it has been reported that aerobic exercise can enhance neurogenesis in the hippocampus and counteract deficits caused by PAE. To determine the effects of each of these treatments on the first stage of neurogenesis, cellular proliferation, a postnatal binge model of ethanol exposure with postnatal choline supplementation and juvenile chronic moderately-paced forced running was employed, and animals were sacrificed during the peri-adolescent period (PD36). Corresponding tissue was subject to immunohistological staining against bromodeoxyuridine (BrdU) and Ki67, and cell-positive densities for each stain were determined. Results showed no significant variations in the densities of proliferating cells of any treatment (ethanol exposure or choline supplementation) except forced running, which showed significant increases in Ki67 cell densities only. Similarly, two-way combinations of treatments and all three treatments showed no significant differences. Although this study provides insight into the relationship between hippocampal neurogenesis and ethanol, choline, and aerobic exercise, it is limited by methodological constraints which may influence the generalizability of the findings.Item Sex chromosomes and sex hormones differently shape microglial properties during normal physiological conditions in the adult mouse hippocampus(2024) Bobotis, Bianca Caroline; Tremblay, Marie-ÈveThe brain presents various structural and functional sex differences, for which multiple factors are attributed: genetic, epigenetic, metabolic, and hormonal. While biological sex is determined by both sex chromosomes and sex hormones, little is known about how these two factors interact to establish this dimorphism. Sex differences in the brain also affect its resident immune cells, microglia, which actively survey the brain parenchyma and interact with sex hormones throughout life. However, microglial differences in density and distribution, morphology and ultrastructural patterns in physiological conditions during adulthood are largely unknown. Here, we investigated these aforementioned properties of microglia using the Four Core Genotypes (FCG) model, which allows for an independent assessment of gonadal hormones and sex chromosomal effects in four conditions: FCG XX and Tg XY- (both ovaries); Tg XXSry and Tg XYSry (both testes). We also compared the FCG results with XX and XY wild-type (WT) mice. In adult mice, we focused our investigation on the ventral hippocampus across different layers: CA1 stratum radiatum (Rad) and CA1 stratum lacunosum-moleculare (LMol), as well as the dentate gyrus polymorphic layer (PoDG). Double immunostaining for Iba1 and TMEM119 revealed that microglial density is influenced by both sex chromosomes and sex hormones. We show in the Rad and LMol that microglia are denser in FCG XX mice compared to Tg XYSry mice, however, microglia were densest in WT XX mice. In the PoDG, ovarian animals had increased microglial density compared to testes animals. Additionally, microglial morphology was modulated by a complex interaction between hormones and chromosomes, affecting both their cellular soma and arborization across the hippocampal layers. Moreover, ultrastructural analysis showed that microglia in WT animals make overall more contacts with pre- and post-synaptic elements than in FCG animals. Lastly, microglial markers of cellular stress, including mitochondrion elongation, dilation of the endoplasmic reticulum and Golgi apparatus were mostly chromosomally driven. Overall, we characterized different aspects of microglial properties during normal physiological conditions that were found to be shaped by sex chromosomes and sex hormones, shading more light onto how sex differences affect brain immunity at steady-state.Item Investigating macropinocytosis as the mechanism of ATP-induced PANX1 internalization in Neuro2a cells(2024) You, Haifei; Swayne, Leigh AnneThe widely expressed pannexin 1 (PANX1) ion and metabolite channel, facilitates the release of ATP release for downstream signaling, and in turn, is regulated by extracellular ATP concentrations. This thesis focuses on the mechanisms underlying ATP-induced PANX1 internalization. Our previous work demonstrated that PANX1 inhibits neuronal differentiation and neurite outgrowth (Wicki-Stordeur & Swayne, 2013) partly through the sequestration of key cytoskeletal proteins (Xu et al., 2018). PANX1 levels peak in the neonatal mouse brain and drop precipitously between the first and second weeks of life, coinciding with peak synapse stabilization (Sanchez-Arias et al., 2019). These findings suggest that the developmental downregulation of PANX1 levels contributes to proper neuronal development, but the mechanisms regulating PANX1 trafficking in neural cells are still relatively unknown (reviewed in Frederiksen et al., 2023). Our prior work suggests that extracellular ATP concentrations