京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22301号 / 医博第4542号 / 新制||医||1040(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 椛島 健治, 教授 林 康紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 新制・課程博士 / 博士(医学) / 甲第23066号 / 医博第4693号 / 新制||医||1049(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邊 直樹, 教授 溝脇 尚志, 教授 藤田 恭之 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

For healthy individuals, wound healing mainly occurs without medical intervention, yet for the growing elderly, diabetic, or obese populations, as well as for those recovering from surgery, disregulated wound healing poses a serious health risk. Therefore, understanding the cellular processes regulating wound healing and correcting them when they go awry is essential for meeting these population’s healthcare needs. Wound healing is a complex process consisting of a suite of injury repair programs executed by cells in the injured tissue. While several of these programs have been previously described, there are many possible cellular signalling pathways that can mediate a given repair program, and its unclear which pathway mediates a specific process. In this work, we aimed to identify the key cellular signaling pathway that regulates the injury contraction process in a dermal microtissue on a chip model. We found that a balance of tissue forces generated via RhoA activation is critical for injury contraction, and that spatially localized RhoA activation can recruit new cells to participate in injury contraction. During our experiments, we also discovered and characterized a novel actin cytoskeleton-plasma membrane topology present in human dermal fibroblasts at the extreme end of cellular contractility. We also developed several technical advances: the real-time imaging and manipulation of calcium in 3D microtissues, the development of a reporter for smooth muscle actin and a labeled cellular fibronectin fusion protein, and the optimization of Forster Resonance Energy Transfer sensors. Taken together, our experimental results demonstrate the importance of RhoA-mediated force balance during injury contraction, which also has implications for scarring wound pathologies, while the tools we developed provide support for future investigations into the cellular signaling mediating injury repair programs.

Episodic memory is an imperfect record of events arranged in time and space. When dealing with the storage of memories, the brain is faced with a predicament: it must retain an acceptably faithful facsimile of transpired events while simultaneously permitting inevitable modifications to accommodate learning new information. In this thesis, I first review contemporary theories of how memories can be stored in a neural substrate within the hippocampus, particularly in regards to how they can be arranged in time. Next, using in vivo calcium imaging, I detail how hippocampal “time cell” sequences could support encoding of behavioral events along multiple temporal dimensions. In this study, I trained mice to run in place on a treadmill, thereby measuring single-cell activity in CA1 as a function of time. Neurons in CA1 formed sequences, each cell firing one after another as if forming a scaffold upon which memories can be laid. These sequences were relatively well-preserved over a period of four days, satisfying the first requirement that information must be stored for a memory to persist. Additionally, these sequences also changed over time, which may be revealing a mechanism for how memories can change over time to assimilate new information. In the next experiment, I describe a collaborative project where we used immunohistochemistry, optogenetics, and calcium imaging to investigate the long-term dynamics of a fear memory. After mice initially associated a context with an aversive stimulus, they were placed in the same context over two days where they gradually relearned that the context was harmless. This produced molecular and neurophysiological signatures consistent with memory modification. However, after re-triggering fear, mice reverted to fearful expression with commensurate neural correlates. Using optogenetics, these behaviors could also be reliably suppressed. Finally, I conclude by synthesizing these findings with hippocampal literature on sequence formation and consolidation by proposing a holistic view of how these features can support episodic memory.

Patients with Alzheimer’s disease (AD) experience sleep disturbances, including disruption in slow-wave sleep (SWS). Slow oscillations (≤1 Hz), a brain rhythm prevalent during SWS, play a role in memory consolidation. Interestingly, patients with AD exhibit slow oscillations of low amplitude, which might contribute to their memory impairments. The mechanisms underlying slow-wave disruptions in AD remain unknown. Slow oscillations originate in the neocortex. Cortical neurons from all layers oscillate between UP and DOWN states during slow oscillations. Astrocytes are known to support neuronal circuit functions, and disruptions in astrocyte activity might contribute to slow-wave aberrations. Here, we investigated astrocytic contributions to slow-wave disruptions in an animal model of beta-amyloidosis (APP mice). First, we monitored astrocytic calcium transients to determine whether astrocytic calcium dynamics were disrupted in APP mice. Fourier transform analysis revealed that the power, but not the frequency of astrocytic calcium transients, was disrupted in young APP mice. This suggested calcium dynamic of astrocytic network was altered and might contribute to the disruption of slow waves in APP mice. Second, we used optogenetics to synchronize cortical astrocyte activity at 0.6 Hz to drive slow oscillations in APP mice. Our results showed that optogenetic activation of ChR2-expressing astrocytes at the endogenous frequency of slow waves restored slow-wave power. Furthermore, chronic optogenetic stimulation of astrocytes at 0.6Hz for 14 or 28 days reduced amyloid plaque deposition, prevented calcium overload in neurites, and improved memory performance in APP mice. These results revealed a malfunction of the astrocytic network driving slow-wave disruptions, and suggested a novel target to restore slow-wave power in APP mice, with translational potential to treat AD.

In the thesis is described the development of two platforms to optogenetically induce single neuron excitation and formation of memory in biological in-vitro neuronal networks.One platform is a photonic chip, with aperiodic grating scatterers able to create a specific light distribution on the surface of the chip. The second platform is a digital light processor device integrated in a microscopy setup, able to create on the sample plane whatever pattern with 3 um resolution. Stimulating simultaneously many neurons, it was demonstrated the ability of this system to induce potentiation on the illuminated cells, and thus engram formation.

