Innovations in neural data recording techniques are revolutionizing neuroscience and presenting both challenges and opportunities for statistical data analysis. This dissertation discusses several recent advances in neural data signal processing, encoding, decoding, and dimension reduction. Chapter 1 introduces challenges in neural data science and common statistical methods used to address them. Chapter 2 develops a new method to detect neurons and extract signals from noisy calcium imaging data with irregular neuron shapes. Chapter 3 introduces a novel probabilistic framework for modeling deconvolved calcium traces. Chapter 4 proposes an improved Bayesian nonparametric extension of the hidden Markov model (HMM) that separates the strength of the self-persistence prior and transition prior. Chapter 5 introduces a more identifiable and interpretable latent variable model for Poisson observations. We develop efficient algorithms to fit each of the aforementioned methods and demonstrate their effectiveness on both simulated and real data.

Le système des noyaux de la base est principalement impliqué dans le contrôle et l'apprentissage moteur.Le rôle de la voie cortico-striatale a été et est toujours fortement étudié mais le striatum reçoit aussides afférences excitatrices du thalamus, souvent considéré comme un simple relais entre les noyaux dela base et le cortex, formant ainsi des boucles de structures sous-corticales. Les principales afférencesthalamostriatales glutamatergiques proviennent des noyaux thalamiques intralaminaires et forment descontacts synaptiques avec les deux types de neurones efférents GABAergiques du striatum (i- et d-MSNs) et les interneurones cholinergiques (INCs). Le complexe thalamique intralaminaire peut sedistinguer en une partie rostrale (ILTr) contenant les noyaux centrolatéral (CL), paracentral (PC) etcentral médial (CeM) et une partie caudale (ILTc) formée du noyau parafasciculaire (Pf) chez lerongeur (équivalent du complexe parafasciculaire-centromedian chez le primate). Le complexe thalamiqueintralaminaire est souvent considéré comme une structure fonctionnelle homogène, cependant de plus enplus d’études mettent en évidence des différences anatomiques, électrophysiologiques et fonctionnelles desparties rostrales et caudales de l’intralaminaire. Le noyau intralaminaire caudal est de mieux en mieuxdécrit et sa projection striatale se montre impliquée principalement dans la réponse aux stimuli sensorielsainsi que dans la flexibilité motrice. Des données obtenues par Marco Diana et collaborateurs à l’EcoleNationale Supérieure (Paris) apportent un éclairage nouveau sur l'importance du noyau intralaminairerostral, en particulier le noyau centrolatéral, dans le contrôle du mouvement, en montrant que la stimulationoptogénétique de la projection glycinergique/GABAergique ponto-intralaminaire thalamique inhibe les neu-rones thalamiques et provoque une hypolocomotion. Ces résultats indiquent que la suppression de laprojection thalamique sur le striatum mène à une perturbation de la fonction des ganglions de la base.Cette dernière décennie se caractérise par une explosion de nouvelles techniques aussi bien dans lesdomaines d’imagerie que dans les techniques de manipulation génique d’animaux permettant de répondreà certaines questions qui ne pouvaient techniquement pas trouver de réponse jusqu’ici. Ce travail dethèse a pour but de mieux comprendre l’importance des afférences du thalamus intralaminaire sur lestriatum, en particulier sa partie rostrale, qui, de manière surprenante, sont très mal caractérisées. Deplus, les noyaux thalamiques intralaminaires sont un relais entre le cervelet et le striatum, par conséquent,l'analyse de ces connexions pourrait améliorer notre compréhension des maladies neurodégénératives tellesque la maladie de Parkinson impliquant à la fois les noyaux gris centraux et le cervelet, mais dont lesinteractions fonctionnelles n'ont pas encore été décryptées.La première partie de ce travail de thèse consiste en une étude anatomique détaillée des projections duthalamus intralaminaire sur le striatum, en particulier sur ses principales sous-populations (d- et i-MSNs,INCs) et sous-régions (dorso-latéral=DLS, dorso-médian=DMS), par l’utilisation combinée d’un marquagerétrograde monosynaptique et d’une technique de transparisation (« clearing ») permettant par la suitede réaliser une imagerie complète du cerveau à l’aide d’un microscope à feuille de lumière. Les analysesanatomiques réalisées ont permis de confirmer l’existence de projections directes des noyaux thalamiquesintralaminaires sur le striatum dorsal, celles-ci présentant un pattern d’innervation préférentiel pour lesINCs (DMS>DLS) suivi par les dMSNs (DLS>DMS). Les cibles postsynaptiques des projectionsthalamostriatales sont similaires aux projections dopaminergiques, suggérant une interaction étroite entreces afférences.La seconde partie de cette thèse, vise a mieux comprendre l’importance fonctionnelle des connexionsthalamostriatales mises en évidence précédemment dans la locomotion spontanée. Pour cela deux ap-proches seront utilisées: une approche modifiant l’activité de ces neurones par l’utilisation de techniquescomme l’optogénétique et la chémogénétique et une approche descriptive par une technique d’imageriecalcique permettant d’enregistrer l’activité neuronale en temps réel sur des animaux libres de se mouvoir.Les résultats obtenus montrent que l’inhibition de l’ensemble des neurones de l’ILTr est nécessaire pourobserver un phénotype moteur d’hypolocomotion. La mise en place d’un système de détection de motricitéfine et l’enregistrement de l’activité calcique des neurones striataux, nous permettront, à l’avenir, de mieux identifier le type de comportement moteur impliqué dans cette hypolocomotion ainsi que d’évaluer l’impactde cette inhibition thalamique sur l’activité des neurones striataux. / Doctorat en Sciences biomédicales et pharmaceutiques (Médecine) / info:eu-repo/semantics/nonPublished

The hippocampus supports the initial formation and recall of episodic memories, as well as the consolidation of short-term into long-term memories. The ability of hippocampal neurons to rapidly change their connection strengths during learning and maintain these changes over long time-scales may provide a mechanism supporting memory. However, little evidence currently exists concerning the long-term stability of information contained in hippocampal neuronal activity, likely due to limitations in recording extracellular activity in vivo from the same neurons across days. In this thesis I employ calcium imaging in freely moving mice to longitudinally track the activity of large ensembles of hippocampal neurons. Using this technology, I explore the proposal that long-term stability of hippocampal information provides a substrate for episodic memory in three different ways. First, I tested the hypothesis that hippocampal activity should remain stable across days in the absence of learning. I found that place cells – hippocampal neurons containing information about a mouse’s position – maintain a coherent map relative to each other across long time-scales but exhibit instability in how they anchor to the external world. Furthermore, I found that coherent maps were frequently used to represent a different environment and incorporated learning via changes in a subset of neurons. Next, I examined how learning a spatial alternation task impacts neuron stability. I found that splitter neurons whose activity patterns reflected an animal’s future or past trajectory emerged relatively slowly when compared to place cells. However, splitter neurons remained more consistently active and relayed more consistent spatial information across days than did place cells, suggesting that the utility of information provided by a neuron influences its long term stability. Last, I investigated how protein synthesis, known to be necessary for long-term maintenance of changes in hippocampal neuron connection strengths and for proper memory consolidation, influences their activity patterns across days. I found that along with blocking memory consolidation, inhibiting protein synthesis induced a profound, long-lasting decrease in neuronal activity up to two days later. These results combined demonstrate the importance of rapid, lasting changes in the hippocampal neuronal code to supporting long-term memory.