regulate PANX1 surface expression in the murine Neuro-2a (N2a) neuroblastoma cell line (Boyce et al., 2015; Boyce & Swayne, 2017). Our recent findings point towards ATP-induced macropinocytosis (‘cell drinking’) of PANX1, as treatment with the macropinocytosis-inhibitor amiloride prevents ATP-induced PANX1 internalization (Boyce et al., 2020); however further experiments are needed to confirm this finding. My thesis investigates the hypothesis that ATP- induced PANX1 internalization occurs through macropinocytosis. I used the N2a cell line, which allows for high transfection efficiency, well-controlled and reproducible culture conditions, and large cytoplasmic volumes ideal for imaging subcellular compartments (Cibelli et al., 2022). Previous work from the lab showed that ATP-dependent macropinocytosis leads to an increase in cell size, which is disrupted by the PANX1 W74A (ATP-insensitive) mutation (Boyce et al., 2020). By modifying extracellular ATP, I quantified macropinocytosis using the uptake of fluorescent dextran (≥70 kDa) (Commisso et al., 2014) and super-resolution confocal/Stimulated Emission-Depletion (STED) microscopy to measure the size of dextran-containing vesicles. In these experiments, I determined that a significant proportion of total PANX1-EGFP localized to dextran-positive vesicles, suggesting that PANX1 is internalized via macropinocytosis in response to extracellular ATP. Additionally, I tested several macropinocytosis inhibitors and observed a significant decrease in the co-localization rate of PANX1-EGFP and dextran. The diameter of PANX1-EGFP/dextran co-positive vesicles was consistent with macropinosome size (0.2 to 8 μm; Swanson & Yoshida, 2019). These results further validate that the endocytosis mechanism involved is macropinocytosis. The outcomes of this work will enhance our understanding of neuronal PANX1 trafficking, with implications for neurodevelopment and disease.Item Light and electron microscopy reveal iron deposition patterns and novel iron-rich cell states across aging and Alzheimer’s disease pathology conditions(2024) Lau, Victor; Tremblay, Marie-ÈveAlzheimer’s disease (AD) involves cognitive decline, possibly via multiple concurrent pathologies associated with iron accumulation. Furthermore, iron accumulation within brain regions affected in AD has been suspected to contribute towards AD progression via cells undergoing ferroptosis-based cell death and maladaptive cellular senescence. To investigate if iron accumulation in AD is more likely due to pathological iron-rich compartments, or a compensatory response of iron within oligodendrocytes to disease progression, we sought to quantify iron-rich staining (via Perl’s diaminobenzidine; DAB). Healthy wild-type (WT) and APPSwe -PS1Δe9 (APP-PS1; amyloid-beta overexpressing) male mice were examined during midde-age, at 14 months. The frontal cortex, a brain region affected over the course of dementia progression, was investigated. Iron-rich compartments were found across genotypes, including oligodendrocytes, and immune cells at the blood-brain barrier, and exclusively amyloid plaques in the APP-PS1 genotype. A semi-automated approach on QuPath was employed to quantify staining intensity of iron-rich compartments with light microscopy. Mouse frontal cortex of each genotype was also assessed qualitatively and ultrastructurally with scanning electron microscopy, to novelly discern and confirm iron-rich staining (via Perl’s DAB). We found parenchymal iron staining corresponding to oligodendrocytes, pericytes, astrocytes, microglia and/or infiltrating macrophages, and amyloid plaques; increased iron deposition and clustering were detected in middle-aged male APP-PS1 vs WT mice, supporting that AD pathology may involve greater brain iron levels and local clustering. Unexpectedly, iron-rich cells were enriched at the central nervous system (CNS) interface and perivascular space in control and APP-PS1 mouse models, with ultrastructural examination revealing examples of these cells loaded with many secretory granules containing iron. Together, our results provide novel exploration and confirmation of iii iron-rich cells/compartments in scanning electron microscopy and reinforce literature that iron deposition is relatively increased in AD over healthy cognitive aging and involves greater local clusters of iron burden. Increased iron burden along the aging trajectory, regardless of cognitive status, may also be attributed to novelly-discovered iron-rich cells secreting granules along the CNS border.Item Improving the efficacy of common cancer treatments via targeted therapeutics towards the tumour and its microenvironment(Pharmaceutics, 2024) Cecchi, Daniel; Jackson, Nolan; Beckham, Wayne; Chithrani, Devika B.Cancer is defined as the uncontrolled proliferation of heterogeneous cell cultures in the body that develop abnormalities and mutations, leading