This thesis describes the development of a minimally invasive integrated platform for all-optical neural stimulation and measurement (OptoSAM). The OptoSAM platform is a single mixed-signal complementary metal-oxide semiconductor (CMOS) chip. After design, the chip is postprocessed to contain the necessary optical filters and emitters to enable both fluorescent detection and neural stimulation. Finally, the chip is packaged in a probe form factor for minimally-invasive implantation into neural tissue. The thesis describes how the OptoSAM is engineered for two applications: optical fluorescent imaging on one hand and optogenetic stimulation on the other. For either application, constraints and tradeoffs are described that guide design specifications. For fluorescent neural detection, this thesis focuses on improvements made in lens-less image reconstruction and optical filtering. It describes circuit design for the lens-less, filter-less fluorescent imaging subsystem and characterizes the resulting imaging performance. The lack of on-chip filters precludes reliable imaging of fluorescent targets both in-vivo and ex-vivo. To address these limitations, the metal-insulator-metal angle sensitive pixel (MIMASP) is introduced, a novel nanophotonic structure that integrates lens-less imaging and optical filtering in an ultrathin (<5μm) frontend. The MIMASP offers three advantages over previously published angle sensitive pixels. First, it orthogonally modulates the detection light field for two arbitrary wavelengths, enabling the separation and detection of colors in the image. Secondly, each layer is constructed from optical long-pass filters, rejecting the blue excitation light. Third, an analytical framework is created that allows to optimize the ensemble image reconstruction resolution as a function of the available per-pixel geometries. The angle sensitive pixels are a promising lens-less imaging method for situations where both the number of pixels and the permitted device dimensions are extremely constrained. Equipped with the MIMASP frontend, the imager is demonstrated in scattering media to successfully separate fluorescent targets based on color, fluorescent lifetime and even environmental pH. The experiments are extended to fluorescent detection in ex-vivo acute brain slices. For optogenetic stimulation, we equip the OptoSAM platform with organic light emitting diodes (OLEDs) as thin-film emitters. In-vivo results show how the OLED probe can evoke neural activity in a fully scalable fashion. Using synchronized groups of OLEDs, large neural populations can be synchronously activated. Simultaneously, single neurons can be manipulated by emission from single OLEDs at a 25μm pitch. We demonstrate single-unit manipulation and separation of both pyramidal and interneurons. A custom flexible, transparent multi-electrode array (MEA) provides the electrophysiological recording for cross-validation in the deep-brain. Measurements show how local field potentials (LFPs) are evoked at both 300μm and 1.2mm deep, and how the LFP magnitude roll-off proves locality of the induced activity. Compared to previously published stateof- the-art, the OLED-on-CMOS approach provides a two orders of magnitude larger field of view (FoV) while improving resolution by 3×. Pixel pitch and count can be fully scaled to provide arbitrary fields of view and resolution. The OptoSAM platform proves a pathway towards behavioral studies in awake mice. These studies could address multiple brain regions independently with a single device insertion. This provides neuroscientists with the tools to study relationship between distant regions with single-neuron resolution. While the detection and stimulation are separately optimized and validated, the chip is a promising platform for future integration of both modalities. To this end, it proposes three future chip designs, each with their respective strengths. The proposals also provide potential solutions to the challenges associated with the design and fabrication. The thesis concludes with recommendations for future experiments, both for the OptoSAM platform and for future designs.

Triplet-triplet annihilation upconversion (TTA-UC) is a process which converts two low energy photons into one higher-energy excited state. TTA-UC has recently received attention for its potential application to many light driven processes, such as improving efficiency in photovoltaic devices and allowing use of low-energy light sources for in vivo applications, including bioimaging, optogenetics, and photochemotherapy. Each of these applications has a different set of energetic requirements, which has created a need for a diverse library of upconverting materials. Additionally, these applications benefit from improved upconversion efficiency in solid-state, a task that has proven challenging for the traditionally solution-phase process. Macromolecular scaffolds are a promising avenue to tune the electronic communication between chromophores and control intermolecular packing in solid-state. Herein, we report the investigation of dendrimers with annihilator-functionalized termini and linear annihilator polymers as frameworks to control local annihilator concentration and communication. We find that multi-annihilator dendrimers exhibit higher upconversion yields at low concentrations compared to similar concentrations of monomer; however, higher generation dendrimers allow strong interchromophore coupling, which promotes parasitic excimer formation, decreasing relative upconversion yields. Linear annihilator copolymers with alternating anthracene and phenyl or naphthyl bridges had ground state optical properties predictive of interchromophore communication based on bridge connectivity, interchromophore length, and polymer planarity. Non-conjugated, naphthyl polymers were observed to be the most efficient at intramolecular TTA-UC in dilute solutions. In this dissertation, we will discuss current efforts in the field towards control and analysis of intramolecular TTA-UC through design of multi-annihilator macromolecules and novel annihilator scaffolds targeting underutilized regions of the electromagnetic spectrum. In Chapter 1, we list important factors to consider about improving TTA-UC and follow with discussion of reported macromolecular systems and their efforts towards intramolecular TTA-UC. Chapter 2 introduces a series of non-conjugated dendrimers functionalized with anthracene annihilators on the periphery and analyzes their upconversion capabilities as a set of macromolecules with controlled molecular structure. In Chapter 3, we investigate the effect of connectivity between annihilators in alternating co-polymer systems, discussing the impact on ground state photophysical properties and upconversion efficiency. Finally in Chapter 4, we introduce an approach for using computational analysis as a high-throughput tool for identifying potential novel annihilator molecules.

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