The medial temporal lobe (MTL) is crucial for episodic and spatial memory, and shows rhythmicity in the local field potential and neuronal spiking. Gamma oscillations (>40Hz) are mediatepd by local circuitry and interact with slower theta oscillations (6-10 Hz). Both oscillation frequencies are modulated by cholinergic input from the medial septum. Entorhinal grid cells fire when an animal visits particular locations in the environment arranged on the corners of tightly packed, equilateral triangles. Grid cells show phase precession, in which neurons fire at progressively earlier phases relative to theta oscillation as animals move through firing fields. This work focuses on the temporal organization of spiking and network rhythms, and their modulation by septal inputs, which are thought to be involved in MTL function. First, I recorded grid cells as rats explored open spaces and examined precession, previously only characterized on linear tracks, and compared it to predictions from models. I identified precession, including in conjunctive head-direction-by-grid cells and on passes that clipped the edge of the firing field. Secondly, I studied problems of measuring single neuron theta rhythmicity and developed an improved approach. Using the novel approach, I identified diverse modulation of rat medial entorhinal neurons’ rhythmic frequencies by running speed, independent from the modulation of firing rate by speed. Under pharmacological inactivation of the septum, rhythmic tuning was disrupted while rate tuning was enhanced. The approach also showed that available data is insufficient to prove that bat grid cells are arrhythmic due to low firing rates. In the final project, I optogenetically silenced cholinergic septal cells while recording from hippocampal area CA1. I identified changes in theta rhythmic currents and in theta-gamma coupling. This silencing disrupted performance when applied during the encoding phase of a delayed match to position task. These data support hypothetical roles of these rhythms in encoding and retrieval and suggest possible mechanisms for their modulation. Together, evidence from these projects suggests a role for theta in the function of spatial and episodic memory. These oscillations have important implications for communication and computation, and they can provide a substrate for efficient brain function.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第22312号 / 医博第4553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 伊佐 正, 教授 林 康紀, 教授 高橋 淳 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The medial temporal lobe supports the encoding of new facts and experiences, and organizes them so that we can infer relationships and make unique associations during new encounters. Evidence from studies on humans and animals suggest that the hippocampus is specifically required for our ability to form these internal representations of the world. The mechanism by which the hippocampus performs this function remains unclear, but electrophysiological recordings in the hippocampus support a general model. One component of this model suggests that the cortex represents places, times, and events separately, and then the hippocampus generates conjunctive representations that connect the three. According to this hypothesis, the hippocampus binds places and events to an existing relational structure. This dissertation explores how item and place associations develop within cortex, and then examines the relational structure that organizes these events within the hippocampus. The first study suggests that contrary to previous models, events and places are bound together outside of the hippocampus in the entorhinal cortex and perirhinal cortex. The second study shows that this relational scaffold may be embodied by a continually changing code that permits both the association and separation of information across the continuum of time. The final study suggests that the hippocampus and entorhinal cortex contain qualitatively different time codes that may act in a complementary fashion to bind events and places and relate them across time. Overall, these studies support a theory wherein time is encoded in a range of brain regions that also contain conjunctive item and position information. In these regions, conjunctive representations of items, places, and times are organized not only by their perceptual similarity but also their temporal proximity.