to their resistance to many forms of treatment. Left untreated, these abnormal cell growths can lead to detrimental and even fatal complications for patients. Radiation therapy is involved in around 50% of cancer treatment workflows; however, it presents significant recurrence rates and normal tissue toxicity, given the inevitable deposition of the dose to the surrounding healthy tissue. Chemotherapy is another treatment modality with excessive normal tissue toxicity that significantly affects patients’ quality of life. To improve the therapeutic efficacy of radiotherapy and chemotherapy, multiple conjunctive modalities have been proposed, which include the targeting of components of the tumour microenvironment inhibiting tumour spread and anti-therapeutic pathways, increasing the oxygen content within the tumour to revert the hypoxic nature of the malignancy, improving the local dose deposition with metal nanoparticles, and the restriction of the cell cycle within radiosensitive phases. The tumour microenvironment is largely responsible for inhibiting nanoparticle capture within the tumour itself and improving resistance to various forms of cancer therapy. In this review, we discuss the current literature surrounding the administration of molecular and nanoparticle therapeutics, their pharmacokinetics, and contrasting mechanisms of action. The review aims to demonstrate the advancements in the field of conjugated nanomaterials and radiotherapeutics targeting, inhibiting, or bypassing the tumour microenvironment to promote further research that can improve treatment outcomes and toxicity rates.Item Molecular mechanisms of reelin in the enteric nervous system and the microbiota–gut–brain axis: Implications for depression and antidepressant therapy(International Journal of Molecular Sciences, 2024) Halvorson, Ciara S.; Sánchez-Lafuente, Carla Liria; Johnston, Jenessa N.; Kalynchuk, Lisa E.; Caruncho, Hector J.Current pharmacological treatments for depression fail to produce adequate remission in a significant proportion of patients. Increasingly, other systems, such as the microbiome–gut–brain axis, are being looked at as putative novel avenues for depression treatment. Dysbiosis and dysregulation along this axis are highly comorbid with the severity of depression symptoms. The endogenous extracellular matrix protein reelin is present in all intestinal layers as well as in myenteric and submucosal ganglia, and its receptors are also present in the gut. Reelin secretion from subepithelial myofibroblasts regulates cellular migration along the crypt–villus axis in the small intestine and colon. Reelin brain expression is downregulated in mood and psychotic disorders, and reelin injections have fast antidepressant-like effects in animal models of depression. This review seeks to discuss the roles of reelin in the gastrointestinal system and propose a putative role for reelin actions in the microbiota–gut–brain axis in the pathogenesis and treatment of depression, primarily reflecting on alterations in gut epithelial cell renewal and in the clustering of serotonin transporters.Item Role of inflammatory mechanisms in major depressive disorder: From etiology to potential pharmacological targets(Cells, 2024) Kouba, Bruna R.; de Araujo Borba, Laura; Borges de Souza, Pedro; Gil-Mohapel, Joana; Rodrigues, Ana Lúcia S.The involvement of central and peripheral inflammation in the pathogenesis and prognosis of major depressive disorder (MDD) has been demonstrated. The increase of pro-inflammatory cytokines (interleukin (IL)-1?, IL-6, IL-18, and TNF-?) in individuals with depression may elicit neuroinflammatory processes and peripheral inflammation, mechanisms that, in turn, can contribute to gut microbiota dysbiosis. Together, neuroinflammation and gut dysbiosis induce alterations in tryptophan metabolism, culminating in decreased serotonin synthesis, impairments in neuroplasticity-related mechanisms, and glutamate-mediated excitotoxicity. This review aims to highlight the inflammatory mechanisms (neuroinflammation, peripheral inflammation, and gut dysbiosis) involved in the pathophysiology of MDD and to explore novel anti-inflammatory therapeutic approaches for this psychiatric disturbance. Several lines of evidence have indicated that in addition to antidepressants, physical exercise, probiotics, and nutraceuticals (agmatine, ascorbic acid, and vitamin D) possess anti-inflammatory effects that may contribute to their antidepressant properties. Further studies are necessary to explore the therapeutic benefits of these alternative therapies for MDD.Item In vitro and in vivo synergetic radiotherapy with gold nanoparticles and docetaxel for pancreatic cancer(Pharmaceutics, 2024) Alhussan, Abdulaziz; Jackson, Nolan; Chow, Norman; Gete, Ermias; Wretham, Nicole; Dos Santos, Nancy; Beckham, Wayne; Duzenli, Cheryl; Chithrani, Devika B.This