The motivation to eat is influenced by both internal physiological demands and by external stimuli with positive or negative associations. A conditioned stimulus (CS) associated with food can potentiate eating in sated subjects, whereas a CS associated with a negative affect can suppress eating in hungry subjects. Although the amygdala has been implicated in these behaviors, the neural mechanisms that underlie this type of non-homeostatic eating are poorly understood. To investigate the role of BLA neurons in CS+ potentiated eating and CS- lick suppression, we developed a behavioral paradigm in mice in which eating behavior could be assessed in conditions of low or high satiety and in relation to CS presentations while recording neural activity using freely-moving endoscopic calcium imaging. We found that satiety partially decreases responses to the CS+, and the neural representation of the CS+ becomes more similar to the CS-. Additionally, we tested the hypothesis that CS-evoked activity is casually involved in CS+ induced licking or CS- lick suppression by using optogenetics during this task. Silencing of BLA glutamatergic neurons labelled by NL189 prevents CS- lick suppression during low satiety and does not affect licking during the CS+. The combination of cellular imaging and optogenetics results indicates that BLA neuronal activity evoked by the CS- is critical for lick suppression, whereas CS+ activity might facilitate appetitive behavior, but this activity is not critical for cue-induced eating.

In this study, I investigate how serotonergic modulation of the medial prefrontal cortex (mPFC) affects neuronal activity and impacts cognitive flexibility, anxiety, and fear extinction (Figure 1). I begin in Chapter 1 with general information on the PFC with a focus on the mPFC, then discuss the role and complexity of serotonin and how manipulation of serotonin affects behavior. I, finally, introduce what is understood about how serotonin modulates the mPFC, the significance of which has implications for cognitive and emotional behaviors. In Chapter 2, I studied the role of serotonin in cognitive flexibility. Specifically, I used retrograde tracing to determine the origin of mPFC and assessed how terminal release of 5-HT affects mPFC pyramidal neuron activity using whole-cell electrophysiology in acute brain slices. Furthermore, through in vivo fiber photometry, I evaluated the activity of 5-HTergic neurons projecting to the mPFC during cognitive flexibility behavior. Lastly, by selectively increasing or decreasing mPFC 5-HTergic terminal release through in vivo optogenetics, I assessed the modulatory role of 5-HTergic input into the mPFC on intradimensional rule reversal and extradimensional rule shift performance in the cognitive flexibility task. Furthermore, I evaluated the activity of 5-HTergic neurons projecting to the mPFC during an open field task using in vivo fiber photometry and, in Chapter 3, used in vivo optogenetics to determine the role 5-HT in the mPFC plays in modulating fear-related behavior. In Chapter 4, I examined a pharmacological screen of a psychedelic drug in the cognitive flexibility task outlined in Chapter 2. I conclude in Chapter 5 with a discussion of the study implications and future directions.

In neuroscience, optical techniques have become the leading method over electrophysiological techniques because of their ability to target defined populations upon tagging both for in vivo recordings using genetically encoded calcium or voltage indicators and stimulation using optogenetic opsins at the single neuronal level. Additionally, optical imaging has a smaller tissue displacement factor, the ratio of displaced neuronal tissue to field of view (FoV), thus accelerating the ability to simultaneously record from a larger volumes of neurons whereas electrophysiology arrays are limited in the total number of recordable neurons by the amount of sustained tissue damage. Conventional optical approaches, however, typically rely on microscopy techniques which require the subject to be head-fixed thus limiting the applicability especially at the chronic setting, raising the need for fully implantable optical interfaces. As a result, multiple lens-based miniature microscopes have been developed in academia and industry. Nevertheless, a truly implantable optical neurotechnology has remained intractable because traditional miniaturized fluorescence microscopes require an opening in the dura and skull that matches or exceeds the FoV and chronically extends outside the skull, resulting in a poor overall displacement factor. To overcome these limitations, I developed and characterized various implantable optoelectronic platforms designed to optically record from large neuronal FoVs in a minimally invasive implantable form factor. These works culminated in the SCOPe (Subdural CMOS Optical Probe) platform which was validated in multiple in vivo demonstrations involving mouse and nonhuman primate.

