Theses (Neuroscience)

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    Microglia change at the micro- and nano-scopic scales in response to therapeutic focused ultrasound blood-brain barrier modulation
    (2023-08-31) Gonçalves de Andrade, Elisa; Tremblay, Marie-Ève
    Transcranial focused ultrasound sonication in combination with intravenously injected microbubbles (FUS+MB) has the unique ability to modulate blood-brain barrier (BBB) permeability with high spatial precision and in a minimally invasive manner. This process induces endothelial mechanical stress and the transient infiltration of blood-derived molecules, collectively triggering an acute inflammation in the targeted region. As the resident immune cells of the central nervous system (CNS), microglia are at the forefront of the acute inflammatory response triggered by FUS+MB BBB modulation. Notably, in inflammatory and non-inflammatory contexts, microglia are highly dynamic and continuously survey the brain parenchyma by extending their processes and interacting with surrounding elements, such as synapses and blood vessels. In response to stressors, microglia rapidly alter their cellular and molecular profile to help facilitate the return to homeostasis. While the underlying mechanisms by which FUS+MB alters microglial function remain largely unknown, several studies in adult mouse models of Alzheimer’s disease pathology have reported changes in the gene expression of microglia, e.g. ionized calcium binding adaptor molecule 1 (Iba1), and in their phagocytic activity, e.g. increased uptake of the toxic protein amyloid beta, in several CNS areas, including the hippocampus where emotional, cognitive and memory processing takes place. Characterizing the baseline responses of microglia to FUS+MB in healthy models is, however, still required to understand possible outcomes of this technology in physiological processes such as microglial parenchymal surveillance, maintenance of the BBB and synaptic plasticity. To address this need, I provide the first cellular (i.e., density, distribution, and morphology) and subcellular (i.e., ultrastructure) description of microglial changes at 1 hour and 24 hours after magnetic resonance imaging-guided focused ultrasound sonication with microbubbles (MRIgFUS) targeting the ipsilateral (ipsi-) ventral hippocampus of adult male mice. Using brightfield imaging of a double immunoperoxidase staining for immunoglobulin G and Iba1, I show that MRIgFUS causes the entering of IgG into the ipsi- Cornu ammonis 1 (CA1) parenchyma at 1 hour and 24 hours, where it correlates with the proximity between Iba1 positive (+) cells. Moreover, I found that specific states within the ipsi- microglial CA1 stratum lacunosum moleculare (LMol) population are more responsive to MRIgFUS compared to the contra- LMol, adjusting their soma, cell body and arborization parameters to a rod-like shape, while collectively, most LMol microglia adopt an elongated soma shape after MRIgFUS. Notably, I observed that the LMol nanoscale structure of microglia changes at 1 hour and 24 hours after MRIgFUS, increasing their interactions with the BBB in vessels with bigger areas and presenting swollen astrocytic endfeet. By contrast, MRIgFUS induces less frequent interactions between microglia, pre-synaptic elements and extracellular space, associated with trogocytosis or nibbling of synaptic elements. Collectively, my research suggests that, at 1 hour and 24 hours after MRIgFUS, ventral hippocampal modifications are mostly restricted to a microglia subset, warranting further mechanistic investigations but simultaneously supporting the overall application of this technology.
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    Postnatal Choline Supplementation Ameliorates Synaptic Plasticity Deficits Following Prenatal Ethanol Exposure in a Sex-Specific Manner
    (2023-07-13) Gräfe, Erin; Christie, Brian R.
    Background: Fetal Alcohol Spectrum Disorder (FASD) is one of the leading causes of neurodevelopmental impairment. FASD is the diagnostic term to encompass the range of physical, cognitive, or emotional impairments due to prenatal ethanol exposure (PNEE). One of the most notable consequences of PNEE are deficits in hippocampal synaptic plasticity, and consequently, learning and memory impairments. Currently, there is no treatment for FASD. However, there are promising data to demonstrate that the essential nutrient choline may improve outcomes following developmental ethanol exposure. Questions remain as to how postnatal choline supplementation improves hippocampal outcomes at a synaptic level, whether these occur in a sex-dependent manner, and if any changes will persist into adulthood. Methods: This dissertation employed a first-two trimester moderate model of PNEE (Gestational day 1-22). Offspring were supplemented with choline chloride (100 mg/kg/day) from postnatal day (PND) 10-30, then tested either immediately following treatment (PND 31-36) or in adulthood (PND 60-90). In juvenile offspring bidirectional plasticity was evaluated, as well as behavioural changes using the Radial Arm Maze. In adulthood, saturating and subthreshold long-term potentiation (LTP) was evaluated, as well as alterations in GluN2B functionality. Results: PNEE reduced the magnitude of LTP in male juvenile offspring, but not in females. Choline treatment increased LTP in both male and female PNEE offspring. However, choline treatment insignificantly decreased the amount of long-term depression (LTD) in male offspring, regardless of prenatal environment. Improvements in PNEE male LTP did not translate to behavioural improvements in the Radial Arm Maze, either in the working or reference memory performance. Female juvenile offspring did not learn the task over the course of the five trials and this lack of learning was not due to differences in search strategy. In adulthood, there were no evident changes in LTP with PNEE or with choline treatment in either sex. Despite the lack of deficit or improvement, choline treatment altered the LTP threshold, such that lower frequency stimulating protocols still resulted in LTP in choline treated offspring. While it is not clear why this change in LTP threshold occurred, it could be due to alterations in GluN2B functionality; GluN2B antagonism increased field excitatory postsynaptic potential (fEPSP) size in control offspring, but not in saline treated PNEE adults. Conclusion & Significance: This dissertation demonstrated that postnatal choline supplementation ameliorated the deficits in LTP in PNEE males and further increased LTP in PNEE females. However, these changes in synaptic plasticity did not persist in adulthood when using a saturating conditioning stimulus. There may still be alterations in hippocampal LTP threshold after treatment cessation, but the exact locus of action remains to be uncovered. These data further support choline as a treatment for PNEE, but also suggest that extended choline treatment may produce more long-lasting changes.