research underscores the potential of combining nanotechnology with conventional therapies in cancer treatment, particularly for challenging cases like pancreatic cancer. We aimed to enhance pancreatic cancer treatment by investigating the synergistic effects of gold nanoparticles (GNPs) and docetaxel (DTX) as potential radiosensitizers in radiotherapy (RT) both in vitro and in vivo, utilizing a MIA PaCa-2 monoculture spheroid model and NRG mice subcutaneously implanted with MIA PaCa-2 cells, respectively. Spheroids were treated with GNPs (7.5 ?g/mL), DTX (100 nM), and 2 Gy of RT using a 6 MV linear accelerator. In parallel, mice received treatments of GNPs (2 mg/kg), DTX (6 mg/kg), and 5 Gy of RT (6 MV linear accelerator). In vitro results showed that though RT and DTX reduced spheroid size and increased DNA DSBs, the triple combination of DTX/RT/GNPs led to a significant 48% (p = 0.05) decrease in spheroid size and a 45% (p = 0.05) increase in DNA DSBs. In vivo results showed a 20% (p = 0.05) reduction in tumor growth 20 days post-treatment with (GNPs/RT/DTX) and an increase in mice median survival. The triple combination exhibited a synergistic effect, enhancing anticancer efficacy beyond individual treatments, and thus could be employed to improve radiotherapy and potentially reduce adverse effects.Item Application of high-Z nanoparticles to enhance current radiotherapy treatment(Molecules, 2024) Jackson, Nolan; Cecchi, Daniel; Beckham, Wayne; Chithrani, Devika B.Radiotherapy is an essential component of the treatment regimens for many cancer patients. Despite recent technological advancements to improve dose delivery techniques, the dose escalation required to enhance tumor control is limited due to the inevitable toxicity to the surrounding healthy tissue. Therefore, the local enhancement of dosing in tumor sites can provide the necessary means to improve the treatment modality. In recent years, the emergence of nanotechnology has facilitated a unique opportunity to increase the efficacy of radiotherapy treatment. The application of high-atomic-number (Z) nanoparticles (NPs) can augment the effects of radiotherapy by increasing the sensitivity of cells to radiation. High-Z NPs can inherently act as radiosensitizers as well as serve as targeted delivery vehicles for radiosensitizing agents. In this work, the therapeutic benefits of high-Z NPs as radiosensitizers, such as their tumor-targeting capabilities and their mechanisms of sensitization, are discussed. Preclinical data supporting their application in radiotherapy treatment as well as the status of their clinical translation will be presented.Item Effects of transition from remote to in-person learning in university students: A longitudinal study(European Journal of Investigation in Health, Psychology and Education, 2024) Siteneski, Aline; de la Cruz-Velez, Melina; Montes-Escobar, Karime; Duran-Ospina, Julia Patricia; Fonseca-Restrepo, Carolina; Barreiro-Linzán, Mónica Daniela; Campos García, Gusdanis Alberto; Gil-Mohapel, JoanaPrevious studies have shown that the transition from the University environment to remote learning impacted student mental health. Our study aimed to investigate the effects of university environment on anxiety and depressive symptoms in health sciences students. Students at the Technical University of Manabí, Ecuador, with 6–10 in-person semesters, who shifted to remote learning and then returned to face-to-face learning were selected. Students responded to the General Anxiety Disorder-7 (GAD-7) and Patient Health Questionnaire-9 (PHQ-9). In addition, questions regarding social interaction, physical exercise, mood and sleep habits were also asked. This longitudinal study tracked 323 students during the return to in-person classes and term end. The results showed similar rates of anxiety (GAD-7, p = 0.011-p = 0.002) and depression (PHQ-9 p = 0.001-p = 0.032) among students at week 1 and week 15. Previous diagnosis of depression (OR, 0.171; CI 0.050–0.579, p < 0.005) was shown to correlate with depression levels in week 1, with no changes seen at follow-up. Anxiety levels were shown to be associated with a previous diagnosis of the disorder at week 1, but not at follow-up (OR 0.233; CI 0.085–0.643, p < 0.005). The return to in-person learning among university students maintained levels of anxiety and depressive symptoms, underscoring ongoing vulnerabilities to mental health disorders in this group.Item Investigating the effects of ischemic stroke and diaschisis on connectomic presynaptic dendritic spine networks(2024) Butterworth, Jenna; Brown, Craig E.Ischemic stroke is a life-threatening medical condition that can lead to dysfunction in brain regions both proximally and distally connected to the stroke site, a phenomenon known as “diaschisis”. Diaschisis can play an important role in recovery after stroke; however, the structural changes