Timothy Murphy

Preclinical drug discovery relies on the identification of appropriate target compounds and usage of effective animal model screening tests. In this work excitability across systems as well as spatial and temporal scales were evaluated to understand long QT syndrome (LQTS) therapeutic targets and develop a behavioral assay that could sensitively characterize motor phenotype onset in a Huntington Disease (HD) mouse model. Case I: The pairing of the tetrameric voltage-gated potassium channel, KCNQ1, with an accessory β-subunit, KCNE1, gives rise to the slow delayed cardiac rectifier current (IKs), known to play an important role in the physiological shortening of the cardiac action potential. With loss-of-function mutations in both subunits found to be associated with LQTS, enhancing IKs has been identified as a therapeutic approach. Here, the NSAID, mefenamic acid, was found to dose- and rate-dependently activate IKs. More KCNE1-saturated IKs channel complexes had a greater response to mefenamic acid treatment. KCNE1 residue K41 was identified as critical for mefenamic acid action, suggesting a potential binding site. Case II: HD is a dominantly inherited neurodegenerative disease with characteristic motor symptoms. Animal models with increasingly better face and construct validity have been developed to understand HD pathophysiology. Although HD gross motor defects have been extensively characterized, less is known about forelimb motor deficits. Using a high-throughput alternating reward/non-reward water-reaching task, HD forelimb movement defects and associated aberrant cortical activity were examined. HD heterozygous-zQ175 mice displayed an event sequence defect at ~5.5 months with progressive forelimb deficits starting at ~6 months. Cortical activity associated with water-reaching increased over time in HD but not wildtype mice. Gross motor defects characterized using the tapered beam and rotarod tasks, as well as post-hoc striatal immunostaining, confirmed HD pathology at ~8 months. Overall, at the nanoscale level, a biophysical and pharmacological characterization of mefenamic acid’s effect on IKs highlighted a binding site and the potential of the NSAID to act as a precursor compound for LQTS therapeutic development. At the mesoscale level, a water-reaching task was developed and used to characterize HD phenotype demonstrating the potential of the behavioral task to examine therapeutic efficacy and intervention windows.

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Stress is a relevant etiological contributor to major mental health conditions, including, but not limited to, major depression. Stress changes brain networks with diverse consequences on arousal, valence systems, cognition, social processes such that the individual’s representation of themselves and the world is altered. Here, I characterize functional networks and changes to activity dynamics that accompany the pathological effects of stress in mouse. I do so using spontaneous activity to align with data collection from human resting state imaging. I capture a wide expanse of dorsal neocortex with millisecond timescale resolution using 1) an acute surgical preparation together with voltage sensitive dye (VSD) incubated over the entire dorsal neocortex and 2) transgenic animals expressing fluorescent sensors (iGluSnFR and GCaMP6s) of neural activity together with chronic cranial windows. I first characterize functional connectivity as measured by zero-lag interregional correlation together with longitudinal real time in vivo sampling of extracellular glutamate signals in conjunction with the chronic social defeat model of stress. This reveals stress susceptibility of a midline network, the murine default mode network, and hyperconnectivity after stress that is selectively restored by the rapid acting antidepressant, ketamine. I then examine how spontaneous cortical dynamics impact faithful representation of external stimuli by isolating a specific set of activity stereotypies resembling sensory experience and processing. Focusing on events resembling limb and whisker sensory experience in spontaneous activity, I show that the occurrence of these motifs has a lasting impact on the magnitude of sensory evoked responses. I further show using multiple models of chronic stress and manipulation of circuitry implicated in negative affect, that these motifs are susceptible to stress and that a dominant motif is upregulated after stress in proportion with emotional behavior. The upregulation of sensory motifs in stressed animals accordingly results in decreased sensory reliability. My data confirms the utility and importance of murine modelling of stress and the potential to identify common and divergent effects of specific stressors, as well as identify putative biomarkers with potential treatment relevance in humans.

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Throughout most of the 20th century the brain has been studied as a reflexive system with ever improving recording methods being applied within a variety of sensory and behavioural paradigms.Yet the brains of most animals (and all mammals) are spontaneously active with incoming sensory stimuli modulating rather than driving neural activity.The aim of this thesis is to characterize spontaneous neural activity across multiple temporal and spatial scales relying on biophysical simulations, experiments and analysis of recordings from the visual cortex of cats and dorsal cortex and thalamus of mouse.Biophysically detailed simulations yielded novel datasets for testing spike sorting algorithms which are critical for isolating single neuron activity. Sorting algorithms tested provided low error rates with operator skill being as important as sorting suite. Simulated datasets have similar characteristics to in vivo acquired data and ongoing larger-scope efforts are proposed for developing the next generation of spike sorting algorithms and extracellular probes.Single neuron spontaneous activity was correlated to dorsal cortex neural activity in mice. Spike-triggered-maps revealed that spontaneously firing cortical neurons were co-activated with homotopic and mono-synaptically connected cortical areas, whereas thalamic neurons co-activatedwith more diversely connected areas. Both bursting and tonic firing modes yielded similar maps and the time courses of spike-triggered-maps revealed distinct patterns suggesting such dynamics may constitute intrinsic single neuron properties. The mapping technique extends previous work tofurther link spontaneous neural activity across temporal and spatial scales and suggests additional avenues of investigation.Synchronized state cat visual and mouse sensory cortex electrophysiological recordings revealed that spontaneously occurring activity UP-state transitions fall into stereotyped classes of events that can be grouped. Single visual cortex neurons active during UP-state transitions fire in a partially preserved order extending previous findings on high firing rate neurons in rat somatosensory and auditory cortex. The firing order for many neurons changes over periods longer than 30-minutes suggesting a complex non-stationary temporal neural code may underlie spontaneous and stimulusevoked neural activity.This thesis shows that ongoing spontaneous brain activity contains substantial structure that can be used to further our understanding of brain function.

