Centre for Neuroscience Studies Graduate Theses

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    Regulation and molecular identification of a lipid-gated cation channel in Aplysia bag cell neurons
    (2024-04-30) Stevens, Elise Mary MacLeod; Neuroscience Studies; Magoski, Neil
    Non-selective cation currents are key to the control of neuronal activity, and can be regulated by numerous intracellular messengers, including lipid metabolites and Ca2+. In neuroendocrine bag cell neurons from the sea snail, Aplysia californica, a diacylglycerol (DAG)-gated cation current supports an ~30 min afterdischarge and hormone secretion to induce egg-laying. To understand how this current is regulated, cultured bag cell neurons were whole-cell voltage-clamped or sharp-electrode current-clamped and exposed to N-(3-trifluoromethyl phenyl)-2,4,6-trimethylbenzenesulfonamide (m-FBS), which direcly stimulates phospholipase C (PLC) to hydrolyse phosphatidylinositol 4,5-bisphosphate (PIP2) into DAG and inositol trisphosphate. Consistent with cation channel opening, m-FBS induced an ~6-fold increase in membrane conductance with a voltage-independent current between -80 and 0 mV that reversed at -25.1 mV. Low extracellular Ca2+, replacing Ca2+ with Ba2+, or low intracellular Ca2+ potentiated the m-FBS-induced current by 2-3 times, suggesting Ca2+-dependent inactivation via channel-mediated Ca2+ entry. PIP2 appeared to be a basal negative regulator, as depletion with wortmannin, which inhibits PIP2 synthesis, augmented the current (+120%) or depolarization (+30%) evoked by the DAG analogue, 1-oleoyl-2-acetyl-sn-glycerol. The m-FBS-induced current may in part be coded for by an Aplysia transient receptor potential channel, ApTRPC5, which our laboratory previously cloned and found responsive to PLC activation with an inward current in a heterologous expression system. Here, real-time quantitative polymerase chain reactions showed ApTRPC5 to be expressed in bag cell neurons and adjacent abdominal ganglion. Human embryonic kidney cells expressing ApTRPC5 responded to m-FBS with a current that reversed at -10.8 mV and, like the native current, was doubled by low extracellular Ca2+. Mutation of Arg627, a residue suspected to be involved in PLC gating of human TRPC5, nearly eliminated the response to m-FBS but did not interfere with ApTRPC5 expression or trafficking as assayed by immunoblotting or epi-fluorescent microscopy, respectively. Moreover, disruption of ApTRPC5 expression in bag cell neurons with double-stranded RNA halved the m-FBS-induced current. In summary, the reproductive afterdischarge is promoted through PLC activation, which generates DAG to open a TRP-like non-selective cation channel, which in turn is both facilitated by PIP2 breakdown and, to prevent aberrant signalling, limited by Ca2+ influx.
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    Investigating Pupil Dynamics in Healthy and Clinical Populations
    (2024-04-30) Huang, Po Yueh; Neuroscience Studies; Munoz, Douglas
    Observing behaviours can provide insight into the underlying functioning of the brain, yielding scientific and clinical utility. The easy-to-measure, involuntary movement of the pupil is under the combined influences of sensory and cognitive processes, including luminance, arousal, sensory-orienting, and executive function, which shift due to changes in the brain across the human lifespan following healthy development and aging as well as clinical disorders. In this thesis, we examined how these changes affect the pupil control circuit and alter pupil behaviour. In the first study, we investigated how the pupil changes across the lifespan related to processes of natural maturation and deterioration in various regions of the brain. Using an oculomotor task that provided sensory cues and engages cognitive control, we assess the processes underlying pupil responses. This study provided insight into the development and aging trajectories of the neural substrates, and established a baseline of normative pupil behaviour to which clinical investigations can be compared. In the second study, we asked how deficits within the pupil circuit affected pupil behaviour by examining pediatric demyelinating disorders. We demonstrated how impairments in sensory and cognitive signalling related to demyelinating injury can affect the latencies of pupil responses associated with structural and functional outcome measures in these disorders. In the final study, we examined changes in pupil behaviour in several neurodegenerative diseases related to the development of dementia. We explored how cognitive impairment is associated with altered pupil responses, and how medications and compensatory mechanisms can affect the pupil in these diseases. Together, these studies give an account of how sensory and cognitive functions can be reflected on the pupil in health and disease. This work will be important for advancing the use of pupillometry in research and clinical applications.
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    The Neurochemical Phenotype of Sensory Somata with Sympathetic Plexuses – The Role of Nerve Growth Factor
    (2024-04-30) Alsaadi, Hanin; Neuroscience Studies; Kawaja, Michael D.
