Ageing is a process correlated with cellular stress and increased risks of neurodegenerative diseases, in particular Alzheimer’s disease (AD), which is accompanied with severe cognitive and memory impairments. Both ageing and AD affect many brain regions and thus induce brain malfunctions. Among the brain regions, the entorhinal cortex (EC) has drawn more and more attentions due to its pivotal role in cognition and memory functions as well as its vulnerability to ageing process and AD neuropathology. Synaptic and neuronal degenerations, which are also manifest features of AD, occur in the EC during the ageing process and at the early stage of AD. In addition, both pathological hallmarks of AD, namely abnormal accumulation of β-amyloid (Aβ) and hyperphosphorlation of tau proteins, initially appear in the EC and then progress to other brain regions such as the hippocampus and the neocortex. Glial alterations in AD and ageing process have been considered as secondary event to neuronal changes. Nevertheless, accumulating evidence indicates the relevant and primary involvement of astroglia, which is responsible for brain homeostasis, in AD and ageing. In this thesis, we have focused on the astroglial alterations in the EC during the progression of AD in an animal model of the disease as well as in ageing process in non-transgenic control mice. We have used the triple transgenic mouse model of AD (3xTg-AD), which is the most relevant animal model of AD and resembles the spatiotemporal progression of human AD pathology. Our results revealed cytoskeletal atrophy of astrocytes in the EC of 3xTg-AD animals (Chapter 3), shown by significant decrease in GFAP surface and volume. This astroglial alteration began at very early age (1 month) and sustained till more advanced age (12 month). Moreover, Aβ plaques did not trigger astrogliosis, and there was rare presence of GFAP labelled astrocytes in the vicinity of Aβ deposition. This may reflect the relative indifference of astroglia in the EC and thus explain the susceptibility of the EC at the early stage of AD. To study whether astroglial atrophy in cytoskeleton compromise astrocytic function in glutamate homeostasis, we investigated the expression of glutamine synthetase (GS), which is specifically expressed in astrocytes and is critical for glutamate balance (Chapter 4). Our results showed constant GS expression and the density of GS positive astrocytes in the EC. However, dual labelling of GS and GFAP revealed 3 different subsets of astrocytes, being GS-, GFAP-, GS/GFAP- positive astrocytes. The morphology of GS-IR cells, measured by surface and volume, did not change in spite of the evident GFAP atrophy. Therefore, GFAP atrophy does not disturb glutamate homeostasis in the EC, suggesting diverse functional populations of astrocytes, which may show distinct responses during AD progression. In addition we also analysed astroglial changes during the ageing process in the EC and its major projection area, the hippocampus (Chapter 5). Astrocytes in the hippocampus exhibited prominent hypertrophy, shown by increased GFAP whereas entorhinal astrocytes in the EC had profound reduction in GFAP expression. This may implicate heterogeneous astrocytic responses to ageing in different brain regions. The general atrophy of astrocytes in the EC of 3xTg-AD mice and aged controls, suggests astroglial atrophy may results in reduced astrocytic coverage and modulation of synapses, accounting for the synaptic dysfunction in ageing and AD.

No description available.

This thesis explores the role of path integration on the firing of hippocampal place cells and medial entorhinal grid cells. Grid cells fire at equidistant locations in an environment, indicating that they keep track of the distance and direction an animal has moved in an environment. One class of model of path integration uses a continuous attractor network to update position information. The first part of this thesis showed that such a network can generate a "look-ahead" of neural activity that sweeps through the positions just visited and about to be visited, on the short time scale that is observed<italic>in vivo</italic>. Adding intrinsic currents to the neurons in the network model allowed this look-ahead to recur every theta cycle, and generate grid fields of a size comparable to data. Grid cells are a major input the hippocampus, and are hypothesized to be the source of the place specificity of place cells. When an animal explores an open environment, place cells are active in a particular location regardless of the direction in which the animal travels through it. While performing a specific task, such as visiting specific locations in the environment in sequence, however, most place cells are active only in one direction. The second part of this thesis studied the development of this directionality. It was determined that upon the initial appearance of place fields in a novel environment, place cells fired in all directions, supporting the hypothesis that the path integration is the primary determinant of place specificity. The directionality of place fields developed gradually, possibly as a result of learning. Ideas about how this directionality could develop are explored.

Kindling involves the progressive development of epileptiform activity that culminates in generalized seizures in response to repeated electrical stimulation of the brain. Kindling induces widespread changes in synaptic sensitivity and neuronal reactivity. These neuroplastic changes are evident in altered memory and behavior. This research was designed to further our understanding of kindling-induced deficits in spatial cognition. Two questions were examined: 1)does entorhinal cortex kindling disrupt spatial cognition; and 2)can bilateral bifocal kindling, of two brain regions known to participate in spatial cognition, produce larger cognitive deficits than unifocal kindling? This research attempted to confirm the spatial cognitive effects produced by unifocal dorsal hippocampal (dHPC) kindling, as a positive control. In contrast, the spatial cognitive effects produce by unifocal entorhinal cortex (EC) and bifocal kindling (i.e., EC kindling with subsequent contralateral dHPC kindling) are unknown and were examined here. Rats were subjected to unifocal EC kindling, unifocal dHPC kindling, or bifocal kindling. Rats exhibited fully generalized seizures prior to Morris water maze training from days 2 to 31. Visible platform trials were used to examine escape motivation and gross motor coordination, and all groups performed adequately. Consistent with previous research, dHPC kindling disrupted performance during acquisition trials; however, EC and bifocal kindling failed to disrupt acquisition. During retention trials, the bifocal kindling group displayed a disruption in performance; however, dHPC and lateral EC kindling failed to affect retention. The bifocal kindled group failed to display larger deficits than the unifocal kindled groups. These data suggest that the number of kindling stimulations given to a particular site may play a critical role in site-dependent disruption of memory.

