Une précision à l’échelle de la milliseconde dans le transfert d'informations entre les neurones est essentielle pour la synchronisation et la plasticité des circuits neuronaux dans le cerveau. Les axones sont des prolongements neuronaux qui assurent la communication via des impulsions électriques ou des potentiels d’action (PA). A cause du manque de myéline et de leur diamètre très fin, les axones de l'hippocampe propagent les PA lentement et ainsi générer des délais de conduction très long (jusqu’à 100 ms) qui sont traditionnellement considérés comme invariants. Cependant, plusieurs études ont montré que l'activité change la morphologie des axones et module le temps de latence de la transmission. Il convient donc de se demander si le diamètre des axones varie en fonction de l'activité pouvant influencer lapropagation des PA.Les diamètres des axones non-myélinisés de l’hippocampe (compris entre 100-350 nm) sont généralement trop petits pour être résolu par la microscopie photonique conventionnelle. Le développement récent de l’imagerie super résolution STED permet désormais l'observation de la dynamique de leur morphologie détaillée dans le tissu vivant. En combinant la microscopie STED, l’électrophysiologie avec enregistrements en champs et patch-clamp dans des tranches de cerveau de souris et des simulations informatiques, nous avons découvert que les axones du CA3 subissent un élargissement de leur diamètre après l'induction de la potentialisation à long terme (PLT). Nous démontrons que cet élargissementde diamètre augmente la vitesse de conduction des PA. Dans l'ensemble, nos résultats indiquent que les axones peuvent réguler leur diamètre de manière dynamique changeant le délai de conduction des PA, ce qui modifie le timing du transfert d’information dans les circuits neuronaux. Cette étude suggère l’existence d’un nouveau type de mécanisme structurel dans le compartiment axonal jouant un rôle pour la plasticité neuronale. / Millisecond timing precision in the transfer of information between neurons is essential for the synchrony and plasticity of neural circuits in the brain. Axons are neuronal extensions that ensure the communication via brief electrical impulses called action potentials (AP). Because they are unmyelinated and are extremely thin, hippocampal axons propagate APsslowly and thus generate long delays of conduction (up to 100 ms) that are traditionally considered invariant. However, recent studies have shown that activity changes the morphology of axons and modulate the latency of transmission, thus raising the question whether axons undergo activity-dependent structural changes that could influence the propagation of APs. The diameter of hippocampal axons (ranging between 100-350 nm) are usually too thin to be properly resolved by conventional light microscopy. However, the development of super resolution STED imaging now enables the observation of their detailed morphological dynamics in living tissue. Using a novel combination of STED microscopy, field recordings, patch-clamp electrophysiology in mouse brain slices and computer simulations we discovered that CA3 axons undergo long-lasting enlargement in their diameter after the induction of long term potentiation (LTP). We provide strong evidence that this diameter enlargement increases AP conduction velocity. Taken together, our findings indicate that axons can dynamically tune AP propagation delays by changing their diameters, thereby altering the timing of information transfer in neural circuits. This study suggests a novel and powerful structural mechanism for neural plasticity.