Imaging of multiple cells has rapidly multiplied the rate of data acquisition as well as our knowledge of the complex dynamics within the mammalian brain. The process of data acquisition has been dramatically enhanced with highly affordable, sensitive image sensors enable high-throughput detection of neural activity in intact animals. Genetically encoded calcium sensors deliver a substantial boost in signal strength and in combination with equally critical advances in the size, speed, and sensitivity of image sensors available in scientific cameras enables high-throughput detection of neural activity in behaving animals using traditional wide-field fluorescence microscopy. However, the tremendous increase in data flow presents challenges to processing, analysis, and storage of captured video, and prompts a reexamination of traditional routines used to process data in neuroscience and now demand improvements in both our hardware and software applications for processing, analyzing, and storing captured video. This project demonstrates the ease with which a dependable and affordable wide-field fluorescence imaging system can be assembled and integrated with behavior control and monitoring system such as found in a typical neuroscience laboratory. An Open-source MATLAB toolbox is employed to efficiently analyze and visualize large imaging data sets in a manner that is both interactive and fully automated. This software package provides a library of image pre-processing routines optimized for batch-processing of continuous functional fluorescence video, and additionally automates a fast unsupervised ROI detection and signal extraction routine. Further, an extension of this toolbox that uses GPU programming to process streaming video, enabling the identification, segmentation and extraction of neural activity signals on-line is described in which specific algorithms improve signal specificity and image quality at the single cell level in a behaving animal. This project describes the strategic ingredients for transforming a large bulk flow of raw continuous video into proportionally informative images and knowledge.

Indiana University-Purdue University Indianapolis (IUPUI) / Non-invasive brain stimulation is valuable for studying neural circuits and treating various neurological disorders in human. However, current technologies of noninvasive brain stimulation usually have low spatial and temporal precision and poor brain penetration, which greatly limit their application. A new class of nanoparticles known as magneto-electric nanoparticles (MENs) is highly efficient in coupling an externally applied magnetics wave with generating local electric fields for neuronal activity modulation. Here, a new type of MENs was developed that consisted of CoFe2O4- BaTiO3 and had excellent magneto-electrical coupling properties. Calcium imaging technique was used to demonstrate their efficacy in evoking neuronal activity in organotyic and acute cortical slices that expressed GCaMP6 protein. For in vivo noninvasive delivery of MENs to brain, fluorescently labeled MENs were intravenously injected and attracted to pass through blood brain barrier to a targeted brain region by applying a focal magnet field. Magnetic wave (~450 G at 10 Hz) applied to mouse brain was able to activate cortical network activity, as revealed by in vivo two-photon and mesoscopic imaging of calcium signals at both cellular and global network levels. The effect was further confirmed by the increased number of c-Fos expressing cells after magnetic stimulation. Histological analysis indicated that neither brain delivery of MENs nor the subsequent magnetic stimulation caused any significant increases in the numbers of GFAP and IBA1 positive astrocytes and microglia in the brain. MENs stimulation also show high efficacy in short-term pain relieve when tested with a tibial nerve injury mouse model. The study demonstrates the feasibility of using MENs as a novel efficient and non-invasive technique of brain stimulation, which may have great potential for translation.

The hippocampus has long been known to play a role in learning and memory as well as spatial navigation. However, studies over the past several decades have shown that the hippocampus is not just a cognitive structure, but is also involved in emotional behaviors, particularly through its ventral pole. Recent experiments, from our lab and others, have revealed that ventral CA1 neural activity is strongly modulated by anxiogenic environments. Furthermore, optogenetic manipulation of ventral CA1 cell bodies and projections modifies anxiety-like behavior in the open field and elevated plus maze. However, it is still unknown if ventral CA1 represents anxiogenic stimuli through a single cell or a population code. Additionally, whether ventral CA1 encodes the moment-to-moment behavioral state changes caused by anxiogenic stimuli is unresolved. I investigate these questions using in-vivo freely moving calcium imaging in combination with neural population decoding analysis and unsupervised behavioral segmentation. My results show that ventral CA1 encodes anxiogenic stimuli through a high dimensional, distributed population code that allows for the separation of aversive stimuli with different sensory properties. I also demonstrate that ventral CA1 represents behavioral states through a low dimensional, distributed population code that generalizes across distinct contexts. Thus, ventral CA1 possesses multiple population codes that represent different kinds of information.