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    Analyzing the effects of high autistic traits on learning: An event-related potential approach
    (2023-04-26) Parsons, Ellis M.; Krigolson, Olav
    It has been established that certain learning differences exist in autistic people in contrast to their neurotypical counterparts in the context of reinforcement learning, decision-making, and working memory as quantified by electroencephalography (EEG) derived event-related potentials (ERPs). However, what is becoming increasingly apparent is the lack of consensus on this matter, which may be attributable to working memory and other executive functioning (EF) differences in autistic people that contribute to differential learning abilities and thus task performance. The present study aimed to answer the question of how and to what extent these learning differences, as quantified by the reward positivity and P300, may appear in a high autistic traits population in contrast to the low autistic traits control by employing the use of both quantitative and qualitative research measures in a concentrated effort to include the perspectives of autistic people. More specifically, it was hypothesized that both a smaller reward positivity and a larger P300 would be found in high autistic traits participants. Furthermore, it was anticipated that the degree of autistic traits expressed by participants as measured by the Autism Quotient (AQ) would be negatively correlated with the reward positivity, and positively correlated with the P300. Though these results were not statistically significant and both groups completed tasks with the same degree of accuracy, it was found that high autistic traits participants perceived their task performance to be significantly worse than those with low autistic traits. These findings shed light on certain methodological considerations that are discussed at length.
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    Investigating the Patterns of Vascular Remodelling in the Healthy Adult Mouse Cortex
    (2023-02-10) Raudales, Alejandra; Brown, Craig E.
    Angiogenesis (the sprouting of new blood vessels from pre-existing ones) is a process that occurs naturally during many physiological and pathological processes. Given that the cerebral vasculature requires tight regulation of homeostatic environments, too much, or too little angiogenesis can severely impact brain function and maintenance. The vast majority of what we know about physiological angiogenesis (particularly in the brain) centers on early embryonic and early postnatal developmental stages where this process is driven by hypoxic signals from un-perfused tissue to develop and expand the vascular network. Once an organism reaches adulthood, the vasculature is still required to sustain the incredibly high metabolic demands of neuronal functioning; however, it remains unclear to what extent the capacity to grow new blood vessels is maintained in the healthy and stable adult vascular network. Previous work from our lab has shown that even in the healthy adult mouse cortex, spontaneous obstructions to capillaries occur on a regular basis. While the vast interconnectedness of the capillary network may be able to adapt to a few lost capillaries, the additive challenge of pruned vessels throughout the lifespan can pose a significant risk to homeostatic efforts. A quantitative analysis of the rates at which vessel density is lost naturally with age across various brain regions found that while many areas experience a significant decrease in vascular density, some areas remain resilient to significant loss. In this thesis I explore the possibility that angiogenesis compensates in a regionally dependent manner to prevent significant vessel density loss. Our results show that while there are no regional differences in the pruning rate of capillaries, angiogenesis is significantly elevated in a graded manner across the cortical surface, with ample evidence of growth of microvessels over lateral-posterior regions. On the contrary, evidence of vessel sprouting along medial-anterior regions is extremely rare. Further probing into molecular mechanisms driving adult angiogenesis, I found that it is not upregulation of pro-angiogenic factors driving regional heterogeneity, but rather an upregulation of pro-stabilizing molecules in medial regions of the cortex that put the brakes on the angiogenic switch. Our data provides concrete evidence that angiogenesis is necessary for the maintenance of vascular systems in the healthy adult mouse brain.
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    Prediction Errors of Decision Demands Influence Cost-Benefit Computations in Reasoning
    (2022-09-28) Williams, Chad; Krigolson, Olave
    For each decision we make, we must first determine the degree of effort that we are going to exert, and this can range from no effort to full effort. To select a reasoning strategy (e.g., withholding or exerting effort), it has been proposed that we must first integrate internal and external factors to compute the degree of effort necessary and solve the problem at hand. In this dissertation, I sought to determine the mechanisms underlying selecting such reasoning strategies by leveraging electroencephalographic imaging techniques. My investigations began by exploring neural correlates of effortful contemplation and evolved to test assumptions of prediction errors as it became apparent that they were an influential factor. I then tied this mechanism to the strategy selection phase of reasoning and cost-benefit computations. From these findings, I proposed that prediction errors of decision demands function to lessen or remove the burden of cost-benefit computations. Specifically, repeated encounters of the same or similar decisions provide an opportunity to develop expectations of the prospective costs and benefits of those judgments and these expectations facilitate the reasoning process. I consider two possible explanations as to how prediction errors may influence reasoning: first, our expectations provide our cost-benefit computations with a starting point to be adjusted if necessary, and second, our expectations act as a gating mechanism for cost-benefit computations. Although more research is needed to test these hypotheses, I hope my work provides grounds for advancing this field of study.