Thesis advisor: Laura Anne Lowery / Axon guidance is a critical process in forming the connections between a neuron and its target. Development of a properly functioning nervous system relies heavily on how accurately an axon is guided to the right target. Defects in the guidance machinery may result in neurological disorders. The growth cone that is formed at the tip of a growing axon is responsible for navigating axons to their final targets. The growth cone steers the growing axon towards the appropriate direction by integrating extracellular guidance cues received by membrane-associated receptors at the growth cone periphery. Upon receiving guidance cues, a number of intracellular signal transduction pathways are initiated downstream of the guidance receptors, that can promote or halt growth cone advance. The growth cone generates these responses by remodeling its cytoskeletal components, which are actin network in the periphery and microtubules in the growth cone center. In this thesis, we focus on understanding the role of microtubule dynamics regulation within the growth cone as it makes guidance decisions. Specifically, we examine the role of TACC3 as a microtubule plus-end binding protein during axon outgrowth and guidance. We show that TACC3 localizes at microtubule plus-ends in embryonic Xenopus laevis growth cones and regulates microtubule growth parameters. We also show that TACC3 is important for promoting axon outgrowth in cultured neural tube explants. Furthermore, our data suggests that TACC3 affects axon guidance in vivo and ex vivo. Examination of embryos depleted of TACC3 revealed guidance defects in the spinal cord neurons, while TACC3-overexpressing cultured spinal neurons showed increased resistance to Slit2-induced growth cone collapse. Finally, in an attempt to delineate the mechanism behind TACC3-mediated axon guidance under Slit2, we studied the importance of tyrosine phosphorylation induced by Abelson tyrosine kinase. We find that retaining phosphorylatable tyrosines within the TACC domain is important for its microtubule plus-end tracking behavior and its impact on microtubule dynamics regulation, axon outgrowth and guidance. Together, this thesis contributes new insights to the understanding of the role of TACC3 as a microtubule plus-end binding protein and identifies TACC3 as a potential regulator of axon outgrowth and guidance during Xenopus laevis embryonic development. / Thesis (PhD) — Boston College, 2019. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Olfactory sensory neurons in the olfactory epithelium are responsible for detecting the odors we smell and are constantly dying. However, in order for the sense of smell to be maintained, the olfactory system has the unique ability to generate new neurons. After an olfactory sensory neuron is born in the olfactory epithelium, it must extend an axon towards the olfactory bulb in the central nervous system. Within the olfactory bulb, these axons make specific synaptic contacts with the dendritic processes of mitral cells, which are the main projection neurons from the olfactory bulbs into higher cortical areas in the brain. In addition to regeneration due to normal turnover, the olfactory system is also capable of recovery after an injury. The olfactory system’s ability to recover is remarkable because it is capable of regeneration after a mild injury (a portion of olfactory epithelium is removed) or a severe injury (in which the entire olfactory epithelium is removed.) A well-established model for producing a severe type of injury in the olfactory epithelium is by inducing a chemical ablation by a single injection of the drug methimazole. A specific interest in the regenerative process after injury is reestablishment of synaptic connections. We hypothesized that expression of synaptic markers will allow for establishing a timeline of functional recovery of the olfactory system after injury. Our lab has studied three synaptic vesicle associated proteins, vesicular glutamate transporter -1 (VGlut-1), vesicular glutamate transporter-2 (VGlut-2), and synaptophysin, as well as one activity-regulated protein, tyrosine hydroxylase. These studies found specific temporal expression profiles at 2, 7 and 14 days post injury. Our initial data show that VGlut-1 and VGlut-2 are decreased after injury, indicative of a reduction in synaptic connectivity in both olfactory sensory neuron axons and in dendrites of mitral cell neurons. These changes in synaptic connectivity help in understanding functional connectivity after an injury and can further be used to correlate histological axonal tracing with behavioral studies.

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There have been many different studies performed in order to examine various properties of neurons. One of the most important properties of neurons is an ability to originate and propagate action potential. The action potential is a source of communication between different neural structures located in different anatomical regions. Many different studies use modeling to describe the action potential and its properties. These models mathematically describe physical properties of neurons and analyze and explain biological and electrochemical processes such as action potential initiation and propagation. Therefore, one of the most important functions of neurons is an ability to provide communication between different neural structures located in different anatomical regions. This is achieved by transmitting electrical signals from one part of the body to another. For example, neurons transmit signals from the brain to the motor neurons (efferent neurons) and from body tissues back to the brain (afferent neurons). This communication process is extremely important for a being to function properly. One of the most valuable studies in neuroscience was conducted by Alan Hodgkin and Andrew Huxley. In their work, Alan Hodgkin and Andrew Huxley used a giant squid axon to create a mathematical model which analyzes and explains the ionic mechanisms underlying the initiation and propagation of action potentials. They received the 1963 Nobel Prize in Physiology/Medicine for their valuable contribution to medical science. The Hodgkin and Huxley model is a mathematical model that describes how the action potential is initiated and how it propagates in a neuron. It is a set of nonlinear ordinary differential equations that approximates the electrical characteristics of excitable cells such as neurons and cardiomyocytes. This work focuses on modeling the Hodgkin and Huxley model using MATLAB extension - Simulink. This tool provides a graphical editor, customizable block libraries, and solvers for modeling and simulating dynamic systems. Simulink model is used to describe the mechanisms and underlying processes involved in action potential initiation and propagation.