Movement is the key to animal behavior. From fighting off predators to reaching for food, our survival relies on movement. Losing the ability to move the body through the world in a purposeful way would be dire. We learn to perform a wide variety of actions, which require exact motor control. How are such skilled actions refined over time? The neural mechanism of motor learning has been posited to arise from integrating neuronal signals about motor commands, environmental context, and outcome through the cortico-basal ganglia-thalamic loop. Here, I investigate the role of two thalamic nuclei — the parafascicular (Pf) and ventroanterior/ventrolateral (VAL) —in the process of motor learning. In an introductory Chapter 1, I introduce some key behavioral signatures of motor learning and the distributed neural circuity for movement through the cortico-basal ganglia-thalamic network. Pf and VAL are at the center of this network. Both receive basal ganglia output but differ in primary projection patterns. Pf sends large excitatory projections directly to the striatum (the main input area of the basal ganglia), while VAL projects back to the cortex. Despite their critical place in the movement system, little is known about their changing roles in motor learning. In Chapter 2, I highlight a novel skilled forelimb joystick target task for mice; the JTT. In the JTT, head-fixed mice learn reaches to spatial targets in 2D space by moving an unrestricted joystick without visual feedback. This task allows for multiple windows of learning and refinement of various reaches in space. Over the learning of targeted reaching movements, mice increase their accuracy and individual trajectories become less variable, showing that they have learned the location of the target in space, and also refine the reaching movements. In Chapter 3, I use 2-photon calcium imaging of the forelimb-related areas of Pf and VAL to investigate how their activity changes over learning of forelimb reaching actions. Both Pf and VAL are highly engaged during movements. Neural population engagement of Pf decreases over time, suggesting a specific role early in learning. Additionally, the underlying neural dynamics of Pf and VAL shift and occupy different state spaces over learning, as shown through principal component analysis. To investigate if neural activity in Pf or VAL encodes behavioral information, we used a ridge regression model to predict the initial direction of movements from neural data. We were able to predict the initial direction from Pf activity on early training days, but not from VAL. In Chapter 4, I performed pre and post-learning lesions to Pf or VAL to investigate if they are needed for learning and/or performance of targeted reaches. Results show that Pf is needed for learning, but not the performance of accurate spatial reaches. VAL, on the other hand, does not affect the learning or performance of target reaches, but does affect the speed of movements. In a discussion-based Chapter 5, I summarize these above experiments, which suggest different roles for PF and VAL over learning of multiple targeted reaches, and reflect on future directions of my findings in the broader context of motor learning research in neuroscience. In particular, my findings highlight a novel and critical role for Pf in learning and processing directional information during early skill learning. This work demonstrates that the thalamus is an essential node of the brain networks involved in motor learning.

Stomata sind kleine Poren in der Blattoberfläche, die Pflanzen eine Anpassung ihres Wasserhaushalts an sich ändernde Umweltbedingungen ermöglichen. Die Öffnungsweite der Stomata wird durch den Turgordruck der Schließzellen bestimmt, der wiederum durch Ionenflüsse über die Membranen der Zelle reguliert wird. Ein Netzwerk von Signaltransduktionswegen sorgt dafür, dass Pflanzen die Stomabewegungen an die Umgebungsbedingungen anpassen können. Viele molekulare Komponenten dieser Signaltransduktionketten in Schließzellen von Angiospermen sind inzwischen bekannt und Calcium spielt darin als Signalmolekül eine wichtige Rolle. Weitgehend unbekannt sind dagegen die Mechanismen, die zur Erzeugung von transienten Erhöhungen der Calciumkonzentration führen. Auch die molekularen Grundlagen der Regulierung der Stomaweite in Nicht-Angiospermen-Arten sind bisher nur wenig verstanden. Um zur Aufklärung dieser Fragestellungen beizutragen, wurden in dieser Arbeit Mechanismen zur Erhöhungen der cytosolischen Calciumkonzentration sowie elektrophysiologische Eigenschaften von Schließzellen untersucht. Der Fokus lag hierbei insbesondere auf der Visualisierung cytosolischer Calciumsignale in Schließzellen. Im ersten Teil der Arbeit wurde durch die Applikation hyperpolarisierender Spannungspulse mittels TEVC (Two Electrode Voltage Clamp) gezielt eine Erhöhung der cytosolischen Calciumkonzentration in einzelnen Schließzellen von Nicotiana tabacum ausgelöst. Um die Dynamik der cytosolischen