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Of all the labs I toured while applying for grad school, the MacVicar lab was the only one where the trainees had absolutely nothing negative to say about their supervisor. Thankful for your support & guidance - you make us better scientists @brian_macvicar #GreatSupervisor #UBC
Can't believe I'm nearing the end of my time as a PhD student. So thankful to my #UBC #GreatSupervisor, @brian_macvicar for being supportive, patient, and encouraging - right to the bitter end!
Whether it’s for science or sailing, @brian_macvicar is always a #GreatSupervisor!
I don't always tweet. But when I do, I have to acknowledge @brian_macvicar for #UBC #GreatSupervisor week!
So privileged to have a #GreatSupervisor at #UBC to mentor me not only in science, but also in life, the universe, and everything. Thanks for your guidance and support @brian_macvicar!
Graduate Student Supervision
Doctoral Student Supervision (Jan 2008 - Nov 2019)
No abstract available.
Astrocyte calcium (Ca²⁺) signaling is involved in the regulation of physiological processes such as synaptic activity and vascular tone in the brain. Recent developments in tools to monitor Ca²⁺ signals have revealed a novel type of spontaneous Ca²⁺ transient that is localized to microdomains in the fine astrocytic processes. However, the molecular mechanisms underlying these signals have not been fully characterized. Based on data from rodent brain transcriptomic and proteomic studies, we identified Piezo1, a mechanosensitive cation channel, as a potential candidate for mediating these Ca²⁺ transients.In Chapter 2, we performed two-photon imaging of the membrane-tethered, genetically encoded Ca²⁺ indicator Lck-GCaMP5 in cultured astrocytes and developed an algorithm for extracting and analyzing the microdomain Ca²⁺ signals. Using a combination of pharmacological and siRNA approaches, we showed that Piezo1 channels contribute to these spontaneous Ca²⁺ transients. We also conducted preliminary imaging experiments in brain slice astrocytes and found that spontaneous Ca²⁺ signals in the endfoot compartments were sensitive to pharmacological modulators of Piezo1. In Chapter 3, we performed immunostaining using strategies that were optimized to target subcellular locations where Piezo1 expression had previously been reported in other cell types. Our results indicated that Piezo1 is localized to subcellular compartments relevant to mechanosensation; Piezo1 immunoreactivity was localized to discrete clusters on the plasma membrane and associated with focal adhesion and actin stress fibers in cultured astrocytes, and Piezo1 expression was observed within the endfoot processes of brain slice astrocytes. Lastly, in Chapter 4, we showed that an osmotic stress model of astrocyte swelling could activate Piezo1-mediated Ca²⁺ microdomain signals in cultured astrocytes.Taken together, the data provide evidence that Piezo1 contributes to spontaneous Ca²⁺ microdomain signals in astrocytes in both cell culture and acute brain slices, and suggest that astrocyte Ca²⁺ signaling may play a role in integrating mechanical stimuli to regulate brain function in physiological and pathological processes involving changes in mechanical force.