    Following peripheral nerve injury, postganglionic sympathetic axons aberrantly sprout towards sensory ganglia where they form perineuronal sympathetic plexuses (basket-like structures) around medium- to large-diameter neurons. This pathological phenomenon is known as sympathosensory coupling and underlies sympathetically-maintained pain. A large body of scholarly literature establishes the critical role of nerve growth factor (NGF) as a primary driver triggering sympathetic sprouting. One important question facing researchers is identifying the precise neurochemical phenotype of the neurons surrounded by sympathetic plexuses. A challenge arises as many phenotypic markers that identify sensory neuronal populations are down-regulated in response to nerve injury. This work utilized a broad range of nociceptive phenotypic biomarkers to neurochemically phenotype those sensory somata surrounded by sympathetic plexuses. The studies employed quantitative immunofluorescence staining analyses in two distinct models: 1) adult transgenic mice that overexpress NGF, which results in the spontaneous formation of sympathetic plexuses (i.e., absence of injury) in the trigeminal ganglia (TG), and, 2) adult rats and adult mice with peripheral nerve injury, which results in the aberrant formation of sympathetic plexuses in the affected dorsal root ganglia (DRG). This thesis shows that sympathetic axons target a precise sub-population of NGF-sensitive and peptidergic nociceptors (i.e., those co-expressing the NGF receptor trkA and calcitonin gene-related peptide, CGRP). These trkA+/CGRP+ nociceptive neurons are immunopositive for ATP/purinergic receptor (P2X3) and for neurofilament heavy chain (NFH). The results also suggest that sympathetic plexuses may trigger shifts in the phenotypic expression of other nociceptive biomarkers. Collectively, this work reveals, for the first time, that sympathosensory sprouting is not a random event but is one that results from sympathetic axons targeting particular subpopulations of nociceptive neurons in the sensory ganglia (i.e., those displaying specific neurochemical features among the trkA+/CGRP+ subgroups). The identification of this specificity is novel and contributes to our understanding of the mechanisms underlying pathological sympathosensory coupling. Ultimately, these findings may contribute to targeted therapeutic interventions in the context of sympathetically-maintained pain, in particular as it relates to the aberrant formation of sympathetic plexuses around subpopulations of nociceptive sensory somata that rely on the availability of target-derived NGF for survival and maintenance in adulthood.
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    On the Use of Robotic Technology to Quantify Sensorimotor Impairments After Stroke
    (2024-02-28) Park, Kayne; Neuroscience Studies; Scott, Stephen
    It is well recognized that sensory feedback plays an important role in allowing us to move and interact in our complex world. However, sensory feedback can be impaired following stroke potentially reducing an individual’s independence and increasing their fall risk. Commonly used clinical assessments do not have the ability to accurately quantify these impairments. The objective of this dissertation is to use robot-based behavioural tasks to accurately quantify impairments in the use of sensory information for guiding motor actions. The first study explored the use of a novel interception task to quantify impairments in the use of proprioceptive and visual feedback to generate rapid motor responses. The Fast Feedback Interception Task (FFIT) was developed to examine the ability to intercept a moving ball with a cursor. Sensory feedback was assessed by either mechanically perturbing the participant’s arm or shifting the visual location of the ball or cursor. Compared to healthy controls, 85% of individuals with stroke displayed impairments using their affected arm. Of note, 75% of individuals with stroke were impaired using their unaffected arm. The second study examined whether individuals with stroke could have distinct impairments in sensory feedback for action and perception. We compared performance on FFIT with a reaching task and an upper limb proprioceptive task. Most individuals with stroke were impaired in FFIT and reaching using their affected arm. Further, most FFIT and reaching task parameters were significantly correlated. We found only a few significant correlations between FFIT and proprioceptive task parameters. The third study examined spatial impairments for individuals with stroke using an 8-target reaching task. Of 265 individuals with stroke, 64% demonstrated impaired reaction time. 35% were impaired in initiating a movement towards a specific region of the workspace and 44% were impaired in all directions of reach. Further, lesions to specific brain regions or white matter tracts were associated with reaction time impairment. Our results highlight the high proportion of individuals with stroke impaired in the use of sensory feedback. Future research is required to understand how these findings can be implemented into clinical practice for guiding diagnosis and tracking stroke recovery.
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    Microbial Ecosystem Therapeutics for Major Depression
    (2024-01-24) Chinna-Meyyappan, Arthi; Neuroscience Studies; Milev, Roumen
    Background: Research suggests gut microbiota repopulation techniques may elicit an improvement in depressive symptoms via the pathways of the gut-brain axis, a bidirectional communication network between the gut and the brain. Objectives: 1) Systematically review the current literature examining fecal microbiota transplantation (FMT) for psychiatric symptoms in clinical and preclinical populations; 2) Examine the efficacy, safety, and tolerability of microbial ecosystem therapeutic-2 (MET-2) on depression and anxiety symptoms in a pilot study; 3) Formulate a protocol for a double-blind, randomized, placebo-controlled trial (DBRCT) to investigate the impact of MET-2 on depression using knowledge obtained from the pilot study 4) Conduct a comparative analysis of the clinical and molecular effects of MET-2 versus placebo. Methods: After systematically reviewing the current literature to evaluate existing evidence for the use of gut repopulation treatments, I conducted a 10-week open-label pilot study examining the effects of MET- 2, an alternative to FMT that is comprised of 40 lyophilized bacterial cultures for oral consumption. Then I developed a protocol for an 8-week DBRCT to examine MET-2’s efficacy in comparison to a placebo and explore changes in immune biomarkers that may be involved in underlying mechanisms of action of MET-2. Results: The systematic review provided relatively strong evidence for the use of gut repopulation techniques for treatment of depression, though there was minimal evidence in clinical populations and a lack of DBRCTs. The pilot results showed promising evidence for using MET-2 in alleviating mood and anxiety symptoms. These findings were not supported by the DBRCT. Though there were significant improvements in mood and related symptoms scores, these were not significantly different between placebo and MET-2 groups. There were also no significant changes in immune biomarkers. However, the response rate data and clinically significant trends seen between the two groups in the clinical data were promising. Conclusions: The findings presented in this thesis are the first to provide evidence for the role of MET-2 in alleviating symptoms of depression and compare its use to a placebo alternative. They provide crucial future directions for gut repopulation research in the context of psychiatric indications.