The hippocampus is one of the most intensely studied brain structures and the oscillatory activity of the hippocampal neurons is believed to be involved in learning and memory consolidation. Therefore, studying rhythm generation and modulation in this structure is an important step in understanding its function. In this thesis, these phenomena are studied via mathematical models of networks of hippocampal interneurons. The two types of neural networks considered here are homogenous and heterogenous networks. In homogenous networks, the input current to each neuron is equal, while in heterogenous networks, this assumption is relaxed and there is a specified degree of heterogeneity in the input stimuli. A phase reduction technique is applied to the neural network model of the hippocampal interneurons and attempts are made to understand the implications of heterogeneity to the existence and stability of the synchronized oscillations. The Existence of a critical level of heterogeneity above which the synchronized rhythms are not stable is established, and linear analysis is applied to derive expressions for estimating the perturbations in the network frequency and timing of the neural spikes. The mathematical techniques developed in this thesis are general enough to be applied to models describing other types of neurons not considered here. Possible biological implications include the application of high frequency local stimulation to alleviate the synchronous neural oscillations in pathological conditions such as epilepsy and Parkinson's disease and the possible role of heterogeneity in controlling the rhythm frequency and switching between various cognitive states.

The hippocampus is one of the most intensely studied brain structures and the oscillatory activity of the hippocampal neurons is believed to be involved in learning and memory consolidation. Therefore, studying rhythm generation and modulation in this structure is an important step in understanding its function. In this thesis, these phenomena are studied via mathematical models of networks of hippocampal interneurons. The two types of neural networks considered here are homogenous and heterogenous networks. In homogenous networks, the input current to each neuron is equal, while in heterogenous networks, this assumption is relaxed and there is a specified degree of heterogeneity in the input stimuli. A phase reduction technique is applied to the neural network model of the hippocampal interneurons and attempts are made to understand the implications of heterogeneity to the existence and stability of the synchronized oscillations. The Existence of a critical level of heterogeneity above which the synchronized rhythms are not stable is established, and linear analysis is applied to derive expressions for estimating the perturbations in the network frequency and timing of the neural spikes. The mathematical techniques developed in this thesis are general enough to be applied to models describing other types of neurons not considered here. Possible biological implications include the application of high frequency local stimulation to alleviate the synchronous neural oscillations in pathological conditions such as epilepsy and Parkinson's disease and the possible role of heterogeneity in controlling the rhythm frequency and switching between various cognitive states.

In humans, damage to the mammillothalamic tract (MTT) as a result of localised strokes, tumours or alcohol abuse has consistently been implicated in the severe anterograde amnesia evident in these patients. This small neural pathway, which connects the mammillary bodies (MB) to the anterior thalamic nuclei (ATN), is thought to provide one important link in a larger extended hippocampal circuit involved in encoding and retrieval of episodic memory. Brain damage in clinical cases is, however, typically diffuse and contributions from additional sites of pathology cannot be ruled out. There are also inconsistencies within a limited animal literature on MTT lesions. The current study made MTT lesions in female rats and used multiple „episodic - like‟ memory tasks relevant to the proposed importance of this pathway. The project also intended to test whether enrichment reduces any impairments after MTT lesions. None of the lesions resulted in complete bilateral disconnection of the MTT, but many had moderate to large bilateral (n = 6) (81% to 50%), or unilateral MTT damage (n = 4). Rats with bilateral lesions were compared to controls (n = 14, including 4 other lesion rats in which no lesion occurred). The severe working memory deficit in the water maze expected for rats with MTT lesion was not found and only a slight deficit in reference memory in the water maze was observed (so enrichment was not implemented). Although none of the bilateral MTT lesions were complete, they are also often incomplete in clinical cases and previous research has shown that lesions to the ATN in excess of 50% are sufficient to induce severe behavioural deficits in rats. Therefore, if the MTT is critical to memory then substantial but not total bilateral disconnection should be sufficient to induce profound deficits in rats, at least on spatial working memory. Taken together these findings suggest a less crucial role for the MTT in memory than previously suggested. Future research needs to resolve the inconsistencies observed in the animal literature by repeating the present study, using larger MTT lesions and both male and female rats.