Calcium (Ca²+) entry through voltage-gated Ca²+ channels in dendrites of hippocampal pyramidal cells (PCs) contributes to synaptic depolarization and activation of downstream pathways that regulate many aspects of synaptic and cellular function. Activated by small depolarizing changes in voltage, T-type Ca²+ channels mediate low-threshold spikes (LTS) that drive the resting membrane potential towards action potential threshold. T-type Ca²+ channels are hypothesized to contribute to subthreshold synaptic depolarization in the CA3 subfield of the hippocampus due to the stratified nature of inputs on CA3 dendrites. While T-type Ca²+ channels are densely expressed in area CA3, their functional characteristics and interactions with postsynaptic receptors are not well understood and LTS have not been reported in CA3 PCs. In Chapter 3, using whole-cell electrophysiology, we demonstrate that LTS in CA3 PCs can be evoked by somatic current injection. LTS were only evoked when 4AP was applied to depress A-type K+ channels. Using specific pharmacological blockers, we show that Cav3.2 channels mediate LTS in CA1 and CA3 PCs. In Chapter 4, using two-photon Ca²+ imaging, we map the subcellular distribution of Cav3.2 channels in hippocampal PCs. Our results show that Cav3.2 channel expression is restricted to the soma and proximal dendrites in CA1 PCs, while Ca²+ influx from Cav3.2 channel activation occurs in distal (>50 μm) regions of CA3 PC dendrites. In Chapter 5, we demonstrate that mAChR stimulation potentiates LTS amplitude and such amplification of Ca²+ influx through Cav3.2 channels is dependent on M-current inhibition. Furthermore, we show that application of t-ACPD causes potent and rapid inhibition of LTS propagation. This inhibition occurs exclusively through mGlu₁ receptors and downstream activation of PKC is necessary for this process. Lastly, in Chapter 6, we show boosting of subthreshold synaptic signals by T-type Ca²+ channels in PCs within area CA3 but not CA1. Taken together, our data identify a new T-type mediated Ca²+ signaling pathway in CA3 PC dendrites that is unlocked by A-type K+ channel blockade, potentiated by mAChR activation, and inhibited by mGluR₁ activation. Furthermore, our study highlights the important involvement of T-type Ca²+ channels in enhancing dendritic depolarization in CA3 PCs.
Cytotoxic brain edema is the principal cause of mortality following brain trauma and cerebral infarct yet the mechanisms underlying neuronal swelling are poorly understood. This thesis aims at identifying cellular mechanisms of neuronal swelling that cause cytotoxic edema (chapter 3) and describes a novel method for highly efficient neuronal transfection using lipid nanoparticle delivery of siRNA in vitro and in vivo (chapter 2). In chapter 2, we demonstrate that neurons accumulate lipid nanoparticles in an apolipoprotein E dependent fashion, resulting in very efficient uptake in cell culture (100%) with little apparent toxicity. In vivo, lipid nanoparticle delivery of siRNA resulted in knockdown of target genes in either discrete regions around the injection site following intracortical injections or in more widespread areas following intracerebroventricular injections with no apparent toxicity or immune reactions from the lipid nanoparticles. Effective targeted knockdown was demonstrated by showing that lipid nanoparticle delivery of siRNA against GRIN1 (encoding GluN1 subunit of the NMDA receptor) selectively reduced synaptic NMDA receptor currents in vivo as compared to synaptic AMPA receptor currents. Therefore, lipid nanoparticle delivery of siRNA rapidly manipulates expression of proteins involved in neuronal processes in vivo, possibly enabling development of gene therapies for neurological disorders.In chapter 3, we show that increasing intracellular sodium concentration ([Na⁺]i) by either activating voltage-gated sodium channels or NMDA receptors triggers a secondary Cl- influx that leads to neuronal swelling and death. Cl- but not Ca²⁺ entry was required for neuronal swelling and cell death. Pharmacological analyses indicated that a DIDS-sensitive HCO₃-/C1- exchanger was responsible for the majority of the Cl- influx. We used lipid nanoparticle-siRNA mediated knockdown (described in chapter 2) to determine the molecular identity of the Cl- influx pathway. Neuronal swelling was attenuated in brain slices by siRNA-mediated knockdown of the Cl-, SO₄²-, HCO₃- exchanger, SLC26A11, but not by knockdown of other HCO₃-/Cl- exchangers examined. We conclude that cytotoxic brain edema can occur when sufficient Na⁺ entry into neurons results in Cl- entry via SLC26A11 to trigger subsequent neuronal swelling.