Calciumkonzentration dabei zeitlich und räumlich hoch aufgelöst zu visualisieren, wurde simultan zu den elektrophysiologischen Messungen ein Spinning-Disc-System für konfokale Aufnahmen eingesetzt. Während der Applikation hyperpolarisierender Spannungspulse wurde eine transiente Vergrößerung des cytosolischen Volumens beobachtet. Diese lässt sich durch einen osmotisch getriebenen Wasserfluss erklären, der durch die Veränderung der Ionenkonzentration im Cytosol verursacht wird. Diese wiederum wird durch die spannungsabhängige Aktivierung einwärtsgleichrichtender Kaliumkanäle in der Plasmamembran der Schließzellen und durch den Kompensationsstrom der eingestochenen Mikroelektrode hervorgerufen. Mit Hilfe des calciumsensitiven Farbstoffs Fura-2 konnte gezeigt werden, dass die Erhöhung der freien cytosolischen Calciumkonzentration während der Applikation hyperpolarisierender Spannungspulse durch zwei Mechanismen verursacht wird. Der erste Mechanismus ist die Aktivierung hyperpolarisationsaktivierter, calciumpermeabler Kanäle (HACCs) in der Plasmamembran, die schon 1998 von Grabov & Blatt beschrieben wurde. Zusätzlich zu diesem Mechanismus der Calciumfreisetzung, konnte ein zweiter bislang unbekannter Mechanismus aufgedeckt werden, bei dem Calcium aus intrazellulären Speichern in das Cytosol freigesetzt wird. Dieser Mechanismus hängt mit der oben beschriebenen Vergrößerung des cytosolischen Volumens zusammen und ist wahrscheinlich durch die Änderungen der mechanischen Spannung der Membran bzw. der Osmolarität innerhalb der Zelle bedingt. Diese könnten zu einer Aktivierung mechanosensitiver, calciumpermeabler Kanäle führen. Der zweite Teil der Arbeit beschäftigt sich mit den molekularen Grundlagen der Regulierung von Stomata in Nicht-Angiospermen. In Schließzellen von Polypodium vulgare konnten durch die Anwendung der TEVC-Technik ähnliche spannungsabhängige Ströme über die Plasmamembran gemessen werden wie in Angiospermen. Ebenso wurden durch die Applikation hyperpolarisierender Spannungspulse an Schließzellen von Polypodium und Asplenium Erhöhungen der cytosolischen Calciumkonzentration ausgelöst, die auf die Existenz spannungsabhängiger, calciumpermeabler Kanäle in der Plasmamembran hinweisen. Die Diffusion von Fluoreszenzfarbstoffen in die Nachbarschließzellen nach der iontophoretischen Beladung in Polypodium, Asplenium, Ceratopteris und Selaginella zeigte, dass in diesen Arten eine symplastische Verbindung zwischen benachbarten Schließzellen besteht, die an Schließzellen von Angiospermen bisher nicht beobachtet werden konnte. Anhand elektronenmikroskopischer Aufnahmen von Polypodium glycyrrhiza Schließzellen konnte gezeigt werden, dass diese Verbindung wahrscheinlich durch Plasmodesmata zwischen benachbarten Schließzellen gebildet wird. Durch die Analyse der Calciumdynamik in benachbarten Schließzellen nach hyperpolarisierenden Spannungspulsen stellte sich heraus, dass die Calciumhomöostase trotz symplastischer Verbindung in beiden Schließzellen unabhängig voneinander reguliert zu werden scheint. Im Rahmen der Untersuchungen an Farnschließzellen wurde desweiteren eine Methode zur Applikation von ABA etabliert, die es erlaubt mithilfe von Mikroelektroden das Phytohormon iontophoretisch in den Apoplasten zu laden. Im Gegensatz zu den Schließzellen von Nicotiana tabacum, die auf eine so durchgeführte ABA-Applikation mit dem Stomaschluss reagierten, wurde in Polypodium vulgare auf diese Weise kein Stomaschluss ausgelöst. Da die ABA-Antwort der Farnstomata aber auch von anderen Faktoren wie Wachstumsbedingungen abhängig ist (Hõrak et al., 2017), kann eine ABA-Responsivität in dieser Farnart trotzdem nicht vollkommen ausgeschlossen werden. Die Freisetzung von Calcium aus intrazellulären Speichern, wie sie in dieser Arbeit gezeigt wurde, könnte eine wichtige Rolle bei der Regulierung der Stomaweite spielen. Zur Aufklärung dieser Fragestellung wäre die Identifizierung der Kanäle, die an der osmotisch/mechanisch induzierten Calciumfreisetzung aus internen Speichern beteiligt sind, von großem Interesse. Weiterführende Studien an Schließzellen von Farnen könnten die physiologische Bedeutung der aus Angiospermen bekannten Ionenkanäle für die Stomabewegungen in evolutionär älteren Landpflanzen aufklären und so maßgeblich zum Verständnis der Evolution der Regulierunsgmechanismen von Stomata beitragen. Außerdem stellt sich die Frage, welche Rolle die hier gezeigte symplastische Verbindung der Nachbarschließzellen durch Plasmodesmata für die Funktion der Stomata spielt. / Stomata are small pores in the leaf surface that allow plants to adapt their water balance to changing environmental conditions. The turgor pressure of the guard cells determines the width of the stomatal aperture and is regulated by ion fluxes in or out of the guard cell. A network of different signal transduction pathways is necessary for the adaption of stomatal movements to ambient conditions. Many of these transduction pathways have been described in detail and many of