Computational models combined with electrophysiological studies have informed our understanding about the role of hippocampal subfields (dentate gyrus, DG; CA subfields, subiculum) and Medial Temporal Lobe (MTL) cortex (entorhinal, perirhinal, parahippocampal cortices) during working memory (WM) tasks. Only recently have functional neuroimaging studies begun to examine under which conditions the MTL are recruited for WM processing in humans, but subfield contributions have not been examined in the WM context. High-resolution fMRI is well suited to test hypotheses regarding the recruitment of MTL subregions and hippocampal subfields. This dissertation describes three experiments using high-resolution fMRI to examine the role of hippocampal subfields and MTL structures in humans during WM. Experiment 1 investigated MTL activity when participants performed a task that required encoding and maintaining overlapping and non-overlapping stimulus pairs during WM. During encoding, activity in CA3/DG and CA1 was greater for stimulus pairs with overlapping features. During delay, activity in CA1 and entorhinal cortex was greater for overlapping stimuli. These results indicate that CA3/DG and CA1 support disambiguating overlapping representations while CA1 and entorhinal cortex maintain these overlapping items. Experiment 2 investigated MTL activity when participants performed a WM task that required encoding and maintaining either low or high WM loads. The results show a load effect in entorhinal and perirhinal cortex during the delay period and suggest that these regions act as a buffer for WM by actively maintaining novel information in a capacity-dependent manner. Experiment 3 investigated MTL activity when participants performed a WM task that required maintaining similar and dissimilar items at different loads. Analysis of a load by similarity interaction effect revealed areas of activity localized to the CA1 subfield. CA1 showed greater activity for higher WM loads for dissimilar, but not similar stimuli. Our findings help identify hippocampal and MTL regions that contribute to disambiguation in a WM context and regions that are active in a capacity-dependent manner which may support long-term memory formation. These results help inform our understanding of the contributions of hippocampal subfields and MTL subregions during WM and help translate findings from animal work to the cognitive domain of WM in humans.

Layer II neurons of the entorhinal cortex (ECII) are selectively vulnerable to Alzheimer’s disease (AD). Investigations into the molecular mechanisms of this ECII vulnerability provide unique opportunities to better understanding the pathology of AD. Preliminary data has suggested teneurin-3 (Tenm3) to have a role in this vulnerability due to its ECII enrichment, genetic variants associated with AD, and altered electrophysiology in Tenm3-knockout (KO) mice. In this study, the impacts of Tenm3- KO in mice were further explored. Electron tomography and immunofluorescent confocal microscopy were utilized to compare wild-type (WT) and KO mice’s perforant pathway synaptic densities and structures. A slight trend was found for increased synaptic density in Tenm3-KO mice. The structural changes in Tenm3-KO mice were more pronounced and encompassed alterations to active zones, bouton volumes, and synaptic vesicle pools. Overall, this work suggests Tenm3’s involvement in structural remodeling of both axonal boutons and dendritic spines thus providing a hypothesis for its role in ECII’s selective vulnerability to AD.

Les réseaux neuronaux, pendant leur période de développement, génèrent des patrons d’activité immatures qui sont supposés participer à la formation des circuits neuronaux. Ces activités synchronisées créent des conditions favorables pour la plasticité hebbienne qui soutient la formation des circuits locaux. Les travaux menés notamment sur les systèmes sensoriels ont montré que les circuits pauci-neuronaux locaux sont capables de présenter une activité synchrone tout en étant isolés du reste des structures cérébrales. La moelle épinière isolée produit des bursts qui sont à l’origine des secousses musculaires, la rétine insensible à la lumière génère des ondes d’activité, d’autres régions cérébrales génèrent des activités synchrones avant de remplir la fonction à laquelle ils sont destinés. De manière similaire, l’hippocampe du rat nouveau-né ou primate prématuré in vitro, ainsi que les néocortex immature in vitro, génèrent une activité neuronale synchronisée, appelée giant depolarising potentials (GDPs). En se basant uniquement sur ces études et en prenant en compte la maturation tardive de certaines projections neuronales à distance, il serait tentant de conclure que le cerveau immature fonctionne comme un ensemble de petits modules fonctionnels qui auto-entretiennent leur activité intrinsèque avant de se connecter entre eux pour créer un cerveau fonctionnel adulte. Cependant, certaines connexions à longue distance sont formées très tôt pendant le développement et permettent la propagation des oscillations immatures entre les structures connectées. En effet, les ondes rétinales se propagent au noyau géniculé latéral et ensuite jusqu’au cortex visuel ; les GDPs hippocampiques se propagent à l’hippocampe controlatéral, septum et cortex entorhinal et finalement, les secousses musculaires, en créant un feed-back sensoriel, déclenchent des oscillations gamma immatures ainsi que les spindle bursts dans le réseau thalamo-cortical. Un fonctionnement similaire est décrit chez le nouveau-né prématuré. Il paraît donc plus probable, que le cerveau soit, dès les stades précoces du développement, organisé en sous-systèmes