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    Mechanisms and circuitry underlying direction selectivity in the mouse retina
    (2022-04-28) Hanson, Laura; Awatramani, Gautam
    Vision is a key sensory modality that is essential for survival. In the vertebrate visual system, light signals detected by photoreceptors are highly processed in the retina itself before being relayed to higher-order visual areas. The retina decomposes visual signals into specific features such as contrast, size, orientation, direction and/or velocity and relays this information via distinct output ganglion cells. For example, the direction-selective ganglion cells (DSGCs) are responsible for encoding the direction of objects moving across the retina. This may occur due to self-motion or as objects move through the environment. DSGCs respond robustly for motion of a particular “preferred” direction while exhibiting little to no response during motion in the opposite or “null” direction. In this dissertation, I examined the synaptic mechanisms involved in generating direction selectivity, both at the level of the DSGC as well as in the dendrites of presynaptic GABAergic/cholinergic starburst amacrine cell, where direction selectivity is first observed. Using mouse genetics to selectively disrupt direction processing capabilities of starburst dendrites, I revealed a second mechanism for generating direction selectivity. This relies on the differential functional wiring of GABA and ACh to DSGCs, which provides a substrate for directional dependent changes in the timing of excitation and inhibition to DSGCs (Chapter 2). In chapters 3 and 4, I turned my focus to the excitatory glutamatergic inputs to the DSGCs mediated by bipolar cells (that bridge the photoreceptor to output ganglion cells). Specifically, I examined the organization and AMPA and NMDA receptor composition (Chapter 3). By analyzing the spontaneous excitatory activity in the DSGC in combination with 2-photon imaging of the NMDA mediated calcium responses and glutamate signaling, I show the ‘silent’ NMDA-rich synapses are able to encode information across a range of contrasts. Finally, I also examined how the bipolar cell output to DSGCs is asymmetrically modified by another class of amacrine cell: the wide-field amacrine cells. In Chapter 4, I discovered that the subtype of nasal coding ON-OFF DSGCs are also orientation-selective (OS)-they respond best to vertically oriented bars. I show this selectivity originates at the level of bipolar cell axon terminals, which appear to be gap junction coupled to the vertically orientated processes of wide field amacrine cells. These finding led me to propose that orientation tuning in bipolar cells may act as a filter to simplify the task of encoding direction of motion for moving edges in the DSGC. This research highlights the mechanisms and circuitry required for the retina to accurately encode the direction of motion across the visual scene and provides us with a deeper understanding of the circuitry and mechanisms involved in direction selectivity in the mouse retina.
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    The antidepressant-like effects of intravenous reelin in the repeated-corticosterone paradigm of chronic stress
    (2022-04-28) Allen, Josh; Caruncho, Hector; Kalynchuk, Lisa
    Depression is an extremely common, devastating psychiatric syndrome with profound effects on the structure of neurons and the proteins that they express. However, the pathophysiology of depression remains unclear despite decades of extensive research efforts, and this lack of understanding makes it difficult to develop effective treatments. It is extremely problematic that conventional antidepressant drugs do not work for many patients, and those that do respond require weeks to months of continuous treatment before adequate therapeutic improvement is achieved. Therefore, there is a clear unmet need to develop mechanistically novel antidepressant compounds that are well-tolerated, more effective, and faster acting. Subjecting rats to repeated-corticosterone (CORT; stress hormone analogous to cortisol for humans) injections produces a depressive-like phenotype that can be used to make inferences about the human condition and screen compounds for antidepressant properties. Our laboratory has previously found that stress downregulates hippocampal reelin in a similar manner to that seen in depression patients, and that drugs with antidepressant actions recover this deficit. This provided a rationale to administer reelin directly into the hippocampus, which rescued behavioral and neurochemical deficits, but intrahippocampal infusions are not clinically viable. Reelin is expressed in the periphery and blood as well as the brain, so the aims of the collection of studies described here are to evaluate the antidepressant-like properties of peripheral intravenous (i.v.) reelin. In the first experiment, the antidepressant-like effects of several dosages of reelin (3/5μg given every 5/10 days) were evaluated in rats that were exposed to 3-weeks of daily CORT (40mg/kg) injections. I found that all the dosages of reelin attenuated CORT-induced despair-like behavior in the forced-swim test (FST) and normalized alterations in serotonin (5-HT) transporter (SERT) membrane protein clustering (MPC) in blood lymphocytes. Reelin treatment also increased reelin-immunoreactive (IR) cell counts in the hippocampal dentate gyrus (DG) subgranular zone (SGZ), but it had less of an effect on neurogenesis as measured by the number and maturation rate of doublecortin (DCX)-IR cells. Interestingly, the lowest dosage used also rescued the number of reelin-IR cells in the hypothalamic paraventricular nucleus (PVN). This suggested that the restoration of SGZ-reelin plays a pivotal role in attenuating depressive-like behavior and that 3μg every 10 days was the most effective dosage that was tested. Using the lowest dosage that showed to be effective in the first experiment, I then evaluated if male and female rats responded similarly to i.v. reelin using a larger battery of behavioral tests. Post-mortem tissue analyses focused on reelin and receptors that bind gamma-aminobutyric acid (GABA) and glutamate in the SGZ, which have been implicated in psychiatric disorders and the mediation of fast-acting antidepressant responses. I found that reelin rescued the FST- behavioral and neurochemical alterations induced by CORT similarly in both sexes, indicating that it may have therapeutic effects by normalizing inhibitory/excitatory transmission. I also evaluated the effect of i.v. reelin on neurogenesis in females and found that, akin to males, the regulation of adult-born cells by peripheral reelin is unlikely to mediate the antidepressant-like effects. The goal of the third experiment was to examine whether the antidepressant-like effects of peripheral reelin are achieved in a rapid manner. I found that a single 3μg injection after 3 weeks of CORT significantly decreased behavioral deficits in the FST 24 hours later in both sexes. Reelin also partially rescued cognitive deficits and expression levels of reelin, GluN2B, and mitochondrial-related pro-apoptotic factors bcl-2 associated X protein (BAX) and cytochrome C (CytC) in the DG. In addition, a single injection of reelin fully recovered the number of GluA1-expressing cells and partially recovered SERT cluster size in males, whereas reelin partially recovered GluA1-IR cell counts and fully recovered SERT cluster sizes in females. Reelin had modest effects on DCX-IR cells in both sexes. The final chapter summarizes and discusses my findings, which suggest that the antidepressant-like effects of peripheral reelin are associated with the recovery of neurochemical deficits that strengthen neurotransmission, at least in the hippocampus. Therefore, developing reelin-based therapeutics with antidepressant activity would be a fruitful area of research, although additional mechanistic, pharmacokinetic, and pharmacodynamic studies are essential.
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    Sensory-evoked activity in somatosensory cortex as a model to probe cortical plasticity in a mouse model of Rett syndrome
    (2021-08-30) Farhoomand, Farnoosh; Delaney, Kerry R.