Cognitive dysfunction and abnormal synaptic transmission are main characteristics of various brain disorders, including stroke, trauma and various neurodegenerative diseases. Neuroinflammation, known to influence synaptic function, plays an important role in all these brain disorders. Hypoxia is usually concurrent with neuroinflammation in these brain disorders. In peripheral systems, inflammation and hypoxia are interdependent on each other by sharing pathways at the initiation of downstream pathways leading to inflammatory responses. However, the interaction of inflammation and hypoxia in the CNS, and more importantly, whether this interaction can affect synaptic function, is still largely unknown.In Chapter 3, I performed field recording, whole cell recording, 2 photon imaging and biochemical measurements in acute hippocampal slices and found that inflammatory stimuli, either LPS or Aβ, interact with hypoxia to trigger a fast-onset (within 15 min) LTD of synaptic transmission. This neuroinflammation+hypoxia LTD is unusual in that it is independent of NMDARs, mGluRs or patterned synaptic activity. Neuroinflammatory stimulus activates NADPH oxidase by triggering subunits assembly and translocation to the plasma membrane. Hypoxia increases the level of lactic acid via glycolysis, resulting in an increase of lactic acid/pyruvate ratio and a consequent increase of NADPH/NADP⁺ ratio, which in turn boosts production of superoxide by NADPH oxidase. Superoxide subsequently activates PP2A, and ultimately leads to GluR2-mediated AMPAR endocytosis, resulting in LTD of synaptic transmission. In Chapter 4, I performed field recording and biochemical measurements in acute hippocampal slices and showed that LPS can impair the induction of LTP in a relatively rapid way (1 h pre-incubation). This blockage of LTP is mediated by the activation of microglial TLR4 and its associated MyD88-dependent signaling pathway. IL-1β released from microglia is the major factor in LPS-induced impairment of LTP. Taken together, our study discovered that neuroinflammation can rapidly induce long-term modulation of synaptic transmission through two distinct pathways. These findings represent novel mechanisms in which environmental stressors modulate synaptic transmission. Furthermore, our study contributes to our understanding of synaptic and cognitive dysfunctions in various brain disorders and suggests new therapeutic targets to alleviate memory loss in these disorders.
Cortical spreading depression (SD) is a slowly propagating wave of brain cell depolarization that manifests in several neurological conditions, including migraine with aura, ischemia and brain trauma. The unique pattern of SD propagation suggests that it arises from an unusual form of intercellular communication. To advance our understanding of SD, we used two-photon imaging, intrinsic optical imaging, electrophysiological recording, and amperometric glutamate biosensor measurement to study the mechanisms underlying SD propagation in acute isolated brain slices.In Chapter 2 we examined and compared the neuronal versus astrocytic changes in cellular processes which are fundamental to both cell types including cell volume, pH and metabolism during SD propagation. We found that SD was correlated in neurons with robust yet transient increased volume, intracellular acidification and mitochondrial depolarization. Our data indicated that a propagating large conductance during SD generated neuronal depolarization, which led to both calcium influx triggering metabolic changes and H⁺ entry. Notably, astrocytes did not exhibit changes in cell volume, pH or mitochondrial membrane potentials associated with SD but they did show alterations induced by changing external [K⁺]. This suggests that astrocytes are not the primary contributor to SD propagation but are instead activated passively by extracellular potassium accumulation. In Chapter 3 we used enzyme based glutamate electrodes to show that NMDA receptors likely at presynaptic sites, trigger SD by evoking glutamate release via vesicular exocytosis, independent of action potentials and voltage gated calcium channels. Both SD- and NMDA-induced vesicular exocytosis of glutamate are triggered by efflux of calcium from mitochondria via the mitochondrial Na⁺/Ca²⁺ exchanger. Through this mechanism NMDAR stimulation evokes a vicious cycle of glutamate-induced glutamate release. Diffusion of glutamate to more distant NMDARs will generate a slowly propagating regenerative glutamate release to cause widespread neuronal depolarization. These data offer support for the hypothesis that neuronal signaling pathways play a crucial role in propagation of SD and the following pathophysiological responses. Particularly, a novel form of NMDAR-dependent regenerative glutamate release is responsible for the cellular mechanisms that promote SD progression. In addition, this research may provide insight into possible clinical targets for treatment of SD-related neurological disorders.