fonctionnels reliés entre eux anatomiquement et fonctionnellement. Au sein des unités fonctionnelles sont générés des patrons d’activité immatures synchrones afin de créer des connexions organisées topographiquement qui serviront de base anatomique de la fonction finale. Si ces étapes développementales sont perturbées pendant les périodes critiques, le système ne pourra pas assurer sa fonction de manière adéquate au stade mature. L’hippocampe mature, ou plus exactement les circuits cortico-hippocampiques, jouant un rôle primordial dans la mémoire déclarative, l’orientation spatiale et l’inhibition du comportement. L’établissement de ces fonctions est progressif au cours du développement. Par exemple les adultes humains n’ont que rarement des souvenirs personnels datant avant l’âge de trois ans. Or, nous savons aujourd'hui que le bébé humain est capable de garder des souvenirs dans la mémoire déclarative (dépendante de l’hippocampe) au cours de la première année de vie avec une efficacité croissante, mais il ne se rappellera pas ces souvenirs à l’âge adulte (Bauer, 2006). Nous ne savons pas s’il s’agit d’un encodage différent d’emblée ou d’un processus secondaire supprimant l’accès à ces souvenirs précoces. Nous pouvons présumer qu’il existe des modifications des activités électrophysiologiques pendant le développement qui soutiennent la modification de ces fonctions. Au cours de ce travail de thèse, nous voulions savoir comment et à partir de quand l’hippocampe, qui reçoit des informations convergentes de nombreuses régions néocorticales, acquiert son mode de fonctionnement adulte. Afin de répondre à cette question nous avons étudié le système cortex entorhinal – hippocampe, le cortex entorhinal étant la principale entrée excitatrice de l’hippocampe et recevant des afférences de nombreuses régions du néocortex. (...) / Neuronal networks spontaneously generate “immature” patterns of activity during development, which are thought to participate on the formation of neural circuits. Local neocortical as well as hippocampal circuits generate synchronised neuronal discharges providing support for Hebbian plasticity. Studies of sensory systems showed that local pauci-neuronal circuits were able to generate synchronous activity while isolated from other brain structures. Isolated spinal cord produces bursts evoking muscle twitching, light insensitive retina generates waves of activity, as well as other brain regions generate synchronous activities before fulfilling the function for which they are intended. Similarly, the hippocampus of newborn rat or premature primate in vitro, as well as immature neocortex in vitro, generates synchronised neuronal activity called giant depolarising potentials (GDPs). Based solely on these studies and taking into account the delayed maturation of certain long-distance neuronal projections, it would be tempting to conclude that the immature brain functions as a set of small functional modules that self-maintain their intrinsic activity before connecting together to create a functional adult brain. However, some long-distance connections are formed very early during development and allow the propagation of oscillations between immature connected structures. Indeed, retinal waves propagate to the lateral geniculate nucleus and then to the visual cortex, hippocampal GDPs propagate to the contralateral hippocampus, septum and entorhinal cortex, and finally, twitching, creating a sensory feedback, triggers immature gamma oscillations and spindle bursts in the thalamo-cortical network. A similar functioning is described in the premature newborn. It therefore seems more likely that the brain is, during the early stages of development, organised into functional subsystems interconnected anatomically and functionally. Within functional units are generated immature patterns of synchronous activity to create topographically organised connections that serve as anatomical basis of the final function. If these developmental stages are disturbed during critical periods, the system cannot perform its function adequately in mature stage. The mature hippocampus, or more precisely the cortico-hippocampal circuits, plays a key role in declarative memory, spatial organisation and behavioural inhibition. The establishment of these functions is progressive during development. For example, human adults rarely have personal memories dating before the age of three years. However, we now know that the human baby is able to keep memories in declarative memory (hippocampus-dependent) during the first year of life with increasing efficiency, but will not remember them in the adulthood. We do not know if the encoding of the memories is different or a secondary process inhibits the access to the early memories. We can assume that changes in electrophysiological activity during development support modification of these functions. In this thesis, we wanted to know how and from when the hippocampus, which receives convergent information from many cortical areas, acquires his adult mode of functioning. To answer this question we studied the entorhinal cortex-hippocampus system, entorhinal cortex being the main excitatory input to the hippocampus and receiving afferents from many parts of the neocortex. We were able to distinguish several periods in the development of the immature hippocampus: Period from P1 till P12 characterised by the sole presence of immature sharp waves triggered by the entorhinal cortex. Period from P13, when two types of sharp waves coexisted: the immature sharp waves and sharp waves as described in the adult animals newly emerged. The mature sharp waves, unlike the immature, can be accompanied by ripples. (...)

