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.

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.

The abuse of methylenedioxymethamphetamine (MDMA) during pregnancy is of concern. MDMA treatment of rats during a period of brain growth analogous to late human gestation leads to neurochemical and behavioral changes. MDMA from postnatal day (P)11–20 in rats produces reductions in serotonin and deficits in spatial and route-based navigation. In this experiment we examined the impact of MDMA from P11 to P20 (20 mg/kg twice daily, 8 h apart) on neuronal architecture. Golgi impregnated sections showed significant changes. In the nucleus accumbens, the dendrites were shorter with fewer spines, whereas in the dentate gyrus the dendritic length was decreased but with more spines, and for the entorhinal cortex, reductions in basilar and apical dendritic lengths in MDMA animals compared with saline animals were seen. The data show that neuronal cytoarchitectural changes are long-lasting following developmental MDMA exposure and are in regions consistent with the learning and memory deficits observed in such animals.

Previous research investigating the neural circuitry underlying memory consolidation has primarily focused on single “nodes” in the circuit rather than the neural connections between brain regions, despite the likely importance of these connections in mediating different aspects or forms of memory. This focus has, in part, been due to technical limitations; however the advent of optogenetics has altered our capabilities in this regard, enabling optical control over neural pathways with temporal and spatial precision. The current set of experiments took advantage of optogenetics to control activity in specific pathways connecting brain regions in rats immediately after different kinds of learning. Chapter 2 first established the use of optogenetics to manipulate activity in the basolateral amygdala (BLA), which has been shown to modulate memory consolidation for a variety of types of learning likely through its connections to various downstream regions. Using a one-trial inhibitory avoidance task, a simple and robust fear learning paradigm, we found that both post-training stimulation and inhibition of BLA activity could enhance or impair later retention of the task, respectively. Enhancement was specific to stimulation using trains of 40, but not 20, Hz light pulses. Chapters 3 and 4 examined the projections from the BLA to the ventral hippocampus (VH) and medial entorhinal cortex (mEC) as the BLA’s ability to influence the consolidation for many types of memory is believed to be mediated through discrete projections to distinct brain regions. Indeed, the BLA innervates both structures, and prior studies suggest that the mEC and VH have distinct roles in memory processing related to contextual and nociceptive (footshock) learning, such as those involved in contextual fear conditioning (CFC). Optogenetic stimulation or inhibition of the BLA-VH or BLA-mEC pathway after training on a modified CFC task, in which the nociceptive or emotional stimulus (the footshock) and the context are separated, enabled experimental manipulations to selectively affect the consolidation for learning about one component and not the other. Optogenetic stimulation/inhibition was given to each candidate pathway immediately after the relevant training to determine its role in influencing consolidation for that component of the CFC learning. Chapter 3 results showed that stimulation of the BLA-VH pathway following footshock, but not context, training enhanced retention, an effect that was specific to trains of 40 Hz stimulation. Post-footshock photoinhibition of the same pathway impaired retention for the task. Similar investigations of the BLA-mEC pathway in Chapter 4 produced complementary findings. Post-context, but not footshock, stimulation of the pathway enhanced retention. In this particular case, only trains of 8 Hz stimulation were effective at enhancing retention. These results are the first, to our knowledge, to find that BLA inputs to different structures selectively modulate consolidation for different aspects of learning, thus enhancing our understanding of the neural connections underlying the consolidation of contextual fear conditioning and providing a critical foundation for future research.

Consolidation of associative memories may depend on communication between the lateral entorhinal cortex (LEC) and hippocampus (HPC) for recently learned memories and the LEC and medial prefrontal cortex (mPFC) for remote memories. To determine whether direct connections between these regions are necessary for the retrieval of a recently or remotely learned memory, rats acquired an associative memory through trace eyeblink conditioning and were tested for memory retention after inactivating the regions of interest with the GABAA agonist, muscimol. Inactivating the LEC-HPC connection did not impair memory retrieval. However, inactivating the LEC-mPFC connection impaired remote, but not recent, memory retrieval. Thus, the LEC and mPFC connection is necessary for the retrieval of a remotely, but not recently learned associative memory. Increased reliance on the entorhinal-prefrontal connection indicates the strengthening of functional connectivity between the two regions, which may be a biological correlate for the proposed reorganization during systems consolidation.

Consolidation of associative memories may depend on communication between the lateral entorhinal cortex (LEC) and hippocampus (HPC) for recently learned memories and the LEC and medial prefrontal cortex (mPFC) for remote memories. To determine whether direct connections between these regions are necessary for the retrieval of a recently or remotely learned memory, rats acquired an associative memory through trace eyeblink conditioning and were tested for memory retention after inactivating the regions of interest with the GABAA agonist, muscimol. Inactivating the LEC-HPC connection did not impair memory retrieval. However, inactivating the LEC-mPFC connection impaired remote, but not recent, memory retrieval. Thus, the LEC and mPFC connection is necessary for the retrieval of a remotely, but not recently learned associative memory. Increased reliance on the entorhinal-prefrontal connection indicates the strengthening of functional connectivity between the two regions, which may be a biological correlate for the proposed reorganization during systems consolidation.