    Rett syndrome (RTT), a severe neurodevelopmental disorder, affects females resulting from loss-of-function mutations in the X-linked transcription factor methyl-CpG-binding protein 2 (MECP2). RTT patients show severe verbal, motor, respiratory, and intellectual impairments. We studied two forms of activity-dependent plasticity in Mecp2 mutant mice to better understand the loss of MECP2 function in neuronal circuit and sensory processing. Sensory deprivation was applied by trimming one whisker to 3 mm to study long-term cortical plasticity in Mecp2-/y mice. Intrinsic optical signaling (IOS) imaging showed the neuronal response to wiggling a non-trimmed was consistent from day 0 to 14 but reduced for the trimmed whisker by 49.0 ± 4.3% in wild type (WT) and 22.7 ± 4.6% (p=0.0135) in RTT mice. Primary hindlimb (HL) somatosensory cortical responses to vibratory stimulation were assessed by IOS and intracortical local field potential (LFP). Responses were assessed before, during and, after 1 hour of repeated HL vibratory stimulation (100Hz,1sec, ISI 6 sec) in symptomatic male (4-6 week), female (10-12 month) and pre-symptomatic young female (4 week) RTT model mice. After 1-hour, cortical responses to test vibrations were reduced by approximately 40% in RTT and WT mice as assessed by both methods. Recovery of the IOS responses (1 sec vibration at 100Hz) and LFP (300µm below pia, 7 stimuli, 100mse ISI) were tested at 15 min intervals for 1 hour after ceasing the repeated stimulation. Reduced responses persisted for at least 60 min in WT but recovered to 90-100% of normal within 15-30 min in RTT. Analysis of the LFP responses within the test train indicated that the reduced cortical sensitivity during and after continuous stimulation resulted primarily from an increase in adaptation during the 7-stimulus test train rather than a reduction in the response to a single vibratory stimulus in all groups. Retention of this increased STA is the primary cause of the persistently reduced tactile response in young WT female mice, while in RTT mice the rapid recovery of tactile sensitivity was due to the return of STA to lower, baseline levels. Male RTT mice exhibited a marked increased excitability to the first stimulus in the test train resulting in hypersensitivity to a single vibration by 45 minutes. Old females exhibited the same pattern of adaptation and recovery but retention of adaptation was less pronounced in both WT and RTT compared to younger animals suggesting an age-dependent reduction in neural plasticity may mask deficits specific to RTT. Recording sciatic nerve sensory afferent activity did not reveal any STA, persistent adaptation or sensitization of peripheral afferent endings in any groups. I propose persistent sensory adaptation mediated by increased short-term adaptation may reflect enhanced feedback by inhibitory elements of circuits within the sensory pathway. The rapid recovery of responsiveness in young female RTT mice may therefore reflect a deficit in the capacity for activity dependent plasticity to consolidate and thus could provide a platform to understand the causes of learning and cognitive deficits in RTT patients.
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    Repeated mild traumatic brain injury is associated with acute microvascular damage in juvenile male and female rats
    (2021-06-25) Trivino Paredes, Juan Sebastian; Christie, Brian R.; Nahirney, Patrick C.
    Traumatic Brain Injury (TBI) is a growing global health problem. Mild forms of TBI (mTBI) such as concussions, represent the most common manifestation of this type of injury with children and youth (< 20 years old) among the most likely to sustain mTBI. There is growing evidence for the cumulative effects of repeated mTBI (rmTBI) suggesting that while a single concussion may not cause evident or long-lasting brain alterations, the summation of multiple mTBI may lead to more severe consequences. In contrast to severe TBI, lesions in mTBI patients are challenging to detect. Despite this, mTBI patients may still present with cognitive and emotional deficits. Cerebral microbleeds (CMBS), a subtle form of vascular damage, have been identified as an early hallmark in brain trauma and several neurodegenerative diseases. The cumulative effects of subtle but sustained microvascular damage could explain the persistent long-term functional deficits observed in mTBI. In this study, the awake closed-head injury (ACHI) model was used to investigate the association between rmTBI and microvascular damage in different brain regions in both male and female juvenile rats at one and seven days after the last injury. The results indicate that the injury paradigm used in this study (i.e. 8 impacts over 4 days) using the ACHI model is associated with an acute increase in sings of microvascular damage in both sexes that is no longer evident at a longer time point. These study is the first to describe the negative impact of rmTBI on CMBs in the juvenile using an awake animal model, and provides evidence for the potential involvement of this subtle form of vascular damage in the development of neurological deficits after rmTBI.
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    Investigating the deleterious effects of type 1 diabetes mellitus on microvascular repair in the mouse cortex
    (2021-05-25) Mehina, Eslam; Brown, Craig E.