that occur at the level of neurons connected to the stroke site are not fully understood. Here, we performed confocal microscopy to visualize dendritic spines after a photothrombotic stroke in the primary somatosensory forelimb cortex (S1FL) of adult mice labeled with a retrograde adeno-associated virus (retro pAAV.CAG.GFP). This allowed for the visualization of presynaptic neurons directly connected to the infarct core in areas such as S1FL, motor cortex (MC), secondary somatosensory cortex (S2), and contralateral primary somatosensory forelimb cortex (Contra-S1FL). We observed a decrease in presynaptic spine density one week after stroke in superficial basilar dendrites within the peri-infarct region, which recovered by six weeks after stroke. An increase in dendritic spine density was also found six weeks after stroke within superficial primary apical dendrites in peri-infarct region, and within S2 in superficial primary and secondary apical dendrites as well as deep basilar dendrites. These results suggest that a retrograde degenerative signal may be localized to the peri-infarct region, whereas other factors may be playing a role in the widespread functional changes seen after stroke. The increase in dendritic spines seen in the peri-infarct and S2 regions six weeks after stroke may be playing an adaptive or compensatory role and aiding in recovery. Using a diaschisis model, these findings add novel information about neuronal structure proximal and distal to the infarct core, as well as elucidate potential degenerative and protective structural processes that may underly recovery after stroke.Item Utilizing the diaschisis model to investigate the effects of stroke on post-synaptic spine densities(2024) Van Sprengel, Myrthe; Brown, Craig E.The brain is highly interconnected, and this is crucial to understanding a wide variety of neurological diseases. Identifying changes in brain connectivity circuits that occur in a disease state, like ischemic stroke, helps explain the deficits observed in both the peri-infarct and distally connected regions to the stroke site. This phenomenon is referred to as ‘diaschisis’. Currently, there is a lack of understanding of the structural changes that occur at the neuronal level in both proximal and distal regions that are synaptically connected to the infarct. In this study, viral vectors and 2-photon laser scanning microscopy were employed to examine both the acute and long-term changes of dendritic spine densities in post-synaptic neurons following a stroke in the primary somatosensory forelimb cortex (S1FL). Results show that dendritic spine density is significantly decreased in the peri-infarct region as well as in the motor cortex (superficial neurons only) after stroke as compared to shams. The secondary somatosensory and contralateral S1FL cortices show no changes in spine density 1-week after stroke. After 6-weeks of recovery (6 weeks after the stroke), spine densities returned to control levels, with the exception of the basilar dendrites on superficial peri-infarct neurons. These findings suggest that not only does stroke cause structural changes to occur in neurons localized in the peri-infarct region, but it also impacts regions heavily reliant on those neurons, supporting the diaschisis model of brain injury. Data suggests that the decreases in spine density 1-week after stroke may be proportionate to the number of synapses they make with neurons in the infarct. In conclusion, these data suggests that changes in dendritic spine densities could play a role in the global dysfunctions observed after stroke and may provide valuable information for therapeutic interventions in the future.Item SERPINE1/PAI-1 role on blood flow, stalling and vessel width in stroke(2024) Narayana, Kamal; Brown, Craig E.The brain relies heavily on the proper function of microvascular hemodynamics such as blood flow for sufficient oxygenation and clearance of metabolic waste. Therefore, it is no surprise that damage to blood vessels can result in heterogenous blood flow and obstructions furthering the effects of injury. Ischemic stroke can lead to a long-lasting disruption of blood flow in microvessels surrounding the infarct site, exacerbating injury beyond the initial insult. Homeostatic blood clotting and proteolytic (clot-busting) pathways are likely fundamental to regulating post-ischemic capillary blood flow, and thus functional recovery. We have recently discovered that the SERPINE1 (Serp1) gene, encoding for Plasminogen Activator-Inhibitor-1 (PAI-1), is highly expressed along blood vessels following photothrombotic stroke in the rodent somatosensory cortex. Therefore, we explored the role of Serp1/PAI-1 on cortical blood flow and its potential downstream effects following experimentally induced ischemic stroke. In the first aim, we examined the spatial and temporal expression of Serp1 post-stroke