The two main forms of hippocampal synaptic plasticity, long-term potentiation(LTP) and long-term depression (LTD) represent a cellular modelfor learning and memory. While synaptic plasticity has been studied extensively, questions still remain on how exogenous and endogenous modulatorscan impact hippocampal LTP and LTD. Here, we use electrophysiology andimaging to investigate the effects of two types of modulators on synapticplasticity. First, we look at the effects of an antagonist of the 5-HT6 receptor on LTP and LTD in two regions of the hippocampus, the CAl andthe dentate gyrus (DG). We find that our 5-HT6 antagonist differentiallyaffects LTP in each region and blocks hippocampal LTD. These findings arethe first report of an involvement of the 5-HT6 receptor in synaptic plasticityand are particularly relevant in light of evidence showing a key role ofthe 5-HT6 receptor in cognition and memory. Second, we look at the effectsof glutathione (GSH) supplementation on LTP in aged animals. We showthat supplementing aged mice with a precursor for GSH formation reversesthe mechanisms underlying hippocampal LTP from L-type calcium channeldependence back to NMDA receptor-dependence. These results suggest animportant role for GSH as a modulator of synaptic plasticity in aging.
The cholinergic system is one of the most important modulatory neurotransmitter systems in theCNS. In this dissertation, I report novel cholinergic modulations of three Ca²⁺ permaable ionchannels, including R-type voltage-gated calcium channels (VGCCs), TRPC5 channels and NMDAreceptors, in hippocampal CA1 pyramidal neurons and the potential functional roles of thesemodulations in both physiological and pathophysiological conditions.I first studied the “toxin-resistant” R-type VGCCs, and found that muscarinic activationspecifically enhances R-type, but does not affect T-type, Ca²⁺ currents in hippocampal CAlpyramidal neurons. The muscarinic stimulation of R-type Ca²⁺ channels is mediated by M1/M3receptors and requires the activation of a Ca²⁺-indepenadent PKC pathway. Furthermore, theenhancement of R-type Ca²⁺ currents resulted in remarkable changes in the firing pattern of the denovo R-type Ca²⁺ spikes, which could fire repetitively in the theta frequency. Therefore, muscarinic enhancement of R-type Ca²⁺ channels could play an important role in the intrinsic resonanceproperties of neurons.Next, I studied the muscarinic-induced prolonged seizure-like depolarizations called plateaupotentials (PPs) in CAl pyramidal neurons. I found that muscarinic stimulation significantly andspecifically triggered rapid translocation of TRPC5 channels into plasma membrane. Moreover,TRPC channels contribute to the generation of PPs, the underlying tail currents (Itail) and theassociated dendritic Ca²⁺ influx in CA1 pyramidal neurons, via a calmodulin- and PI₃K-dependentpathway. Thus the muscarinic-induced membrane insertion of TRPC5 channels could contribute tothe generation of PPs and the prolonged neuronal depolarization during the ictal discharges inepilepsy.And finally, I report that muscarinic modulation of NMDA-evoked current (INMDA) in CA1pyramidal neurons is age-dependent. I found that muscarinic stimulation potentiated INMDA in bothyoung and old animals. However, in young animals, muscarinic stimulation potentiated INMDAthrough a [Ca²⁺]i-independent but PKC- and Src-dependent pathway. While in old animals,muscarinic stimulation potentiated INMDA through a [Ca²⁺]i-dependent but PKC-independentpathway. Interestingly, the activity of the Gαq -coupled M1-like muscarinic receptors was required for the potentiation of INMDA in both cases. These findings may provide a crucial mechanism bywhich cholinergic input modulates learning and memory.