    Microglia and brain-resident macrophages are the sentinel immune cells of the central nervous system (CNS), and are ideally situated to respond to any damage to the brain parenchyma or vasculature. Circulating leukocytes are generally excluded from the CNS environment under homeostatic conditions but can gain access to this region in diseases that disrupt immune system function and blood-brain barrier integrity. Although these diverse immune cells exhibit properties that may engender them to be well-suited to resolve microcirculatory insults, their relative contributions to the recanalization of capillary rupture in the cortex, known as cerebral microbleeds (CMBs), has yet to be described. CMBs are particularly concerning in conditions, such as diabetes mellitus (DM), in which these insults occur more frequently and potentially underlie the onset and progression of cognitive decline. Using in vivo 2-photon microscopy and confocal imaging, here I highlight the compromised repair of CMBs in a mouse model of type 1 DM and characterize the robust, heterogeneous macrophage response to these insults. Specifically, 20% of damaged capillaries were eliminated from the circulation in the diabetic cortex and chronic insulin treatment failed to prevent this microvascular loss. Administration of interferon-α or interferon-γ neutralizing antibodies to dampen inflammatory signalling, or dexamethasone to reduce global inflammation, also failed to improve repair rates of damaged microvessels in diabetic mice. In contrast, CMBs in nondiabetic mice repaired without exception. Interestingly, depletion of CNS macrophages using the colony stimulating factor-1 receptor antagonist PLX5622 resulted in microvascular elimination in nondiabetic mice. Given the robust depletion of brain macrophage populations with this treatment, at first these data suggested that these cells were necessary for microvascular repair since their elimination produced vessel loss. However, by parsing the data I identified that microvessels repaired in all cases where macrophages were not identified at the CMB; when CX3CR1+ aggregate was localized to the injury, ~20% of microvessels were eliminated. These findings show that CNS macrophages are not required for microvascular repair following CMB. Immunofluorescent co-labelling of various microglial and macrophage markers within the diabetic CMB milieu revealed a novel population of Mac2+/TMEM119- cells, distinct from homeostatic TMEM119+ microglia. These cells reliably localized to CMBs that failed to repair and rarely associated with vessels that recanalized; Mac2+/TMEM119- cells were not found within nondiabetic CMBs. Treatment of diabetic mice with clodronate liposomes (CLR) to deplete circulating phagocytic leukocytes prevented aggregation of Mac2+/TMEM119- cells to CMBs and improved capillary repair rates. The efficacy of CLR in excluding these cells from the CMB aggregate, coincident with eradication of monocytes from circulation, indicated that these cells likely arose from the periphery. In vivo 2-photon imaging revealed significant increases in lipofuscin at the site of diabetic CMBs relative to the nondiabetic context; other phagocytic markers including CD68 and TREM2 were also upregulated. Mac2+/TMEM119- cells showed elevated lipofuscin content relative to homeostatic microglia; their association with CMBs may thus signal an increase in phagocytosis that contributes to capillary pruning. Taken together, these data identify a novel Mac2+/TMEM119- macrophage associated with pathological microvascular elimination following CMB in the diabetic neocortex. These findings highlight the diversity of immune cell responses to CNS injury and provide insights into the cellular mechanisms of capillary pruning. Furthermore, these advances in our understanding of the regulation of microvascular elimination in the diabetic brain may have clinical implications for patients with DM as they provide evidence for putative adjuvant anti-inflammatory treatments, such as CLR, in mitigating cerebrovascular pathology.
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    A novel preclinical pediatric concussion model causes neurobehavioural impairment and diffuse neurodegeneration
    (2021-05-03) Meconi, Alicia Louise; Christie, Brian R.
    Concussions are the injury and symptoms that can result from transmission of a biomechanical force to the brain. They represent a significant global health burden, and are the subject of a growing body of medical research. A concussion can only be definitively diagnosed by a medical professional based on symptoms, although advanced neuroimaging and biomarker-based approaches are promising future diagnostic tools. There is no treatment for concussion beyond following return-to-work or -play guidelines, which recommend avoiding strenuous physical and cognitive activities until they no longer exacerbate symptoms. Preclinical models of concussion have been used to examine pathophysiological processes underlying symptoms, which is an important step in developing tools for diagnosis and treatment. Historically the clinical translation of preclinical concussion research has been limited, and the use of anaesthesia, and preference for adult male rats may contribute to this. These means of reducing variability are justified, but preclinical research moving forward should address these limitations to translatability by including more clinically relevant subjects and avoiding anaesthesia. To this end, we developed a new preclinical model for pediatric concussion. Our awake closed head injury (ACHI) model is well-suited to this purpose because it produces a helmeted closed-head injury involving vertical and rotational displacement of the head, and does not require anaesthesia. Before the ACHI model can be used to investigate concussion mechanism, diagnosis, and treatment, it needs to be characterized to demonstrate that it produces clinically relevant neurobehavioral and pathological changes. We developed a modified neurologic assessment protocol to test neurologic function immediately after each injury. The Barnes maze, elevated plus maze, open field, and Rotarod were used to measure injury-related changes in cognition, anxiety, and motor function. The Barnes maze reversal task was used to detect more subtle cognitive impairments of executive function. Structural MRI was used to search for visible lesion, hemorrhage, or atrophy; and silver-stain histology was used to detect neurodegeneration. We determined repeated ACHI produced acute neurologic impairment with the NAP, and a mild spatial learning deficit potentially mediated impaired cognitive flexibility in the Barnes maze and reversal training. These were accompanied by neurodegeneration in the optic tract, hippocampus, and ipsilateral cortex during the first week of recovery. Thus, following the internationally recognised definition developed by the concussion in sport group, we demonstrated 1) an “impulsive” force transmitted to the head results in 2) the rapid onset of short-lived neurologic impairment that resolves spontaneously. This occurs 3) with normal structural neuroimaging, and 4) produces cognitive impairment, and LOC in a subset of cases. The ACHI model is the first in Canada to forego anaesthesia, and this is the first demonstration of neurocognitive impairment accompanied by diffuse neurodegeneration in the absence of structural MRI abnormalities after mild traumatic brain injury in juvenile male and female rats.
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    Sex differences in hippocampal cell proliferation and inflammation following repeated mild traumatic brain injury in adolescent rats
    (2020-08-05) Neale, Katie J.; Christie, Brian R.
    Traumatic brain injury (TBI) is becoming increasingly recognized as a global health issue. Each year over 160,000 Canadians experience some form of TBI, which can be caused by sport-related injuries, motor vehicle accidents, or assault. Adolescents are especially susceptible to repeat head injury and represent an at-risk population for sustaining sports-related concussions. The hippocampus, known for its role in learning and memory, is vulnerable to this injury. Although most TBI studies exclude females, there are important sex differences in outcomes and recovery following brain injury. A greater understanding of how sex differences contribute to the heterogeneity of this disease is critical for clinical care and potential treatments. Currently, few preclinical studies have assessed sex differences in adolescents following repeated mild traumatic brain injury (rmTBI). This study uses an awake closed head injury (ACHI) paradigm in male and female adolescent rats to investigate acute injury-induced changes to the hippocampus after rmTBI. A neurological assessment protocol (NAP) administered immediately after each impact showed that the ACHI acutely alters state of consciousness, and results in deficits after each impact. Following 8 ACHIs spaced 2 hours apart, adolescent rats were injected with the thymidine analogue BrdU and perfused 2 hours later on either post injury day (PID) 1 or 3. BrdU was used to identify cells undergoing DNA synthesis, and Ki-67 - expressed during all active phases of the cell cycle - was used as an endogenous marker for proliferation. Results indicate a robust and diffuse increase in cellular proliferation in male rmTBI animals that was not present to the same extent in female rmTBI animals. Triple labeling experiments revealed a higher proportion of microglia/macrophages in the subgranular zone of rmTBI animals, indicating an immediate inflammatory response in both sexes. This study shows sex differences in the pathophysiology of rmTBI in adolescent rats. Further investigation will reveal the detrimental versus neuroprotective contributions of this effect on learning and memory.