Kanalrhodopsine (ChRs) sind eine Gruppe von lichtgesteuerten Ionenkanälen, die ursprünglich aus motilen Algen stammen. In ihren nativen Organismus vermitteln sie die Bewegung zu optimalen Lichtbedingungen. In der biologischen Forschung hingegen werden ChRs eingesetzt, um die Erregbarkeit spezifischer Zellen mit hoher räumlicher und zeitlicher Auflösung optisch zu steuern, ein Forschungsfeld, was als Optogenetik bezeichnet wird. Es wurden zahlreiche ChRs mit unterschiedlichen Eigenschaften charakterisiert und entwickelt, darunter solche, die selektiv für H+, Na+, K+ und Anionen sind. Im Gegensatz dazu sind bisher keine Ca2+-selektiven ChRs bekannt. In Anbetracht der Dominanz der von Kalzium in zellulären Signalwegen in allen Reichen des Lebens, würde ein Ca2+-leitendes ChR präzise Photokontrolle einer Vielzahl von zellulären Prozessen ermöglichen. In dieser Arbeit wurden Chlamydomonas reinhardtii channelrhodopsin 2 (CrChR2) Mutanten, die mit einer Erhöhung der Ca2+-Leitfähigkeit einhergehen, elektrophysiologisch charakterisiert und systematisch verglichen. Von den getesteten Varianten zeigten diejenigen, die eine Erhöhung der negativen Ladung am Selektivitätsfilter des Kanals, dem zentralen Tor, verursachen, erhebliche Auswirkungen auf die Leitfähigkeit für Ca2+ bei negativen Membranspannungen. Daraufhin wurden gezielt homologe Mutationen an mehreren verwandten ChRs eingeführt wodurch erfolgreich zwei Kalzium-durchlässige Kanalrhodopsine (CapChR1 und 2) erzeugt werden konnten. Die erweiterte Charakterisierung der CapChRs ergab eine unterdrückte Na+-Leitfähigkeit und eine erhöhte Ca2+-Durchlässigkeit bei negativen Spannungen. Bei niedrigen extrazellulären Konzentrationen des zweiwertigen Kations zeigten Kalzium-Imaging Experimente die Überlegenheit von CapChR2 bei der Vermittlung des durch Licht ausgelösten Ca2+-Einstroms in kultivierten Zellen. / Channelrhodopsins (ChRs) constitute a group of light-gated ion channels originating from motile algae. In their native organisms, they mediate movement towards optimal light conditions. In biological research, ChRs are employed to optically control excitability of specific cells with a high spatiotemporal resolution in a field commonly referred to as optogenetics. Numerous ChRs with varying properties have been characterized and engineered, including members that are selective for H+, Na+, K+ or anions. In contrast, no Ca2+-selective ChRs have been reported to date. Given the dominance of calcium signaling across the kingdoms of life, a Ca2+-conducting ChRs would enable precise photocontrol of a multitude of cellular processes. In this work, mutants of Chlamydomonas reinhardtii channelrhodopsin 2 (CrChR2) associated with an increase in Ca2+-conductance were characterized via electrophysiology and compared systematically. Out of the tested variants, those increasing the negative electric charge at the selectivity filter of the channel, the central gate, were found to have substantial effects on the conductance for Ca2+ at negative membrane voltages. Subsequently, targeted mutations on several related ChRs were introduced in order to produce two calcium-permeable channelrhodopsins (CapChR1 and 2). Extended characterization of the engineered CapChRs revealed suppressed Na+ conductance and increased Ca2+ permeation at negative voltages. At low extracellular concentrations of the divalent cation, calcium imaging experiments demonstrated the superiority of CapChR2 in mediating light-triggered Ca2+-influx in cultured cells.

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24669号 / 農博第2552号 / 新制||農||1099(附属図書館) / 学位論文||R5||N5450(農学部図書室) / 京都大学大学院農学研究科応用生命科学専攻 / (主査)教授 菅瀬 謙治, 教授 小川 順, 教授 森 直樹 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

No description available.

No description available.

No description available.

No description available.

Gliotransmitters dérivés de l'astrocyte glutamate et l'ATP modulent l'activité neuronale. Cependant, il reste à savoir comment les astrocytes contrôlent la libération et coordonnent les actions de ces gliotransmetteurs. Dans la première partie de ma thèse, en utilisant l'expression transgénique de la canalrhodopsine 2 (ChR2) sensible à la lumière dans les astrocytes, nous avons observé que la photostimulation augmentait de manière fiable le potentiel d'action des neurones pyramidaux de l'hippocampe. Cette excitation repose principalement sur une libération de glutamate dépendant du Ca2+ par les astrocytes qui active les NMDR neuronaux extrasynaptiques. Remarquablement, nos résultats montrent que l'augmentation de Ca2+ induite par ChR2 et la libération ultérieure de glutamate sont amplifiées par l'activation autocrine induite par l'ATP/ADP des récepteurs P2Y1 sur les astrocytes. Ainsi, l'excitation neuronale est favorisée par une action synergique de la signalisation glutamatergique et purinergique autocrine dans les astrocytes. Ce nouveau mécanisme peut être particulièrement pertinent pour les conditions pathologiques dans lesquelles la concentration extracellulaire d'ATP est augmentée et agit comme un signal de danger majeur. Dans la seconde partie de ma thèse, nous rapportons que la photostimulation sélective des astrocytes ChR2 dans le gyrus denté facilite la transmission synaptique excitatrice sur les cellules granulaires via l'activation des NMDR pré-synaptiques contenant GluN2B. De plus, nous avons découvert que l'élévation intracellulaire du Ca2+ induite par l'ATP et dérivée de l'ATP contrôlait étroitement la libération du glutamate par les astrocytes au cours de la photostimulation des astrocytes. Nos résultats fournissent des preuves d'une relation étroite entre l'ATP dérivé d'astrocyte et le glutamate. / Astrocyte-derived gliotransmitters glutamate and ATP modulate neuronal activity. It remains unclear, however, how astrocytes control the release and coordinate the actions of these gliotransmitters. In the first part of my thesis, using transgenic expression of the light-sensitive channelrhodopsin 2 (ChR2) in astrocytes, we observed that photostimulation reliably increases action potential firing of hippocampal pyramidal neurons. This excitation relies primarily on a Ca2+-dependent glutamate release by astrocytes that activates neuronal extra-synaptic NMDRs. Remarkably, our results show that ChR2-induced Ca2+ increase and subsequent glutamate release are amplified by ATP/ADP-mediated autocrine activation of P2Y1 receptors on astrocytes. Thus, neuronal excitation is promoted by a synergistic action of glutamatergic and autocrine purinergic signaling in astrocytes. This new mechanism may be particularly relevant for pathological conditions in which ATP extracellular concentration is increased and acts as a major danger signal. In the second part of my thesis, we report that selective photostimulation ChR2 positive astrocytes in dentate gyrus facilitates excitatory synaptic transmission onto granule cells via the activation of pre-synaptic GluN2B-containing NMDRs. Moreover, we discovered that astrocyte-derived ATP-mediated intracellular Ca2+ elevation tightly controls glutamate release from astrocytes during astrocyte photostimulation. Our results provide evidence for a close relationship between astrocytic-derived ATP and glutamate.