Die Neurone der Hirnrinde sind in Mikroschaltkreisen, Modulen und Arealen organisiert. In dieser Doktorarbeit habe ich die Neurobiologie und Hirnrindenstruktur der Etruskerspitzmaus - ein neues Modelltier für neurobiologische Forschung - und die modulare Struktur des entorhinalen Kortex der Ratte untersucht. Die geringe Größe des Gehirns der Etruskerspitzmaus bietet besondere Vorteile für das Verständnis kortikaler Aktivität von Zellgruppen. Die entorhinale Kortex enthält sowohl gut definierte funktionelle als auch anatomische Module und bietet daher eine einzigartige Gelegenheit für das Studium ihrer Wechselbeziehungen. Die Organisation der Hirnrinde der Etruskerspitzmaus reflektiert die Spezialisierung als schnelle, auf taktile Reize spezialisierte Jäger. Mehrere kortikale Regionen, die ein Drittel des kortikalen Volumens ausmachen, reagieren auf taktile Reize. Eine kortikale Hemisphäre enthält nur etwa eine Million Neuronen. Basierend auf der Zellarchitektur und histochemischen Färbungen haben wir 13 kortikale Regionen definiert - eine große Zahl angesichts der geringen Größe des Spitzmausgehirns. Pyramidenzellnester in Schicht 2 des medialen entorhinalen Kortex sind einfach zu identifizieren und eignen sich als Bezugssystem für die verschiedenen modulären Strukturen dieser Hirnregion. Diese Pyramidenzellen bündeln ihre Dendriten hin zu einem Punkt, der sich mit erhöhten Konzentrationen von präsynaptischen cholinergen Markern überschneidet. Cholinerge Transmission ist ein wichtiger Bestandteil des Theta-Rhythmus und unsere Ergebnisse zeigen, daß Pyramidenzellen im Vergleich zu Sternzellen doppelt so stark Theta-moduliert sind. Da fast alle Gitterzellen stark Theta-moduliert sind, ist anzunehmen dass Pyramidenzellen eine wichtige Rolle für die räumliche Navigation spielen. In dieser Arbeit wurden an der Hirnrinde der Etruskerspitzmaus sowie der entorhinalen Hirnrinde der Ratte modellhaft Struktur-Funktions-Beziehungen in der Großhirnrinde aufgeklärt. / Neurons of the cerebral cortex are collectively organized into microcircuits, modules and cortical areas. Here, I study the neurobiology and cortical structure of the Etruscan shrew - a new model animal for neurobiological research - and the modular structure of the entorhinal cortex of the rat. The small size of the Etruscan shrew''s brain offers particular advantages for understanding cortical activity at the multi-cell level, due to its small number of cortical neurons and its intrinsic advantages for optical imaging approaches. The entorhinal cortex contains well-defined functional and anatomical modules and provides a unique opportunity for studying their interrelation. The organization of the cerebral cortex of the Etruscan shrew reflects their behavioral specialization as fast touch-and-kill hunters. Several cortical areas comprising a third of the cortical volume respond to vibrissal touch. One cortical hemisphere contains only about 1 million neurons. Cytoarchitecture and histochemical staining revealed 13 cortical regions - a large number considering the small size of the shrew''s brain. Pyramidal cell clusters in layer 2 of medial entorhinal are reliably identifiable and thus provide common anatomical framework for entorhinal cortex modularity. These cells form geometrically arranged clusters and bundle their dendrites towards a common point overlapping with presynaptic markers of cholinergic inputs. Cholinergic drive is an important component of theta-rhythmicity which we found to be two-fold stronger in pyramidal than in stellate neurons. Since nearly all grid cells are strongly theta modulated, we suggest that pyramidal cells may play an important role in microcircuits for spatial navigation. In this work, we studied the areal architecture of the Etruscan shrew cortex and the modular architecture of the rat medial entorhinal cortex as contributions towards understanding structure-function relations in the cerebral cortex.