Kindling involves the progressive development of epileptiform activity that culminates in generalized seizures in response to repeated electrical stimulation of the brain. Kindling induces widespread changes in synaptic sensitivity and neuronal reactivity. These neuroplastic changes are evident in altered memory and behavior. This research was designed to further our understanding of kindling-induced deficits in spatial cognition. Two questions were examined: 1)does entorhinal cortex kindling disrupt spatial cognition; and 2)can bilateral bifocal kindling, of two brain regions known to participate in spatial cognition, produce larger cognitive deficits than unifocal kindling? This research attempted to confirm the spatial cognitive effects produced by unifocal dorsal hippocampal (dHPC) kindling, as a positive control. In contrast, the spatial cognitive effects produce by unifocal entorhinal cortex (EC) and bifocal kindling (i.e., EC kindling with subsequent contralateral dHPC kindling) are unknown and were examined here. Rats were subjected to unifocal EC kindling, unifocal dHPC kindling, or bifocal kindling. Rats exhibited fully generalized seizures prior to Morris water maze training from days 2 to 31. Visible platform trials were used to examine escape motivation and gross motor coordination, and all groups performed adequately. Consistent with previous research, dHPC kindling disrupted performance during acquisition trials; however, EC and bifocal kindling failed to disrupt acquisition. During retention trials, the bifocal kindling group displayed a disruption in performance; however, dHPC and lateral EC kindling failed to affect retention. The bifocal kindled group failed to display larger deficits than the unifocal kindled groups. These data suggest that the number of kindling stimulations given to a particular site may play a critical role in site-dependent disruption of memory.

No description available.

Our ability to recall information and events is astounding and dependent on the medial temporal lobe (MTL) memory system. The synaptic interconnections between the neocortex (nCTX), entorhinal cortex (EC), and hippocampus (HPC) are the anatomical basis of this memory system. The electrophysiological basis of memory formation in this system is largely unknown, but the activity patterns that occur during slow wave sleep (SWS) are thought to play an important role. One prominent activity pattern that occurs during SWS is the slow oscillation (SO). It is a large-amplitude rhythm of ~1Hz that was first described in the nCTX and only occurs during SWS and deep anaesthesia. Using the urethane-anaesthetised rat, I provide the first description of the SO in the HPC in Chapter 2. I found that the SO in the HPC was dynamically coordinated with that in the nCTX. Because the EC is the anatomical interface between the nCTX and HPC, I hypothesised that it could be responsible for this coordination. Chapter 3 characterises the SO in the EC and its coordination with both the nCTX and HPC. My results suggested that the synaptic interconnections between the nCTX and HPC via the EC were not solely responsible for SO coordination across these structures. Another possibility is that SO coordination across the nCTX, EC, and HPC occurs via the nucleus reuniens thalami (NReu). In Chapter 4, I delivered trains of electrical stimulation to the frontal cortex (fCTX) to enhance the SO in the nCTX and assess any effect in the HPC. In addition, I delivered the same stimulation trains directly to the medial prefrontal cortex (mpfCTX) and NReu. I found that repeated stimulation in each structure entrained the hippocampal SO. I also found that repeated stimulation of the fCTX and mpfCTX enhanced SO coordination across the nCTX and HPC, but repeated stimulation of the NReu did not. My results suggested that SO coordination across the nCTX and HPC occurs via both the EC and NReu. Understanding the coordination of SO activity across these structures will provide insight to the electrophysiological basis of the MTL memory system and the role of SWS in its function.

Memory formation and recall depend on a complex circuit that includes the hippocampus and associated cortical regions. The goal of this thesis was to understand how two of the cortical connections, the medial entorhinal cortex (MEC) and medial prefrontal cortex (mPFC), influence spatial and nonspatial activity in the hippocampus. Cells in the MEC exhibit prominent spatially selective activity and have been hypothesized to drive place representation in the hippocampus. In Experiment 1 the MEC was transiently inactivated using the inhibitory opsin ArchaerhodopsinT (ArchT), and simultaneous recordings from CA1 were made as rats ran on an elliptical track. In response to MEC disruption some cells in the hippocampus shifted the preferred location of activity, some changed firing rate and others were unaffected. The new representation that developed following MEC disruption remained stable despite the fact that inhibition was transient. If the MEC is the source of spatial activity in the hippocampus the activity would be either time-locked to periods of inhibition or unstable throughout the period of inconsistent input. These results show that the MEC guides spatial representation in the hippocampus but does not directly drive spatial firing. The mPFC is generally thought to guide behavior in response to contextual elements. Experiment 2 examined the interaction between the mPFC and the hippocampus as rats performed a contextual discrimination task. Recordings were made in CA1, and the mPFC was disrupted using ArchT during the odor sampling phase of the discrimination. As animals perform this task neurons in the hippocampus respond to a conjunction of odor and location which indicates an association of what and where information in the hippocampus. Optogenetic disruption of the mPFC led to a decrease in nonspatial representation. Individual cells showed lower levels of odor selectivity, but there was no change in the level of spatial representation. This indicates that the mPFC is important for determining how the hippocampus represents nonspatial information but does not alter the spatial representation. The results are discussed within a model of memory formation that includes binding spatial and nonspatial information in the hippocampus.

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