Browsing by Author "Raimondo, Joseph"

Now showing 1 - 5 of 5
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    Exploring the effects of classical immune activation on circuit excitability and cell viability in the mouse brain
    (2021) Tinelli, Sasha; Raimondo, Joseph; de Lange, Anja
    Epilepsy directly affects approximately 50 million people globally and is the most common neurological disorder in sub-Saharan Africa, mainly due to high rates of neuroinfections and head trauma experienced by people in the region. A common factor in these causes of acquired epilepsy is their association with significant neuroinflammation, which is thought to drive the epileptogenic process. Although epilepsy exerts a heavy toll on the health, wellbeing and socio-economic outcomes of Africans, there are still major deficits in our understanding of how infections and inflammatory processes drive seizure development. Using the hippocampal organotypic brain slice culture model in mouse brains, I investigated the effects of classical immune activation on circuit excitability and cell viability. To initiate inflammation, I administered lipopolysaccharide (LPS), an endotoxin derived from gramnegative bacteria, and interferon-gamma (IFNy), a cytokine typically released by lymphocytes, to brain slices on varying time scales. I used enzyme-linked immune-sorbent assays to show that this reliably induced the release of the proinflammatory cytokines TNFα and IL-6 from the brain slices. I used patch-clamp electrophysiology to assess both the intrinsic electrical characteristics as well as the synaptic strength between pyramidal neurons after immune activation. I found no changes in the basic membrane properties of pyramidal neurons after short term neuroinflammation, but I did observe changes to the function of hippocampal networks at intermediate (24 hours) and lengthy (72 hours) time scales of immune activation in the form of significantly reduced spontaneous excitatory and inhibitory postsynaptic current frequencies and amplitudes. In addition, I developed an assay to determine neuronal survival to monitor the health of neurons in brain slices after immune activation and report that hippocampal organotypic brain slice cultures that were immuneactivated for 72 hours do not appear to experience either apoptotic or necrotic cell death. Taken together, these data constitute a valuable contribution towards understanding how inflammatory mechanisms drive changes to neuronal function, which could be relevant for understanding epileptogenesis in infectious and inflammatory causes of epilepsy.
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    Investigating cestode modulation of host neuronal excitability and cell-type-specific gene expression in an in vitro mouse model of neurocysticercosis
    (2023) Steyn, Teresa; Raimondo, Joseph
    Neurocysticercosis (NCC), caused by Taenia solium larvae, is the leading helminthic brain infection in humans, with epilepsy as its most common manifestation. Interestingly, seizures are rare during early stages when viable larvae are thought to suppress host inflammation. Seizures tend to occur when larvae die and immune suppression ceases, which has led to the hypothesis that the host's immune response contributes to seizures in NCC. Further research is required to better understand the effects that immune activation and Taenia larvae have in the brain. In this thesis, I exposed mouse hippocampal organotypic brain slice cultures (OBSCs) to lipopolysaccharide (LPS) and a Taenia crassiceps homogenate and used singlenucleus RNA sequencing and whole-cell patch-clamp electrophysiology to investigate potential links between inflammation and network excitability at a transcriptomic and electrophysiological level. In the first part of the thesis, I found that while LPS significantly affected cell-type specific gene expression associated with inflammatory pathways, it had little impact on genes modulating neuronal excitability. This was corroborated by whole-cell patchclamp data demonstrating no LPS-induced changes in the intrinsic electrical properties of pyramidal neurons. In the second part of the thesis, I exposed mouse hippocampal OBSCs to a T. crassiceps homogenate alone or in combination with LPS to evaluate its immunomodulatory activity. The T. crassiceps homogenate blocked the induction of pro-inflammatory transcriptional activity across different cell types when added to LPS. This suggested that it likely acts up-stream of the toll-like receptor 4 proinflammatory cascade. The homogenate had minimal influence on the expression of neuronal excitability genes, and whole-cell patch-clamp experiments confirmed no significant differences in pyramidal neuron electrical properties among the treatment groups. My data indicate that both LPS and a homogenate made from viable Taenia larvae drive cell-type-specific immunomodulatory changes but have limited effects on basic neuronal excitability, at least over a relatively short period of exposure and in the absence of an adaptive immune response. My findings are relevant for understanding how Taenia larvae and inflammatory responses relate to the emergence of seizures in NCC.