across multiple days using immunohistochemistry and confirmed with whole tissue RNA sequencing. We discovered that Serp1/PAI-1 expression is highly upregulated 3-days post-stroke (i.e., subacute), and interestingly, this expression was brain-wide. We also obtained RNA levels at 3-days confirming an upregulation in Serp1 post-stroke. We then successfully performed an endothelial-specific knockdown (KD) of Serp1 which we confirmed using RNA seq. In the second aim, using in vivo 2-photon imaging, we longitudinally imaged superficial cortical blood flow in adjacent and distant areas to the infarct in both Serp1+/+ and cerebral endothelial Serp1 KD mice (Serp1-/-). We discovered reduced blood flow in Serp1-/- mice in the penumbra and distant regions following stroke in the subacute (3d) and chronic phase (35d). The effects of the reduced blood were prominent in arteriole capillaries, and surprisingly, blood flow did not recover in Serp1-/-. In the third aim, we measured vessel width in the penumbra and distant regions across the imaging days and separated them by arteriole and venule capillaries. Surprisingly, we found that arteriole and venule capillaries in Serp1-/- were constricted in the subacute and chronic phase post-stroke, whereas in Serp1+/+, capillaries were dilated in the subacute phase only. These respective effects mimicked the observed heterogenous blood flow (increase in Serp1+/+ and decrease in Serp1-/-) between the genotypes. In the fourth aim, we used in vivo 2-photon imaging to longitudinally label leukocytes with anti-CD45.2 to distinguish between the type of stalling. Surprisingly, we found that, alongside Serp1+/+, the total number of stalls increased in Serp1-/- mice, despite the presumed coagulant role of PAI-1, which was mediated by a greater number of leukocyte stalls over red-blood cell (RBC) stalls. In the fifth aim, we analyzed neuroinflammatory gene expression in both Serp1+/+ and Serp1-/- mice in the subacute phase post-stroke. We found an asymmetrical distribution in favor of upregulating neuroinflammatory genes as an effect of stroke with Serp1 as one of the top genes in Serp1+/+. Interestingly, the effect of Serp1 KD overall reduced the fold change of the genes highly expressed in Serp1+/+ indicating for a potential beneficial and protective role of the KD. These findings reveal that Serp1 KD significantly affects microvascular hemodynamics and leukocyte recruitment, suggesting that merely increasing clot degradation (i.e., tissue plasminogen activator (tPA) treatment) following stroke may not necessarily improve local blood flow to penumbra. In combination with the genomic changes observed, Serp1 KD shifted the distribution of the neuroinflammatory genes, which may also suggest for a protective role in KD post-stroke. Altogether, Serp1 KD elucidated a complex set of microvascular-related and genomic changes that requires further investigation into its role in stroke recovery.Item Effects of transition from remote to in-person learning in university students: A longitudinal study(European Journal of Investigation in Health, Psychology, and Education, 2024) Siteneski, Aline; de la Cruz-Velez, Melina; Montes Escobar, Karime; Durán Ospina, Patricia; Fonseca-Restrepo, Carolina; Barreiro-Linzán, Mónica Daniela; Campos García, Gusdanis Alberto; Gil-Mohapel, JoanaPrevious studies have shown that the transition from the University environment to remote learning impacted student mental health. Our study aimed to investigate the effects of university environment on anxiety and depressive symptoms in health sciences students. Students at the Technical University of Manabí, Ecuador, with 6–10 in-person semesters, who shifted to remote learning and then returned to face-to-face learning were selected. Students responded to the General Anxiety Disorder-7 (GAD-7) and Patient Health Questionnaire-9 (PHQ-9). In addition, questions regarding social interaction, physical exercise, mood and sleep habits were also asked. This longitudinal study tracked 323 students during the return to in-person classes and term end. The results showed similar rates of anxiety (GAD-7, p = 0.011-p = 0.002) and depression (PHQ-9 p = 0.001-p = 0.032) among students at week 1 and week 15. Previous diagnosis of depression (OR, 0.171; CI 0.050–0.579, p < 0.005) was shown to correlate with depression levels in week 1, with no changes seen at follow-up. Anxiety levels were shown to be associated with a previous diagnosis of the disorder at week 1, but not at follow-up (OR 0.233; CI 0.085–0.643, p < 0.005). The return to in-person learning among university students maintained levels of anxiety and depressive symptoms, underscoring ongoing vulnerabilities to mental health disorders in this group.Item The emerging neuroimmune hypothesis of bipolar disorder: An updated overview of neuroimmune and microglial findings(Journal of Neurochemistry, 