In mammals, spatial learning and memory depend on neural processing carried out in the hippocampal formation. Interestingly, extracellular recordings from behaving animals have shown that cells in this region exhibit spatially modulated activity patterns, thus providing insights into the neural activity underlying spatial behaviour. One area within the hippocampal formation, layer II of the medial entorhinal cortex, houses cells that encode a grid-like map of space using a firing rate code. At the same time, oscillatory signals at distinct theta (4–12 Hz) and gamma (30–120 Hz) frequencies are also present in layer II, providing a substrate for a timing code. To understand how layer II of the medial entorhinal cortex produces these outputs I sought to characterise the electrical properties and functional computational architecture of its microcircuitry. The functionality of any neural circuit depends on the electrical properties of its constituent cells. Because the grid cells in layer II are likely to be stellate cells, I used the perforated patch-clamp technique to accurately assess the intrinsic excitable properties of this cell type. Compared to whole-cell recordings, these recordings indicate that some intrinsic properties of stellate cells, such as spike clustering, which is revealed to be robust, are more likely to play a functional role in circuit computation. Conversely, other intrinsic properties, such as spontaneous membrane potential fluctuations, which are confirmed to be insufficiently stable to support reliable interference patterns, are revealed to be less likely than other, more robust electrical properties to play a direct role in circuit function. The characteristic connectivity profiles of different cell types are also critical for circuit function. To investigate cell type-specific connectivity in layer II I used optogenetic stimulation in combination with in vitro electrophysiology to record synaptic activity in different cell types while selectively activating distinct subpopulations of cells with light. Using this method I found that connections between stellate cells are absent or very rare and that communication between stellate cells is instead mediated by strong feedback inhibition from fast-spiking interneurons. Dissecting oscillatory activity in neural circuits may be important for establishing functionally relevant circuit architecture and dynamics but is difficult in vivo. I accomplished this in vitro by recapitulating the interacting theta and gamma rhythms that are observed in vivo with an optogenetic method. I found that locally driving a subset of neurons in the layer II microcircuit at theta frequency with a light stimiulus produced a nested field rhythm at gamma frequency that was also evident as rhythmic inhibition onto stellate cells. Critically, these interacting rhythms closely resembled those recorded from behaving animals. In addition, I found that this thetanested gamma is sufficiently regular to act as a clock-like reference signal, indicating its potential role in implementing a timing code. To functionally dissect the circuit I performed multiple simultaneous whole-cell patch-clamp recordings during circuit activation. These recordings revealed how feedback interactions between stellate cells and fast-spiking interneurons underpin the theta-nested gamma rhythm. Together, these results suggest that feedback inhibition in layer II acts as a common substrate for theta-nested gamma oscillations and possibly also grid firing fields, thereby providing a framework for understanding how computations are carried out in layer II of the medial entorhinal cortex.

The hippocampus is crucial for episodic memory and certain forms of spatial navigation. Firing activity of hippocampal principal neurons contains environmental information, including the presence of specific objects, as well as the animal’s spatial and temporal position relative to environmental and behavioral cues. The organization of these firing correlates may allow the formation of memory traces through the integration of object and event information onto a spatiotemporal framework of cell assemblies. Characterizing how external inputs guide internal dynamics in the hippocampus to enable this process across different experiences is crucial to understanding hippocampal function. A body of literature implicates the medial entorhinal cortex (MEC) in supplying spatial and temporal information to the hippocampus. Here we develop a protocol utilizing bilaterally implanted custom designed triple fiber optic arrays and the red-shifted inhibitory opsin JAWS to transiently inactivate large volumes of MEC in freely behaving rats. This was coupled with extracellular tetrode recording of ensembles in CA1 of the hippocampus during a novel memory task involving temporal, spatial and object related epochs, in order to assess the importance of MEC activity for hippocampal feature selectivity during a rich and familiar experience. We report that inactivation of MEC during a mnemonic temporal delay disrupts the existing temporal firing field sequence in CA1 both during and following the inactivation period. Neurons with firing fields prior to the inactivation on each trial remained relatively stable. The disruption of CA1 temporal firing field sequences was accompanied by a behavioral deficit implicating MEC activity and hippocampal temporal field sequences in effective memory across time. Inactivating MEC during the object or spatial epochs of the task did not significantly alter CA1 object selective or spatial firing fields and behavioral performance remained stable. Our findings suggest that MEC is crucial specifically for temporal field organization and expression during a familiar and rich experience. These results support a role for MEC in guiding hippocampal cell assembly sequences in the absence of salient changing stimuli, which may extend to the navigation of cognitive organization in humans and support memory formation and retrieval.

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.