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Global ischemia occurs during cardiac arrest and has been implicated as a complication that can occur during cardiac surgery. It induces delayed neuronal death in human and animal models, particularly in the hippocampus, while it also can affect the cortex. Other than morphology and measures of cell death, relatively few studies have examined neuronal networks and motor-sensory function following reversible global ischemia in vivo. Optogenetics allows the combination of genetics and optics to control or monitor cells in living tissues. Here, I adapted optogenetics to examine neuronal excitability and motor function in the mouse cortex following a transient global ischemia. Following optogenetic stimulation, I recorded electrical signals from direct stimulation to targeted neuronal populations before and after a 5 min transient global ischemia. I found that both excitatory and inhibitory neuronal network in the somatosensory cortex exhibited prolonged suppression of synaptic transmission despite reperfusion, while the excitability and morphology of neurons recovered rapidly and more completely. Next, I adapted optogenetic motor mapping to investigate the changes of motor processing, and compared to the changes of sensory processing following the transient global ischemia. I found that both sensory and motor processing showed prolonged impairments despite of the recovery of neuronal excitability following reperfusion, presumably due to the unrestored synaptic transmission. Interestingly, motor processing recovered faster and more completely than sensory processing. My results suggest a uniform suppression of synaptic transmission, both in excitatory and inhibitory network, despite the rapid recovery of neuronal excitability and morphology, following a global ischemia and reperfusion. This prolonged suppression of synaptic transmission might impede the recovery of sensory and motor processing with differential severity. Besides, I extended tools for mesoscopic imaging using novel optogenetic sensors, including genetically encoded Ca2+ indicators - GCaMPs, and extracellular glutamate sensor - iGluSnFR. I found that iGluSnFR has fastest kinetics for reporting both sensory and spontaneous activity in the cortex, which can resolve temporal features of sensory processing that were not readily observed with GECIs. I suggest that iGluSnFR tools have potential utility in normal physiology, and neurologic pathologies in which abnormalities in glutamatergic signaling are implicated, such as stroke.

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One of the major goals in neuroscience is to understand and map the connectivity of the brain. While this is no small undertaking, recent technological advances have allowed brain mapping to reach unprecedented levels. Optogenetic tools have been developed that permit selective manipulation and investigation of neural systems. Here, we have mapped in vivo intracortical activity in the mouse by combining arbitrary point optogenetic stimulation and regional voltage-sensitive dye (VSD) imaging. We first show that optogenetic photostimulation using channelrhodopsin-2 (ChR2) led to cortical maps that were similar to the maps generated with sensory stimulation. ChR2-evoked maps confirmed known intrahemispheric relationships (such as between barrel cortex and motor cortex) and known interhemispheric relationships (such as between homotopic areas). We used ChR2 point stimulation to map a number of cortical areas and used network analysis to examine relationships between cortical areas. We found asymmetrical connections between primary and secondary sensory cortex and defined the parietal association cortex as a hub node. We then applied this mapping method to map altered cortical connectivity in the early and late stages after a targeted cortical stroke (1 week post-stroke and 8 weeks post-stroke, respectively). Network analysis based on ChR2-evoked responses revealed a symmetrical bilateral sham network that was disrupted after stroke. At 1 week post-stroke, we observed wide-spread depression of ChR2-evoked activity that extended to the contralesional hemisphere. By 8 weeks post-stroke significant recovery was observed. When we considered the network as a whole, we found that scaling the ChR2-evoked activity from the stroke groups to match the sham group mean resulted in a relative distribution of responses that was indistinguishable from the sham group, suggesting network-wide down-scaling and connectional diaschisis after stroke. When connections within the peri-infarct were isolated, we did not observe equal down-scaling of responses after stroke. Our findings suggest that during recovery, most cortical areas undergo homeostatic upscaling, resulting in a relative distribution of responses that is similar to the pre-stroke (sham) network, albeit still depressed. However, recovery within the peri-infarct zone is heterogeneous and these cortical points do not follow the recovery scaling factor expected for the entire network.