As both the major input and output of the hippocampal formation, the entorhinal cortex (EC) occupies a pivotal position in the medial temporal lobe. The discovery of grid cells in the medial entorhinal cortex (mEC) has led to this region being widely implicated in spatial information processing. Importantly, the EC is also the first area affected by dementia pathology, with neurons appearing particularly susceptible to degeneration. Despite this, little is known about how pathology affects the functional output of mEC neurons, either in their ability to coordinate firing to produce network oscillations, or to represent information regarding the external environment. This thesis will use electrophysiological techniques to examine how dementia pathology contributes to the breakdown of mEC neuronal networks using the rTg4510 mouse model of tauopathy. The first 2 results chapters will show how the anatomical organisation along the dorso-ventral axis of the mEC has profound influence on the network activity that can be observed both in brain slices and awake-behaving mice. It will further show how deficits in network activity in rTg4510 mice occur differentially across this axis, with dorsal mEC appearing more vulnerable to changes in oscillatory function than ventral. The third results chapter will begin to explore the relationship between global network activity and the external environment, showing that rTg4510 mice display clear deficits in the relationship between oscillation properties and locomotor activity. Finally, the underlying basis for these changes will be examined, through the recording of single-unit activity in these mice. It will show a decreased tendency for mEC neurons to display firing rates modulated by running speed, as well as an almost complete breakdown of grid cell periodicity after periods of tau overexpression. Understanding how dementia pathology produces changes to neuronal function and ultimately cognition is key for understanding and treating the disease. This thesis will therefore provide novel insights into the dysfunction of the EC during dementia pathology.

Spatial memory and navigation relies on estimation of location. This can be achieved through several strategies, including the use of landmarks and by path integration. The latter involves inferring location from direction and distance moved relative to a known start point. The neural mechanisms of path integration are not well understood and implementation of experiments that dissociate path integration from alternative strategies is challenging. The roles of specific cell types are also unknown. Although grid cells in layer 2 of the medial entorhinal cortex (MEC) are theorised to be involved given their periodic and repeating firing fields that form a grid-like map that tiles the environment. Two excitatory cell populations have been identified in layer 2 of the MEC. Clusters of pyramidal cells that project to the CA1 are surrounded by dentate gyrus (DG) projecting stellate cells. Both populations have been shown to exhibit grid-like activity. The extent to which these cell types contribute to path integration or other strategies for solving spatial tasks is unknown. To investigate these issues, I developed a spatial memory task for mice, which uses virtual reality to generate sensitive measures of an animal’s ability to path integrate. In this task mice are trained to locate a reward zone marked with a visual cue within a virtual linear track. Use of path integration strategies can be tested in trials in which the reward zone is unmarked. In this task mice can locate the reward zone using either a local beaconing cue or path integration strategies. To assess whether self-motion derived motor information or visual feedback is used for path integration, I manipulated the translation between physical and virtual movement, putting optic and motor feedback in conflict. These manipulations suggest that mice use motor information to locate the reward zone on path integration trials. To test roles of stellate cells in the task I injected adeno-associated virus expressing the light chain of tetanus toxin, conditionally on the presence of Cre, into the MEC of mice expressing Cre specifically in stellate cells. This abolishes synaptic output from stellate cells therefore preventing them from influencing downstream neurons. I find mice with dorsal expression of the tetanus toxin virus in layer 2 stellate cells are unable to locate the reward zone using a local beaconing cue or path integration strategies. In contrast, mice with expression of green fluorescent protein (GFP) were able to locate the reward zone using both strategies. Locating the