Chronic intermittent ethanol exposure (CIE) is associated with degeneration of hippocampal neurons. The present study used hippocampal cultures to examine the loss of NeuN immunoreactivity, a relaible marker or neuronal density, after 1, 2, or 3 cycles of 5 days EtOH exposure (50 mM), followed by a 24-hour period of EWD or continuous EtOH exposure. NeuN immunoreactivity was decreased by 13%, 19%, and 16% in the CA1, CA3, and dentate gyrus after 3 cycles of CIE respectively; thionine staining confirmed significant cellular losses within each hippocampal subregion. Two cycles of CIE in aged tissue cultures resulted in significant decreases in NeuN immunoreactivity in all hippocampal subregions; however continuous ethanol exposure or exposure to one cycle of CIE did not. Further, exposure to the N-Methyl-D-aspartate receptor (NMDAR) antagonist 2-amino-7-phosphonvaleric acid (APV) (30 uM) during periods of EWD attenuated the loss of NeuN in all hippocampal subregions, while exposure to APV (40 uM) prevented the loss of NeuN in the CA1 and dentate gyrus. These results suggest that the loss of mature neurons after CIE is associated with the overactivation on the NMDAR.

Master of Philosophy (Medicine) / The actin cytoskeleton is important in many cellular processes such as motility, and establishing and maintaining cell morphology. Members of the tropomyosin protein family associate with the actin cytoskeleton along the major groove of actin filaments (F-actin), stabilising them and regulating actin-filament dynamics. To date over 40 non-muscle tropomyosin isoforms have been identified, which are encoded by 4 different genes (α, β, γ, δ). Individual tropomyosin isoforms define functionally distinct F-actin populations. Previous studies have shown that tropomyosins sort to distinct subcellular compartments at different stages of development in polarised cells. Neuronal growth cones are highly dynamic polarised structures, dependent on a constant reorganisation of the actin cytoskeleton. By eliminating tropomyosins in a knockout (KO) mouse model, we investigated the role of two tropomyosin isoforms, Tm5NM1 and Tm5NM2 (γTm gene products) in growth cone dynamics and neurite outgrowth. Growth cone protrusion rates were significantly increased in one day old Tm5NM1/2 KO hippocampal neurons compared to WT controls. Neuritogenesis was significantly affected by the elimination of Tm5NM1/2, with a slight decrease in neurite length and an increase in neuronal branching in neurons cultured for four days. At the molecular level, the depletion of Tm5NM1/2 had no impact on the protein levels and activity of ADF/cofilin in hippocampal neurons while in cortical neurons a subtle but significant increase in ADF/cofilin activity was observed. The subtle phenotype in the early stages of neuritogenesis observed from eliminating Tm5NM1/2 may be explained with functional compensation by other tropomyosin isoforms. Functional compensation for the loss of Tm5NM1/2 may be provided by isoforms Tm5a/5b, TmBr2 and Tm4 as they localise to the growth cones, structures where Tm5NM1/2 are normally found. These results suggest that Tm5NM1/2 may not be required for early stages of neuritogenesis but may still play a fine-tuning role for this process.

Master of Philosophy (Medicine) / The actin cytoskeleton is important in many cellular processes such as motility, and establishing and maintaining cell morphology. Members of the tropomyosin protein family associate with the actin cytoskeleton along the major groove of actin filaments (F-actin), stabilising them and regulating actin-filament dynamics. To date over 40 non-muscle tropomyosin isoforms have been identified, which are encoded by 4 different genes (α, β, γ, δ). Individual tropomyosin isoforms define functionally distinct F-actin populations. Previous studies have shown that tropomyosins sort to distinct subcellular compartments at different stages of development in polarised cells. Neuronal growth cones are highly dynamic polarised structures, dependent on a constant reorganisation of the actin cytoskeleton. By eliminating tropomyosins in a knockout (KO) mouse model, we investigated the role of two tropomyosin isoforms, Tm5NM1 and Tm5NM2 (γTm gene products) in growth cone dynamics and neurite outgrowth. Growth cone protrusion rates were significantly increased in one day old Tm5NM1/2 KO hippocampal neurons compared to WT controls. Neuritogenesis was significantly affected by the elimination of Tm5NM1/2, with a slight decrease in neurite length and an increase in neuronal branching in neurons cultured for four days. At the molecular level, the depletion of Tm5NM1/2 had no impact on the protein levels and activity of ADF/cofilin in hippocampal neurons while in cortical neurons a subtle but significant increase in ADF/cofilin activity was observed. The subtle phenotype in the early stages of neuritogenesis observed from eliminating Tm5NM1/2 may be explained with functional compensation by other tropomyosin isoforms. Functional compensation for the loss of Tm5NM1/2 may be provided by isoforms Tm5a/5b, TmBr2 and Tm4 as they localise to the growth cones, structures where Tm5NM1/2 are normally found. These results suggest that Tm5NM1/2 may not be required for early stages of neuritogenesis but may still play a fine-tuning role for this process.