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    Investigating gene expression differences in acute and organotypic mouse hippocampal brain slices as models for studying the brain
    (2025) Kundieko, Sagel; Raimondo, Joseph; Steyn, Teresa
    Organotypic brain slice cultures (OBSCs) are widely used to study neural circuit function, but their molecular and cellular composition relative to acute brain slices remains poorly characterized. Here, using single-nucleus RNA sequencing, I performed a comprehensive comparison of gene expression between OBSCs and acute brain slices prepared from postnatal day 7 mouse hippocampus. I identified significant differences in cellular composition, with OBSCs showing an overrepresentation of glial cells and a relative underrepresentation of neuronal populations. Notably, oligodendrocytes were almost exclusively found in OBSCs, while dentate progenitor cells were predominantly present in acute slices, reflecting ongoing developmental processes in culture. Differential gene expression analysis revealed over 4200 unique differentially expressed genes across major cell types, with excitatory neurons and astrocytes showing the most substantial transcriptional changes. Gene ontology analysis demonstrated upregulation of pathways involved in neuronal development and cell signalling alongside downregulation of axon guidance pathways in OBSCs. Specific cell-type analysis revealed distinct adaptations, including altered inflammatory responses in microglia and astrocytes as well as modified synaptic signalling in dentate gyrus cells. These findings provide crucial insights into how brain slice cultures adapt to ex vivo conditions and highlight important considerations for their use as experimental models in neuroscience research.
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    Investigating the effects of impermeant anions on the electrical and computational properties on neurons
    (2023) Shorer, Eran; Raimondo, Joseph
    Impermeant anions (proteins, amino acids, etc.) are negatively charged ions that are unable to traverse the cell membrane. Impermeant anion quantities and their average charge vary with metabolism as well as protein and nucleic acid synthesis/turnover.The effect of spatiotemporal changes to impermeant anions on neurons is poorly understood. Using a multicompartment electrodiffusion-based computational model I investigated the influence of impermeant anions on neuronal cellular physiology, passive cable properties, and synaptic integration. Spatial differences in the average charge of impermeant anions result in a nonisopotential dendrite with ionic microdomains. At steady state local discrepancies in membrane potentials and ion concentrations do not impact the passive or active electrical properties of neurons as ionic driving forces are unchanged, irrespective of impermeant anion mean charge. These findings explain how electrical signalling remains consistent in the face of an ever-changing impermeant anion milieu with implications related to our understanding of both normal and pathological neuronal physiology.
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    Using single nucleus RNA-seq to determine the effect of immunosuppression on the transcriptional activity of resident CNS cells in a mouse model of CNS-TB
    (2025) Amien, Ilyaas; Hockman, Dorit; Jacobs, Muazzam; Raimondo, Joseph
    Tuberculosis (TB) is the leading infectious cause of death worldwide and is particularly prevalent in South Africa. Central Nervous System TB (CNS-TB), specifically TB Meningitis (TBM) is the deadliest complication of the disease. Given the high co-prevalence of HIV and CNS-TB, many transcriptional studies have examined the immunocompromised response to CNS-TB, but none have done so atsingle cell resolution. Single nucleus RNA Sequencing (snRNA-seq) was used to determine the effect of immunosuppression on the transcriptional activity of resident CNS cells in a mouse model of CNS-TB. The model consisted of two mouse genotypes, namely Wildtype and TNF knockout (TNF-/- ). Mycobacterium Tuberculosis was injected into the somatosensory cortex of the treatment groups, with control samples receiving a saline injection. Brains were harvested at 9- and 17-days post inoculation. Following library generation, removal of ambient RNA and data processing using Seurat, differential gene expression analysis was used to identify differentially expressed genes (DEGs). Oligodendrocytes had the most DEGs in the general TB response, whereas excitatory neurons had the highest DEGs in the immunocompromised TB response and with disease progression. Potential mechanisms by which these cell types contribute to the pathogenesis of CNS-TB were explored, such as excitotoxicity, demyelination and neurodegeneration. Pathways related to neurogenesis were identified in the immunocompromised TB response. The study adds to the body of knowledge by identifying cell type specific resident gene expression responses in the context of CNS-TB.