2024) Chaves-Filho, Adriano José Maia; Eyres, Capri; Blöbaum, Leonie; Landwehr, Antonia; Tremblay, Marie-ÈveBipolar disorder (BD) is a severe and multifactorial disease, with onset usually in young adulthood, which follows a progressive course throughout life. Replicated epidemiological studies have suggested inflammatory mechanisms and neuroimmune risk factors as primary contributors to the onset and development of BD. While not all patients display overt markers of inflammation, significant evidence suggests that aberrant immune signaling contributes to all stages of the disease and seems to be mood phase dependent, likely explaining the heterogeneity of findings observed in this population. As the brain's immune cells, microglia orchestrate the brain's immune response and play a critical role in maintaining the brain's health across the lifespan. Microglia are also highly sensitive to environmental changes and respond to physiological and pathological events by adapting their functions, structure, and molecular expression. Recently, it has been highlighted that instead of a single population of cells, microglia comprise a heterogeneous community with specialized states adjusted according to the local molecular cues and intercellular interactions. Early evidence has highlighted the contribution of microglia to BD neuropathology, notably for severe outcomes, such as suicidality. However, the roles and diversity of microglial states in this disease are still largely undermined. This review brings an updated overview of current literature on the contribution of neuroimmune risk factors for the onset and progression of BD, the most prominent neuroimmune abnormalities (including biomarker, neuroimaging, ex vivo studies) and the most recent findings of microglial involvement in BD neuropathology. Combining these different shreds of evidence, we aim to propose a unifying hypothesis for BD pathophysiology centered on neuroimmune abnormalities and microglia. Also, we highlight the urgent need to apply novel multi-system biology approaches to characterize the diversity of microglial states and functions involved in this enigmatic disorder, which can open bright perspectives for novel biomarkers and therapeutic discoveries.Item Neuronal computations supporting direction selectivity in the mouse retina(2024) deRosenroll, Geoff; Awatramani, GautamBy the time visual information leaves the eye, it has already passed through multiple layers of neurons organized into feature-selective circuits that work to distill the analogue light signal received by photoreceptors down into diverse spike rate codes in the ganglion cells of the retina, whose axons make up the optic nerve. Taking advantage of the accessibility of the retina relative to the rest of the brain, dissecting these circuits provides great opportunities for the study of neuronal computations. One such circuit is centred around the direction-selective ganglion cell, which spikes robustly when objects move through their receptive field in particular directions and weakly or not at all in the opposite directions owing to inhibition from presynaptic starburst amacrine cells. This is supported by multiple complementary and redundant computations, both in the presynaptic starburst amacrine cells and postsynaptically in the DSGCs. In this thesis, I use computational modelling methods to complement and formalize theories based on empirical studies of these directional mechanisms as well as to form new predictions at the edge of our current understanding of the circuit. I start by modelling the "space-time wiring" directional mechanism involving the systematic distribution of kinetically distinct bipolar cell inputs along starburst amacrine cell dendrites using physiologically derived bipolar release transients for the first time. Then, moving downstream, I demonstrate how the asymmetric wiring of starburst dendrites to DSGCs is sufficient to drive DS spiking, even in the absence of directional release of neurotransmitters from starbursts. After exploring two mechanisms generating direction-selective responses in DSGCs, I focus on improving our understanding of how the non-directional glutamatergic and cholinergic sources of excitation to DSGC dendrites support the reliable computation of direction. I show that the mediation of glutamatergic inputs by voltage-dependent NMDA receptors at low-contrasts enables a context-dependent switch between modes of neuronal arithmetic: nearly flat addition over contrast and tuning-preserving multiplication over direction. Finally, I examine how the multi-directed corelease of acetylcholine alongside GABA from starbursts means that the dominant excitation to DSGCs is highly spatiotemporally correlated with inhibition in the null directions, ensuring reliable suppression of spiking. Overall, this research highlights multiple examples of how circuit structure