In the 1980s, it was widely accepted that new neurons are continuously generated in the dentate gyrus of the mammalian hippocampus. Since its acceptance, researchers have employed various techniques and behavioral paradigms to study the proliferation, differentiation, and functional role of adult-born neurons. This literature thesis aims to discuss how optogenetics is able to overcome the limitations of past techniques and provide the field with new insights into the functional role of neurogenesis. We will review the current knowledge on both adult hippocampal neurogenesis and optogenetics, present representative studies using optogenetics to investigate neurogenesis and discuss potential limitations and concerns involved in using optogenetics.

Old-age hippocampal sclerosis (HS), characterised by severe neuron loss in hippocampal CA1, is a poorly understood cause of dementia. At present no objective pathological HS criteria exist. In life HS is commonly diagnosed as Alzheimer's disease. HS aetiology is unclear, although it has been associated with both ischaemia and TAR-DNA-binding protein-43 (TDP-43)-related neurodegeneration. Variations in genes GRN, TMEM106B and ABCC9 are proposed as HS risk factors. The aim of this thesis was to investigate epidemiological, clinical, pathological and genetic characteristics of HS in older European populations. 976 brains donated for the Cambridge City over-75s Cohort, the Cognitive Function and Ageing Study and the Finnish Vantaa 85+ study were available for evaluation -including bilateral hippocampi from 302 individuals. A protocol capturing the extent and severity of hippocampal neuron loss was developed, establishing objective HS diagnosis criteria and allowing observation of distinct neuron loss patterns associated with ischaemia and neurodegeneration. 71 HS cases (overall prevalence: 7.3%) were identified. HS was significantly associated with an advanced age at death as well as dementia at the end of life. Neuropsychological and cardiovascular characteristics were similar between HS and AD, except for a longer duration of dementia and more disability in HS. HS was not associated with neurofibrillary tangles, amyloid plaques, or vascular pathologies, but all HS cases evaluated for TDP-43 showed neuronal inclusions in the hippocampal dentate and a high frequency of other glial, neuronal and neurite TDP-43 pathologies. GRN and TMEM106B but not ABCC9 variations were linked to HS. A moderating effect of TDP-43 on this association was detected. HS presented pathologically similarly to frontotemporal dementia cases with TDP-43 (FTLD-TDP) caused by mutations in GRN, but differed from other FTLD-TDP subtypes. Results of this thesis reveal the importance of HS in the oldest old in the population, the key role of TDP-43, as well as providing robust methods to capture HS characteristics for an area that has been under-researched but is clearly vital to understanding dementia in the oldest old.