High precision is critical for normal neural circuit function, but that precision is not innate. The location, strength, and number of inputs in a neural circuit are modified in early postnatal development in a process called refinement. The refinement of long-range excitatory projections is well-known, but less is known about the refinement of long-range inhibitory projections. What we do know about inhibitory projection refinement comes from the glycinergic medial nucleus to the trapezoid body to lateral superior olive (MNTB-LSO) projection of the auditory brainstem. During early postnatal life, the MNTB-LSO projection undergoes morphological and physiological refinement. Notably, the MNTB-LSO projection transiently expresses vesicular glutamate transporter 3 (VGLUT3) and synaptotagmin 1 (Syt1), transiently releases glutamate, and undergoes glutamate-dependent refinement. However, it remains uncertain whether glutamate release is specific to the auditory brainstem or could be a more general phenomenon of inhibitory projections. To shed light on this question, I investigated another inhibitory projection of the hindbrain, the GABAergic Purkinje projection of the cerebellum. The Purkinje projection shares key characteristics with the MNTB-LSO projection, including its inhibitory nature, location in the hindbrain, obvious topographic organization, heterogeneity of the target cells, and expression of VGLUT3 transcript and protein. In this thesis, I sought to determine: 1) whether the expression profile of VGLUT3 and Syt1 in the Purkinje projection matches that of the MNTB-LSO projection, and whether the Purkinje projection also releases glutamate, 2) whether the expression profile of synaptic vesicle protein 2 (SV2) isoforms, SV2B and SV2C, matches the expression profile of other synaptic vesicle proteins in the Purkinje and MNTB-LSO projection, and 3) whether the Purkinje projection undergoes postnatal morphological refinement like the MNTB-LSO projection. I found that like the MNTB-LSO projection, the Purkinje projection transiently expresses VGLUT3 and Syt1, releases glutamate in early postnatal life, and may undergo morphological refinement. / Dissertation / Doctor of Philosophy (PhD) / Everything you do, whether it be playing your favorite sport or begrudgingly reading this thesis, requires neural circuits, which are the basic functional unit of the nervous system. How neurons are wired together is crucial for their role in executing a task. But how these neurons fine-tune their connections – in a process called refinement, by getting the right connections to the right location, of the right strength, and of the right number – is an open-ended question in neuroscience. Refinement is more well-studied in excitatory projection neurons, but we know very little about how refinement occurs in inhibitory projection neurons. I compare some of the unusual characteristics of what we do know about inhibitory refinement in the auditory brainstem to another famous projection of the hindbrain, the Purkinje projection. Understanding more about the refinement of inhibitory projections gives key insights into how neural circuits function and how they facilitate complex behaviours.

Spinal cord injury (SCI) often leads to irreversible damage, and permanent paralysis inferior to the injury is common (Leibinger et al., 2013). Injury to the spinal cord occurs in two phases. In the first phase, components of the spinal cord are subject to mechanical trauma causing direct damage. In the second phase, damage spreads from the area of injury through molecular processes. Several studies have linked M1 "pro-inflammatory" macrophages to exacerbation of damage by inducing dieback of dystrophic axons, but not healthy axons, through direct cellular contact. Several studies have identified the presence of macrophage subtypes at specific time. A literature review was conducted in order to summarize these findings (Busch, Horn, Silver, & Silver, 2009; Evans et al., 2014; Horn, Busch, Hawthorne, van Rooijen, & Silver, 2008; Kigerl et al., 2009; Shechter et al., 2013). Although the full mechanism behind the process of M1 macrophage-mediated dieback of dystrophic axons is unclear, matrix metalloproteinase-9 (MMP-9) produced by these macrophages has been shown to play a role. However, the specific interaction between MMP-9 and neurons is under investigation. The research described explores the relationship between MMP-9 and fractalkine (CX3CL1), a surface protein expressed by CNS neurons. SDS-PAGE and western blot were used to determine whether the presence of MMP-9 increases the cleavage of fractalkine at several time intervals. At a concentration of 300ng/ml, MMP-9 was not found to demonstrate cleavage of fractalkine.

Central Nervous System (CNS) injury may lead to irreversible damage to cognitive and motor abilities when injured. This is due to the inability of axons to regenerate. This thesis focuses on two methods of promoting axonal regeneration: microtubule stabilization and upregulation of the intrinsic growth capacity of the neuron via the mechanistic target of rapamycin (mTOR) pathway. Both have shown promising results in potentially being a therapeutic treatment for CNS trauma. This research seeks to (1) test a combinatorial method of axonal regeneration utilizing both methods simultaneously and (2) compare microtubule stabilization and upregulation of the mTOR pathway as neuronal regeneration methods. Aim 1 serves to test the combinatorial treatment of Taxol, a microtubule stabilizer, and cRheb transfection, which upregulates the mTOR pathway, on neuronal cell cultures. Cells were cultured in either a growth-promoting substrate or a mix of growth-promoting and growth-inhibitory substrates. The results of this study revealed combinatorial treatment of 2DIV Taxol application with cRheb transfection as a promising treatment that yielded significantly greater axonal outgrowth than either treatment alone. Aim 2 serves to compare the two established methods of axonal regeneration in the scientific community. Based off of a meta-analysis, results of this aim indicate upregulation of mTOR is more effective at promoting axonal regeneration than microtubule stabilization.

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