their components have been identified. It is a well known fact that calcium acts as a second messenger in pathways regulating stomatal movements. However, the mechanisms that lead to transient elevations of the cytosolic calcium concentration are largely unknown. The molecular basis of the regulation of stomatal aperture in non-angiosperm species is also poorly understood. In order to gain new insights into these topics, mechanisms of calcium elevation and electrophysiological properties of guard cells were studied, focussing especially on the visualization of the cytosolic calcium concentration in guard cells. In the first part of this study, the application of hyperpolarizing voltage pulses by means of TEVC (Two Electrode Voltage Clamp) was used to specifically trigger an increase in the cytosolic calcium concentration in individual guard cells in the angiosperm model plant Nicotiana tabacum. To visualize the dynamics of the cytosolic calcium concentration with high temporal and spatial resolution, a spinning disc system for confocal imaging was used simultaneously with the electrophysiological recordings. During the application of hyperpolarizing voltage pulses a transient increase in cytosolic volume was observed. This increase can be explained by an osmotically driven water flux caused by changes of the cytosolic ion concentration. These in turn are caused by the voltage-dependent activation of inward rectifying potassium channels in the guard cell plasma membrane and by the compensating current from the impaled microelectrode. Using the calcium-sensitive dye Fura-2, it could be shown that two mechanisms lead to the elevation of the cytosolic calcium concentration during the application of hyperpolarizing voltage pulses. The first mechanism is the activation of hyperpolarization-activated calcium permeable channels (HACCs) in the plasma membrane, which has already been described in 1998 by Grabov & Blatt. In addition to this mechanism of calcium release, a second previously unknown mechanism was discovered in which calcium is released into the cytosol from intracellular stores. This mechanism is related to the increase in cytosolic volume we described above and is probably caused by changes in membrane tension or osmolarity within the cell. These changes could lead to an activation of mechanosensitive calciumpermeable channels. The second part of this thesis deals with the molecular basis of the regulation of stomata in non-angiosperms. In guard cells of Polypodium vulgare voltage-dependent currents across the plasma membrane similar to those described in angiosperm model plants could be measured using TEVC. Furthermore, the application of hyperpolarizing voltage pulses induced increases in cytosolic calcium concentration in guard cells of Polypodium and Asplenium indicating the existence of voltage-dependent calcium permeable channels in the plasma membrane. The diffusion of iontophoretically injected fluorescent dyes into the neighboring guard cells in Polypodium, Asplenium, Ceratopteris and Selaginella showed that in these species a symplastic connection between neighboring guard cells exists, which could not be observed in guard cells of angiosperms. Electron microscopic images of Polypodium glycyrrhiza guard cells showed that this connection is probably formed by plasmodesmata between adjacent guard cells. Analysis of the calcium dynamics in neighboring guard cells after hyperpolarizing voltage pulses revealed that calcium homeostasis seems to be regulated independently in both guard cells despite their symplastic connection. As part of the investigations on guard cells of ferns, a new method for the application of ABA was established, which allows the phytohormone to be charged iontophoretically into the apoplast with the aid of microelectrodes. In contrast to the guard cells of Nicotiana tabacum, which reacted with loss of turgor and subsequential stomatal closure to this method of ABA-application, no closure of the stomata could be induced in Polypodium vulgare in this way. However, since the ABA response of fern stomata is also dependent on other factors such as growth conditions (Hõrak et al., 2017), an ABA-responsiveness in this fern species can still not be completely excluded. The release of calcium from intracellular stores, as shown in this work, could play an important role for the regulation of stomatal aperture. To clarify this question, the identification of the channels involved in osmotically/mechanically induced calcium release from internal stores would be of great interest. Further studies on fern guard cells could clarify the physiological significance of ion channels known from angiosperms for the stomatal movements in early land plants, and thus contribute significantly to the understanding of the evolution of stomatal regulation. In addition, the question arises as to what role the symplastic connection of the neighboring guard cells through plasmodesmata plays for the function of stomata.