reward zone using path integration strategies first requires animal’s to learn the reward zone location, as denoted in trials with a beacon cue. To distinguish the role of stellate cells in learning versus execution of the tasks, I temporally modified the activity of stellate cells after mice had learnt to locate the reward zone using both strategies. Temporal control was achieved by use of cre-dependent adeno-associated viruses expressing mutant human muscarinic 4 receptor (hM4). When activated by clozapine - N - oxide (CNO), this receptor opens G-protein inwardly rectifying potassium (GIRK) channels and attenuates neuronal firing. Using this method, the activity of stellate cells can be temporally controlled during task execution and potentially distinguish their involvement in learning and execution of spatial memory tasks. No effect on behavioural performance was seen under these conditions. This may indicate stellate cells are required for learning but not execution of spatial memory tasks that require the use of local beaconing cues or path integration.

Place cells in the hippocampus fire in specific locations within an environment. The aim of this thesis is to investigate the different inputs to the hippocampus and what they contribute to place cell activity and performance of hippocampus-dependent tasks. Place cell activity can also be modulated by relevant features of a task such as a future destination or trajectory. Initial experiments investigated the origin and function of this trajectory-dependent activity and later experiments targeted the medial entorhinal cortex inputs to the hippocampal formation and investigated what they contributed to place cell activity and behaviour. The purpose of the first study was to determine whether trajectory dependent activity occurs in CA3 in a hippocampus-dependent serial-reversal task on the double-Y-maze and to compare it with that seen in CA1. Place cells in both CA3 and CA1 were recorded in rats trained on a serial-reversal task on a double-Y-maze. Rats were trained to run from a start box through two Y-junctions to one of four goal locations. After 10 trials the reward was moved to a new location, until all the boxes had been rewarded. Previous research has found that 44% of CA1 place cells with fields in the start areas of the maze show trajectory-dependent activity in rats trained on the task. This study found that a similar proportion of CA3 place cells also show trajectory-dependent activity in rats trained on this task and that this activity develops at the same time point as the task is learned. This result suggests that trajectory-dependent activity may be generated earlier in the circuit than CA1. Secondly, the contribution of the nucleus reuniens (N.Re) to spatial tasks was investigated. Previously, trajectory-dependent activity has been found to reach the hippocampus via N.Re, however this was shown in a hippocampus-independent task. To investigate the possible role that this input may play in behaviour, N.Re was lesioned and animals were tested on acquisition and performance of the double-Y-maze serial-reversal task described previously. Surprisingly, lesions had no effects on either learning or performance. Taken together with previous data from other studies, this suggests that trajectory dependent activity is not one unique phenomenon but is rather multiple similar phenomena which may originate in different brain regions and fulfil different roles in navigation depending on the demands of the task. In addition, animals were tested on tasks involving allocentric or egocentric navigation. Results suggest that N.Re may have a role in the selection or performance of allocentric navigation but not egocentric navigation. Thirdly, the role of inputs from the medial entorhinal cortex (MEC) to place cells was investigated. Consistent with previous research, MEC lesions resulted in larger, less precise place fields in CA1 place cells. By performing cue-rotation experiments using either distal or proximal cues it was observed that place fields in the MEC lesion animals were not anchored to distal cues but were either stable or anchored to other aspects of the environment. However, place cells in the MEC lesion group still followed proximal cues suggesting that the deficit is restricted to distal landmarks. This suggests that the MEC may process distal landmark information allowing the use of distal landmarks for orientation and self-location within an environment. This thesis contributes a better understanding of the role and origins of trajectory dependent activity as well as a novel finding that the MEC contributes information about distal landmarks to the hippocampus.