As the principal immune cell of the brain, microglia cells are responsible for monitoring the activity of other cells in the CNS, and are able to respond to harmful stimuli with a myriad of supportive and defensive mechanisms. With these capacities, microglia are involved in numerous diseases of the CNS, ranging from acute damage and infection to chronic neurodegeneration.In chapter 2 we examine the motile functions of microglia, with particular focus on theresponse of microglia to damage. Microglia cells exhibit two forms of motility, constantmovement of filopodia probing surrounding brain tissue, and outgrowth of larger processes in response to nearby damage. The mechanisms and functions of these motile processes are not well characterized. Using two photon microscopy we investigated microglia motility, and explored the relationship between process outgrowth and filopodia movement. We found that fiolopodia sensing and rapid process outgrowth activities of microglia are mediated by distinct mechanisms, but both require actin polymerization. We also showed that rapid outgrowth of microglia processes contacted the damaged area and resulted in a decrease in lesion volume, whereas inhibition of process outgrowth allowed lesion volume to increase and spread into the surrounding tissue.In chapter 3 we examine the response of microglia to immune stimuli and use specificinterfering peptides to modulate this response. We used peptides blocking LPS-induced activation of the innate immune Toll-Like Receptor 4 (TLR4) to prevent downstream signaling in the brain, and thereby suppress sickness behavior. Interfering peptides blocked TLR4 signalling and prevented second messenger activation and cytokine production normally induced by LPS treatment. These peptides also blocked morphological changes in microglia induced by LPS. Further, injections of interfering peptides prevented LPS-induced sickness behavior, as assessed in novel homecage behavior and with the intracranial self-stimulation paradigm.Taken together our studies demonstrate distinct responses of microglia to different types of pathological insults: acute damage, and immune stimuli. Further, these studies provide means of regulating the dynamic responses of microglia to different stimuli. Ultimately, this research reveals pathways by which microglia can be manipulated, and potentially provides therapeutic targets to enhance the recovery of the brain from acute injury or infection.
Master's Student Supervision (2010 - 2018)
Spreading depolarization (SD) is a wave of intense depolarization that propagates within the gray matter of the brain. It is implicated in migraine and acute brain injuries such as stroke, subarachnoid hemorrhage, and trauma. SD is thought to contribute to the expansions of infarct volume in energy compromised brains. Understanding SD and the associated cellular changes will provide basic knowledge to the field of neurophysiology, and will help identify potential therapeutic targets. The experimental methodologies designed to examine SD properties in acutely prepared brain slices are described in Chapter 2. Techniques used in this study involved simultaneous two photon laser scanning microscopy, intrinsic optical signal (IOS) imaging, electrophysiology, pharmacology, and siRNA delivery by lipid nanoparticles (LNP). In Chapter 3, experimental investigations that tested the contribution of neuronal swelling to the IOS changes in SD, as well as the potential role(s) for SLC26A11 chloride channel to neuronal swelling are described. The pharmacological agent GlyH-101 (an inhibitor of SLC26A11 and other chloride channels) reduced neuronal swelling while other properties of SD were unaltered. This suggests that neuronal swelling likely occurs downstream, or in parallel with other cellular processes. Interestingly, the temporal profiles of neuronal swelling and IOS were not correlated, suggesting that other cellular mechanisms generate IOS changes during SD. I also demonstrated that LNP-mediated delivery of siRNA was effective in gene knock-down in vitro but not in vivo. This was further supported by the lack of functional effect during SD in Slc26a11 siRNA treated samples compared to control siRNAs. Finally, I mimicked situations of mild energy failure in brain slices in parallel to SD induction; however, I observed no differences in SD electrical and optical properties compared to control conditions.These data supported the importance of cellular chloride entry in mediating neuronal swelling associated with SD. However, the role for SLC26A11 and the exact molecular mechanism(s) of swelling that are inhibited by GlyH-101 remain unidentified. My data provide insights into the molecular processes during SD, and work towards identifying potential therapeutic targets in neurological disorders associated with SD.