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.

Despite being integral to the practice of surgery, the mechanisms by which general anesthetics mediate their effects remain unknown. For this reason, it is difficult to predict adverse side-effects and to determine how treatment should be modified for specific patient populations. Recent clinical studies have reported post-operative neuropsychological and behavioral abnormalities in children and protracted periods of post-operative cognitive decline in elderly patients. Definitively linking these post-operative consequences to the agents used to induce anesthesia has been difficult, due to a lack of proper clinical controls and an abundance of confounding health factors. Animal studies, have repeatedly shown that general anesthetics can be neurotoxic and lead to lasting impairments in learning and memory acquisition in both the very young and old. However, the scope and causes of these post-exposure impairments and the reasons why age seems to measurably affect outcomes remain unclear. Here we employ multi-neuronal fluorescence imaging in the nematode Caenorhabditis elegans to measure changes in neuronal activity and connectivity across the animal’s nervous system, following exposure to the volatile anesthetic isoflurane during neurodevelopment and senescence. Employing transgenic expression of the fluorescent calcium indicator GCaMP6s, we measure neuronal activity of specific command interneurons as well as across the majority of the nervous system with single cell resolution. Isoflurane exposure during developing, results changes in the transition rate between neuronal activity states and an overall increase in excitatory connectivity. Importantly these effects are dependent on cellular stress pathways involved mTOR and daf-16 but not on apoptotic cell death (medatied by ced-3). Measuring neuronal activity across the animals lifespan, we identify substantial age-related alterations to neural activity, connectivity and functional organization of the system. These include a progressive loss of system-wide organization and a corresponding shift in individual neuron activity toward higher frequencies. We also observe a specific loss of anti-correlative (i.e. inhibitory) signaling between neurons, resulting in an overall shift in the excitatory/inhibitory balance of the system. In support of this, we find that application of the GABAA agonist muscimol diminishes certain aspects of nervous system decline in aged animals. We further identify genes that either hasten or delay the progression toward senescent neural activity patterns, including the presynaptic voltage-gated Ca2+ channel UNC-2/CaV2, and also CED-4, a key mediator of the conserved cell-death pathway. Finally, imaging post-exposure consequences of isoflurane during senescence reveals long term effects on neuronal signaling that involved a decrease in excitatory connectivity, the opposite of what is observed during development. We conclude that anesthetic exposure during development cause permanent alteration in neuronal activity and signaling which involves cellular stress pathways but that these effects are distinct from long-term effect of anesthetic exposure we observe in age animals. Our studies also begin to define the changes in neuronal dynamics with age and demonstrate the importance of excitatory/inhibitory balance in this processes. Through comprehensive multi-neuronal imaging in C. elegans, we are able to measure the progressive breakdown of neuronal activity and system dynamics with age and isoflurane exposure and begin to identify the cellular processes and changes in synaptic signaling that contribute to these declines. Moreover, we leverage this platform to gain insight into the age-dependency of isoflurane-induced insult to neural systems.