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While the body rests, the mind remains active. In fact, the brain exhibits a rich pattern of structured activity despite having few immediate sensory or motor tasks. During infancy, this brain activity appears tailored to assist in the maturation of neural systems. In the adult, it influences memory consolidation and maintenance of synaptic connections. In this thesis, I address these differences by using voltage-sensitive dye imaging to record spontaneous cortical activity in rodents during development and adulthood.In the adult, I examine slow-wave activity, a key form of spontaneous activity. I show that functionally related regions of the cortex activate synchronously, forming a core set of structures that underlie spontaneous activation. I also show that sensory connections shape the patterns of this activity. These effects hold true in the quietly awake mouse, to a lesser extent. These findings are consistent with an active role for slow-wave activity in the maintenance of cortical connections. In the infant, I examine a dominant form of brain activity, the spindle burst. This pattern of activity follows spontaneous sensory inputs generated by the developing sensory systems, including small twitches in the limbs and tail. It is generally thought to remain localized with the appropriate cortical sensory system, but using wide-field imaging, I show it spreads medially across the cortex. This suggests a potential role in the maturation of connections between sensory and motor regions. I explored this possibility more closely by recording activity in early life from the whisker system of the rat. In the adult, connections exist between the sensory and motor regions of the whisker system. To gain insight into whether the spontaneous activation of these systems contributed to their development, I compared the activity evoked by stimulation to the spontaneous activation of these systems. I found synchronized spontaneous activation of motor and sensory areas that were not yet functionally connected. This suggests that other structures synchronize these areas to promote the maturation of connections between them. Overall, this work reveals details about spontaneous activity that provide clues to why the brain devotes time and energy to activity disconnected from the outside world.

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The motor cortex controls voluntary, skilled movements. It possesses a basic somatotopic organization, but its fine structure remains controversial. Although the reorganization of motor cortex after brain injury is believed to be a critical part of behavioral recovery, our understanding of this process has been constrained by technical limitations. Conventional methods for mapping the motor cortex rely on the insertion of intracortical stimulating electrodes into the brain. We developed a new method for light-based motor mapping that is faster, less invasive, and better suited to repeated mapping in both acute and longitudinal studies. Using this technique, we identified a functional subdivision of the mouse forelimb motor representation according to the direction of movement. Pharmacological and anatomical experiments revealed that the expression of complex movements requires the intact function of the intracortical circuitry, whereas the basic topography of movement in motor cortex may arise primarily from the arrangement of output projections. We also refined methods for intrinsic optical signal sensory imaging, which can be combined with light-based motor mapping to obtain a more complete map of sensorimotor cortex. Performing light-based mapping of sensorimotor cortex for weeks before and after targeted photothrombotic stroke allowed us to monitor cortical reorganization on an unprecedented timescale. We found that if forelimb somatosensory cortex was destroyed by targeted stroke, the forelimb motor cortex was able to incorporate a reorganized sensory map. As a consequence of this increased integration of sensory and motor function after stroke, however, the structure of the motor map was altered, evidenced by a decrease in spatial autocorrelation. Strokes in motor cortex caused increased motor output in peri-infarct cortex, but had no effect on the location or sensitivity of the forelimb somatosensory representation. Light-based motor mapping has provided new insights into both the functional organization of motor cortex and its capacity for spontaneous reorganization after stroke.

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In the mammalian brain, excitatory (glutamatergic) synapses are mainly located on dendritic spines; bulbous protrusions enriched with F-actin. Dendritic filopodia are thin protrusions thought to be involved in the development of spines. However, limited evidence illustrating the emergence of spines from filopodia has been found. In addition, the molecular machinery required for filopodia induction and transformation to spines is not well understood. Paralemmin-1 has been shown to induce cell expansion and process formation and is concentrated at the plasma membrane, in part through a lipid modification known as palmitoylation. Palmitoylation of paralemmin-1 may also serve as a signal for its delivery to subcellular lipid microdomains to induce changes in cell morphology and membrane dynamics making it a candidate synapse-inducing molecule. Using live imaging as well as loss and gain-of-function approaches, our analysis identifies paralemmin-1 as a regulator of filopodia induction, synapse formation, and spine maturation. We show neuronal activity-driven translocation of paralemmin-1 to membranes induces rapid protrusion expansion, emphasizing the importance of paralemmin-1 in paradigms that control structural changes associated with synaptic plasticity and learning. Finally, we show that knockdown of paralemmin-1 results in loss of filopodia and compromises spine maturation induced by Shank1b, a protein that facilitates rapid transformation of newly formed filopodia to spines. To investigate the role of filopodia in synapse formation, we contrasted the roles of molecules that affect filopodia elaboration and motility, versus those that impact synapse induction and maturation. Expression of the palmitoylated protein motifs found in growth associated protein 43kDa, enhanced filopodia number and motility, but reduced the probability of forming a stable axon-dendrite contact. Conversely, expression of neuroligin-1 (NLG-1), a synapse inducing cell adhesion molecule, resulted in a decrease in filopodia motility, but an increase in the number of stable axonal contacts. Moreover, siRNA knockdown of NLG-1, reduced the number of presynaptic contacts formed. Postsynaptic scaffolding proteins such as Shank1b, a protein that induces the maturation of spine synapses, reduced filopodia number, but increased the stabilization of the initial contact with axons. These results suggest that increased filopodia stability and not density may be the rate-limiting step for synapse formation.

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No abstract available.