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    Pannexin 1 regulates dendritic spines in developing cortical neurons
    (2020-05-04) Sanchez-Arias, Juan C.; Swayne, Leigh Anne
    Sensory, cognitive, and emotional processing are rooted in the cerebral cortex. The cerebral cortex is comprised of six layers defined by the neurons within them that have distinctive connection, both within cortex itself and with other subcortical structures. Although still far from solving the mysteries of the mind, it is clear that networks form by neurons in the cerebral cortex provide the computational substrate for a remarkable range of behaviours. This neuron to neuron activation is mediated through dendritic spines, the postsynaptic target of most excitatory synapses in the cerebral cortex. Dendritic spines are small protrusions found along dendrites of neurons, and their number, as well as structural changes, accompany the development of synapses and establishment of neuronal networks. In fact, dendritic spines undergo rapid structural and functional changes guided by neuronal activity. This marriage between structural and functional plasticity, makes dendritic spines crucial in fine-tuning of networks in the brain; not surprisingly, dendritic spine aberrations are a hallmark of multiple neurodevelopmental, neuropsychiatric, and neurodegenerative disorders. With this in mind, I considered Pannexin 1 (Panx1) an interesting novel candidate for a regulatory role on cortical neuronal network and dendritic spine development, for the following reasons. First, Panx1 transcripts are enriched in the brain and in the cortex are most abundant during the embryonic and early postnatal period. . This timing roughly corresponds to a period of synaptogenesis in the postnatal brain. Second, Panx1 localizes to postsynaptic compartments in neurons and its disruption leads to enhance excitability and potentiation of neuron-to neuron communication. Third, disruption of Panx1 (either knockdown or pharmacological blockade) leads to neurite outgrowth in neuron-like cells. Lastly, genetic variants in PANX1 have been associated with neurodevelopmental disorders. This dissertation explores the role of Panx1 in the development of dendritic spines and neuronal networks, furthering our understanding on cortical development and placing Panx1 as a novel regulator of structural and functional plasticity in the brain. Chapter 1 discusses core concepts on cortical development, with an emphasis on pyramidal neuron, the most abundant and only known projection neurons in the cerebral cortex. Here, I review the embryonic origin of pyramidal neurons, their postnasal development, and how cortical circuits are assembled. I finish this chapter with a brief review on Panx1 and its known expression and involvement in neuronal function. In Chapter 2 I explore the functional properties of neuronal networks and synaptic composition of cortical neurons in the absence of Panx1. Using live cell imaging and analysis of Ca2+ transients in dense primary cortical cultures, revealed that Panx1 knock-out (KO) cultures exhibit more and larger groups of significantly co-activated neurons, known as network ensembles. These network properties were not explained by differences in cell viability or cell-type composition. Examination of protein expression from cortical synaptosome preparations revealed that Panx1 is enriched in synaptic compartments, while also confirming a developmental downregulation. This analysis also revealed increased levels of the postsynaptic scaffolding protein PSD-95, along with the postsynaptic glutamate receptors GluA1 and GluN2A. Lastly, ex vivo tracing of dendritic spines on apical dendrites of Layer 5 pyramidal neurons in global and glutamatergic-specific Panx1 KO brain slices revealed higher spine densities in early and late postnatal development, with no differences in spine length. Analysis of dendritic spine densities in fixed cultured cortical neurons revealed an increase associated with Panx1 KO. Altogether, this work presents for the first time a connection between Panx1 and structural development of dendritic spines and suggest that Panx1 regulates cortical neuronal networks through changes in spine density. Chapter 3 examines the influence of Panx1 on spiny protrusions growth and movement. Spiny protrusion are long, thin, highly dynamic spine precursors. Taking advantage of a fluorescent signal localized to the plasma membrane, I visualized spiny protrusions and quantified their dynamics in wildtype (WT) and Panx1 KO developing cortical neurons, both under fixed and live conditions. I found that transient Panx1 expression is associated with decreased spiny protrusion density both in WT and Panx1 KO neurons. Using live cell imaging, I found that spiny protrusions are more stable and less motile in Panx1 KO neurons, while its transient expression reversed both of these phenotypes. These results suggest that Panx1 regulation of dendritic spines development is rooted partly in the regulation of spiny protrusion dynamics. Overall, this dissertation demonstrates that Panx1 negatively regulates dendritic spines in part by influencing spiny protrusion dynamics. It is reasonable to speculate that Panx1 regulation of dendritic spines underlies its newly discovered role in the formation network ensembles, providing a putative mechanism for previously described roles of Panx1 in synaptic plasticity. Likewise, this body of work furthers our understanding of Panx1 by filling some of the gaps attached to its developmental expression and association with neurodevelopmental disease.
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    Longitudinal calcium imaging of VIP interneuron circuits reveals shifting response fidelity dynamics in the stroke damaged brain
    (2020-01-29) Motaharinia, Mohammad; Brown, Craig E.