and function work together to support consistent neuronal computations over space and time.Item Containment of neuroimmune challenge by diosgenin confers amelioration of neurochemical and neurotrophic dysfunctions in ketamine-induced schizophrenia in mice(Brain Disorders, 2024) Ben-Azu, Benneth; Adebayo, Olusegun G.; Fokoua, Aliance R.; Onuelu, Jackson E.; Asiwe, Jerome N.; Moke, Emuesiri G.; Omogbiya, Itivere A.; Okpara, Oghenemarho L.; Okoro, Jennifer E.; Oghenevwerutevwe, Omadevuaye M.; Uruaka, Christian I.Inhibition of neuroinflammation through N-methyl-D-aspartate receptor (NMDAR) regulation can provide normalization of neurochemical homeostasis and neurotrophic support in the pathogenesis of psychiatric disorders with complex symptoms such as schizophrenia. Accordingly, the preventive and reversal effects, and potential mechanisms of diosgenin, a phyto-steroidal sapogenin with anti-inflammatory functions, was evaluated in ketamine (an NMDAR antagonist) model of schizophrenia in mice. Adult male mice were allotted into 5 groups. In the preventive protocol, mice received saline (10 mL/kg), diosgenin (25 and 50 mg/kg) and risperidone (0.5 mg/kg) orally for 14 days, with additional injection of ketamine (20 mg/kg/day/i.p.) from days 8–14. In the reversal protocol, mice took ketamine injection consecutively for 14 days prior to diosgenin and risperidone treatments from days 8–14. Thereafter, schizophrenia-like behavior, therapeutic extrapyramidal adverse effect, neuroimmune, neurochemical and neurotrophic consequences in important brain areas affected in the disorder were assayed. Diosgenin prevented and reversed stereotypy behavior, cognitive impairment, and psychotic-depression relative to ketamine groups. Complementarily, diosgenin prevents and reverses ketamine-induced dopamine and serotonin alterations in the striatum, prefrontal-cortex, and hippocampus relative to ketamine groups. Except for the cortical regions, diosgenin prevented and reversed glutamic acid decarboxylase depletion in these brain regions by ketamine, suggesting improved GABAergic system. Additionally, ketamine-induced elevation of neuroinflammatory markers: myeloperoxidase, tumor necrosis factor-alpha and interleukin-6, were inhibited in the striatum, prefrontal-cortex, and hippocampus. Also, diosgenin improved the levels of neurotrophic factor in the three brain regions in both protocols respectively. Among other mechanisms, the antipsychotic effect of diosgenin might be associated with attenuation of neurochemical and neuroimmune alterations.Item Alterations in intrinsic and synaptic properties of hippocampal CA1 VIP interneurons during aging(Fronteirs in Cellular Neuroscience, 2020) Francavilla, Ruggiero; Guet-McCreight, Alexandre; Amalyan, Sona; Hui, Chin Wai; Topolnik, Dimitry; Michaud, Félix; Marino, Beatrice; Tremblay, Marie-Ève; Skinner, Frances K.; Topolnik, LisaLearning and memory deficits are hallmarks of the aging brain, with cortical neuronal circuits representing the main target in cognitive deterioration. While GABAergic inhibitory and disinhibitory circuits are critical in supporting cognitive processes, their roles in age-related cognitive decline remain largely unknown. Here, we examined the morphological and physiological properties of the hippocampal CA1 vasoactive intestinal peptide/calretinin-expressing (VIP+/CR+) type 3 interneuron-specific (I-S3) cells across mouse lifespan. Our data showed that while the number and morphological features of I-S3 cells remained unchanged, their firing and synaptic properties were significantly altered in old animals. In particular, the action potential duration and the level of steady-state depolarization were significantly increased in old animals in parallel with a significant decrease in the maximal firing frequency. Reducing the fast-delayed rectifier potassium or transient sodium conductances in I-S3 cell computational models could reproduce the age-related changes in I-S3 cell firing properties. However, experimental data revealed no difference in the activation properties of the Kv3.1 and A-type potassium currents, indicating that transient sodium together with other ion conductances may be responsible for the observed phenomena. Furthermore, I-S3 cells in aged mice received a stronger inhibitory drive due to concomitant increase in the amplitude and frequency of spontaneous inhibitory currents. These age-associated changes in the I-S3 cell properties occurred in parallel with an increased inhibition of their target interneurons and were associated with spatial memory deficits and increased anxiety. Taken together, these data indicate that VIP+/CR+ interneurons responsible for local circuit disinhibition survive during aging but exhibit significantly altered physiological properties, which may result in the increased inhibition of hippocampal interneurons and distorted mnemonic functions.