    Although inhibitory cortical interneurons play a critical role in regulating brain excitability and function, the effects of stroke on these neurons is poorly understood. In particular, interneurons expressing vasoactive intestinal peptide (VIP) specialize in inhibiting other classes of inhibitory neurons, and thus serve to modulate cortical sensory processing. To understand how stroke affects this circuit, we imaged VIP neuron responses (using GCaMP6s) to low and high intensity forepaw stimulation, both before and after focal stroke in somatosensory cortex. Our data show that the fraction of forelimb responsive VIP interneurons and their response fidelity (defined as a cell’s number of responsive trials out of eight trials at a certain imaging week) was significantly reduced in the first week after stroke, especially when lower intensity forepaw stimulation was employed. The loss of responsiveness was most evident in highly active VIP neurons (defined by their level of responsiveness before stroke), whereas less active neurons were minimally affected. Of note, a small fraction of VIP neurons that were minimally active before stroke, became responsive afterwards suggesting that stroke may unmask sensory responses in some neurons. Although VIP responses to forepaw stimulation generally improved from 2-5 weeks recovery, the variance in response fidelity after stroke was comparatively high and therefore less predictable than that observed before stroke. Lastly, stroke related changes in response properties were restricted to within 400μm of the infarct border. These findings reveal the dynamic and resilient nature of VIP neurons and suggest that a sub-population of these cells are more apt to lose sensory responsiveness during the initial phase of stroke, whereas some minimally responsive cells are progressively recruited into the forelimb sensory circuit. Furthermore, stroke appears to disrupt the predictability of sensory-evoked responses in these cortical interneurons which could have important consequences for sensory perception.
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    Determinants of brain region-specific age-related declines in microvascular density in the mouse brain
    (2020-01-27) Schager, Benjamin; Brown, Craig E.
    It is emerging that the brain’s vasculature consists of a highly spatially heterogeneous network; however, information on how various vascular characteristics differ between brain regions is still lacking. Furthermore, aging studies rarely acknowledge regional differences in the changes of vascular features. The density of the capillary bed is one vascular feature that is important for the adequate delivery of nutrients to brain tissue. Additionally, capillary density may influence regional cerebral blood flow, a parameter that has been repeatedly correlated to cognitive-behavioural performance. Age-related decline in capillary density has been widely reported in various animal models, yet important questions remain concerning whether there are regional vulnerabilities and what mechanisms could account for these regional differences, if they exist. Here we used confocal microscopy combined with a fluorescent dye-filling approach to label the vasculature, and subsequently quantified vessel length, tortuosity and diameter in 15 brain regions in young adult and aged mice. Our data indicate that vessel loss was most pronounced in white matter followed by cortical, then subcortical gray matter regions, while some regions (visual cortex, amygdala, insular cortex) showed little decline with aging. Changes in capillary density are determined by a balance of pruning and sprouting events. Previous research showed that capillaries are naturally prone to plugging and prolonged obstructions often lead to vessel pruning without subsequent compensatory vessel sprouting. We therefore hypothesized that regional susceptibilities to plugging could help predict vessel loss. By mapping the distribution of microsphere-induced capillary obstructions, we discovered that regions with a higher density of persistent obstructions were more likely to show vessel loss with aging and vice versa. Although the relationship between obstruction density and vessel loss was strong, it was clear obstruction rates were insufficient to explain vessel loss on their own. For that reason, we subsequently used in vivo two-photon microscopy to track microsphere-induced capillary obstructions and vascular network changes over 24 days in two areas of cortex that showed different magnitudes of vessel loss and obstruction densities: visual and retrosplenial cortex. Surprisingly, we did not find evidence for differences in vessel pruning rates between areas, as we would have expected. Instead, we observed brain region-specific differences in recanalization times and rates of angiogenesis. These findings indicate that age-related vessel loss is region specific and that regional susceptibilities to capillary plugging and angiogenesis must be considered to explain these differences. Altogether, this work supports the overarching hypothesis that regional differences in vascular structure and function contribute to a regionally heterogeneous phenotype in the aging brain.
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    Effects of mild traumatic brain injury on hippocampal synaptic plasticity and behaviour in juvenile rats
    (2019-12-11) Pinar Cabeza de Vaca, Cristina; Christie, Brian R.
    Traumatic Brain Injury (TBI) is a global health problem and concussion, or mild TBI (mTBI), accounts for up to 75% of all brain injuries occurring annually in the US. There is also growing awareness that repeated mild traumatic brain injury (r-mTBI) can result in cumulative neuropathology and learning and memory deficits, however there is a paucity of preclinical data as to the extent these deficits manifest. R-mTBI in juvenile populations is of special interest as not only is this a high risk group, but this is also a time period when the human brain continues to mature. The hippocampus is a brain region important for learning and memory processes, and r-mTBI during the juvenile period may particularly disrupt the development of cognitive processes. To examine this issue we used a model of awake closed head injury (ACHI), and administered 8 impacts over a 4 day period to juvenile male and female rats (P25-28). At 1 or 7 days after the last injury, a cohort of rats was used for behavioural testing to study anxiety and risk-taking behaviours and cognitive abilities. From a different cohort, hippocampal slices were generated and used for in vitro electrophysiological recordings, and the capacity for long-term depression (LTD) and long-term potentiation (LTP) was examined in the medial perforant path (MPP)-dentate gyrus (DG) synapse. Our results showed that r-mTBI impaired hippocampal-dependent spatial learning and memory and that r-mTBI significantly impaired the capacity for LTD but not LTP in both sexes. These data are the first to describe the negative impact of r-mTBI on LTD in the juvenile DG in both males and females, and provide evidence for the delayed development of neurological deficits with r-mTBI.
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    Regulation of Cav2.1 by Ankyrin B and its variants
    (2019-08-19) Choi, Catherine S.W.; Swayne, Leigh Anne
    Ankyrin B (AnkB) is a scaffolding protein, acting as a bridge between ion channels and cytoskeleton networks. AnkB variants are associated with cognitive disorders including autism spectrum disorder and epilepsy. In the brain, AnkB interacts with Cav2.1, the pore-forming subunit of P/Q type voltage gated calcium channels. However, how AnkB regulates Cav2.1 is not fully understood. Using HEK293T cells, we discovered that AnkB increases Cav2.1 expression levels but does not change Cav2.1 surface levels. AnkB p.S646F increases Cav2.1 to an even greater level of expression, again without impacting Cav2.1 surface levels. Looking at a partial loss of AnkB in glutamatergic neurons, overall Cav2.1 levels decreased at P30 but the synaptosomal fraction was not impacted. Our findings indicate that AnkB plays a role in regulating an intracellular pool of Cav2.1 but does not affect the surface or the synaptosomal pools of Cav2.1. This intracellular pool of Cav2.1 may play an important role in neuronal function and homeostasis, suggesting a mechanism for neuronal pathogenicity of AnkB variants.
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    Exploiting evolutionarily conserved pathways to promote plasticity of human spinal circuits
    (2019-06-18) Pearcey, Gregory E. P.; Zehr, E. Paul
    Humans evolved from species that walked on all four limbs, which means that experiments in quadrupeds can guide and support experiments in humans. This is particularly helpful for neural rehabilitation because the central nervous system is plastic in nature, meaning that activities promoting central nervous system activity can alter subsequent output properties. This is known as neuroplasticity and can be measured as changes in spinal cord excitability through reflexes as a proxy. By targeting evolutionarily conserved pathways that act on similar interneurons within the spinal cord to either increase or decrease excitability, it may be possible to preferentially modulate spinal cord excitability based on a desirable outcome. For example, rhythmic movement reduces spinal cord excitability whereas brief sensory input to cutaneous afferents increases spinal cord excitability. Alterations in spinal cord excitability have been shown to outlast the activity duration, suggesting that neuroplasticity is not transient. This evidence suggests that both rhythmic movement and sensory input can induce acute neuroplasticity of spinal cord excitability. The overall purpose of this dissertation was two-fold; 1) to provide reviews of how evolutionarily conserved pathways are studied in humans and how they contribute to human rhythmic movement, and 2) experimentally examine how these conserved pathways, which converge onto similar interneuron circuitry, can be exploited to cause bidirectional changes in spinal cord excitability. Reviews indicate that humans have retained characteristics of quadrupedal locomotion and, in particular, activity of the arms affects the excitability of the legs, and vice versa. Cutaneous input is integrated throughout the body during locomotion, such that cutaneous sensations elicit neuromechanical responses that are nerve-specific and modulated according to the phase of movement. In experiment 1, there was increased spinal cord excitability following patterned stimulation of cutaneous afferents innervating the bottom of the foot. In experiment 2, stimulation to cutaneous afferents innervating both the top and bottom of the foot amplified voluntary plantar- and dorsiflexion. In experiment 3, cervicolumbar connections were exploited to amplify plasticity in spinal cord excitability induced by rhythmic movement. Finally, in experiment 4, there were interactions of rhythmic movement and fatigue, which both reduce spinal cord excitability, with cutaneous stimulation, which increases spinal cord excitability, such that reductions in spinal cord excitability associated with fatigue were mitigated by cutaneous stimulation. Taken together, these experiments suggest that cutaneous stimulation can increase spinal cord excitability, whereas quadrupedal locomotor activity can decrease spinal cord excitability. These conserved pathways can be exploited to intentionally modify spinal cord excitability in a bidirectional fashion, which provides fruitful information for the exploration of rehabilitation and sport performance practices.
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    Ethanol modulation of NMDA receptors and NMDAr-dependent long-term depression in the developing juvenile dentate gyrus
    (2019-05-01) Sawchuk, Scott D.; Christie, Brian R.
    Long-term depression (LTD) induced by low frequency stimulation (LFS; 900x1Hz) at medial perforant path (MPP) synapses in the rat dentate gyrus (DG) has been described as both developmentally regulated and N-methyl D-aspartate receptor (NMDAr) independent, yet sufficient evidence suggest that the processes is not entirely independent of NMDAr activity. In the present study, in vitro DG-LTD LFS was induced in hippocampal slices prepared from rats at postnatal day (PND) 14, 21 and 28 to investigate how the sensitivity of DG-LTD~LFS to the NMDAr antagonist amino-5-phosphonovaleric acid (AP5; 50µM) changes throughout the juvenile developmental period (jDP; PNDs 12-29) that occurs immediately after the period of peak neurogenesis. We further examined the acute effects of the partial NMDAr antagonist ethanol (EtOH) on DG-LTD LFS and NMDAr excitatory post synaptic currents (NMDAr-EPSCs) in dentate granule cells (DGCs) using 50 and 100mM concentrations (50mM ~0.2%BAC) of EtOH. The magnitude of LTD induced at all three time points was not statistically different between age groups, but the probability of successfully inducing LTD did decrease with age. We found that AP5 was insufficient to inhibit DG-LTD LFS at PND14, but significantly inhibited DG-LTD LFS at PND21 and PND28. We also found that 50mM EtOH, but not 100mM EtOH, significantly attenuated the mag-nitude of DG-LTD LFS induced at each time point. Acute effects of 50mM EtOH had relatively little effect on NMDAr-EPSCs at PND14, and showed a slight potentiation of the response at PND21. 50mM EtOH at PND28, and 100mM EtOH at all three developmental time points showed inhibition of the NMDAr-EPSC. These findings provide insight on how developmental changes to the DG network and dentate gran-ule cells (DGCs) influences mechanisms and processes involved in the induction and expression of synaptic plasticity in the DG.
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    Role of a novel C-terminal motif in Pannexin 1 trafficking and oligomerization
    (2019-04-24) Epp, Anna; Swayne, Leigh Anne
    Pannexin 1 (Panx1) is a metabolite channel enriched in the brain and known to localize to the cell surface, where it is involved in a variety of neuronal processes including cell proliferation and differentiation. The mechanisms through which Panx1 is trafficked or stabilized at the surface, however, are not fully understood. The proximal Panx1 C-terminus (Panx1CT), upstream of a caspase-cleavage site has been demonstrated to be required for Panx1 cell-surface expression. We discovered a previously unreported putative leucine-rich repeat (LRR) motif within the proximal Panx1CT. I investigated the involvement of this putative LRR motif on Panx1 localization and oligomerization. Deletion of the putative LRR motif or uniquely the highly conserved segment of the putative LRR motif resulted in a significant loss of Panx1 cell surface expression. Finally, ectopic expression of Panx1-EGFP in HEK293T cells increased cell proliferation, which was not recapitulated by a Panx1 deletion mutant lacking the putative LRR motif. Overall the findings presented in this thesis provide new insights into the molecular determinants of Panx1 trafficking and oligomerization.