This experiment explored whether a long-term aerobic exercise program may induce significant structural and functional changes in the hippocampus, an area of the brain that is important for spatial navigation and memory formation. Based on existing rodent studies, we hypothesize that exercise will cause a shift to allocentric processing, away from a less robust egocentric learning strategy. It is possible that exercise-induced relief of chronic stress, which contributes to improved hippocampal function, will increase reliance on allocentric spatial navigation. Neurogenesis, which occurs in the dentate gyrus region of the hippocampus, is another indicator of hippocampal function that may influence this shift to allocentric learning. The current study examines whether six weeks of aerobic exercise enhances allocentric processing in healthy young adults. Forty-nine young adults (35 female; age range 18-29 years) were randomly assigned to one of three groups: 1) High intensity interval training group, 2) Moderate intensity training group, or 3) Non-exercising control group. Hippocampus-dependent memory was assessed before and after the intervention on a Virtual Reality Water Maze task, and a high interference memory task, the Mnemonic Similarity Task (MST) which may be dependent on hippocampal neurogenesis. Levels of chronic stress and depression were measured using the Beck Depression Inventory II. It was expected that exercise would improve spatial memory performance on the water maze task, and that performance would improve in proportion to enhanced fitness levels. This improvement in spatial memory performance was expected to correlate with the two indicators of hippocampal function that were assessed in the current study—chronic stress and performance on the high interference memory task. Six weeks of regular aerobic exercise resulted in a 21.5% improvement in spatial memory performance on the water maze task, indicating improved hippocampus-mediated spatial memory function. Improvements displayed by high intensity exercisers were greater than those observed in the moderate intensity exercisers, suggesting that higher intensity exercise may be more effective in enhancing hippocampal function. Importantly, low responders to exercise exhibited a 30% improvement in water maze performance, suggesting that even minor fitness improvements can lead to significant cognitive gains. Chronic stress and depression, and performance on the MST were not significantly associated with changes in spatial memory performance; however trends observed may offer some explanation to the aforementioned changes in spatial memory. Findings from the current study have important implications for treatment options in populations that are currently, or at risk of suffering from impaired hippocampal function. / Thesis / Master of Science (MSc)

The domestic chick (Gallus gallus domesticus) has been widely used as an animal model to investigate spatial orientation and the neural mechanisms underlying this function. In all vertebrate species the hippocampus plays an essential role in spatial orientation. Since the hippocampus is a bilateral structure, it is important to investigate the specific role of the left and the right hippocampi in spatial processing. Although, the domestic chick has been often used as animal model to assess cognitive lateralization, the involvement of the left and the right hippocampal formation in spatial orientation has been poorly investigated in this model. Behavioral studies using monocular eye occlusion have shown that in chicks the left eye-system (right hemisphere) is involved in the elaboration of spatial relational information, while the right eye-system (left hemisphere) processes local information. However, while visual lateralization in chicks had been traditionally considered to be induced by embryonic light exposure, recent studies suggest the presence of structural and behavioural asymmetries also in dark-incubated chicks. Thus, the main aim of this thesis was to test the lateralization of hippocampal functions in dark incubated chicks, both in spatial and non-spatial tasks. In the first study dark-incubated chicks were trained to orient in a large circular arena using spatial relational information provided by free-standing objects. Once chicks reached a learning criterion they were tested binocularly or under a monocular eye-occlusion condition. This study provided the first demonstration that domestic chicks are able to orient by relational spatial information provided by free-standing objects, in binocular vision conditions. However, if either one of the two eyes was occluded, chicks failed the orientation task. These results show that at least in dark-incubated chicks binocular integration is needed to solve this spatial orientation task. We also investigated if chicks have a preference to orient by local or spatial information provided by free-standing objects and if this ability is influenced by eye occlusion. Chicks preferred to use local over spatial cues to orient, both in binocular and monocular conditions (independently of which eye was occluded). These results indicate that local cues are processed by both eye-systems and do not require access to information from both eyes, contrary to relational spatial cues. Using the same setup, in the second study we directly investigated the involvement of chicks’ left and right hippocampal formation during orientation by free-standing objects. For this purpose we performed an immunohistochemical staining of the immediate early gene product c-Fos (a neural activity marker). Two independent groups of dark-incubated chicks were trained to find food in the large circular arena and the level of hippocampal activation was compared between the two groups. One group was trained to orient exclusively by local cues, while the other was orienting by spatial relational information provided by free-standing objects. This revealed selective activation of the right hippocampus during orientation by spatial relational information in dark-incubated chicks. While monocular occlusion has often been used to test lateralization of spatial functions in chicks, it is still unclear whether this manipulation affects hippocampal activation. The aim of the third study was to clarify this issue, by exposing dark-incubated chicks to a novel environment in conditions of monocular occlusion or binocular vision. Activation of the hippocampal formation was once again measured by c-Fos expression. Exposure to a novel environment is known to trigger hippocampal activation in different animals, including domestic chicks. As expected, exposure to the novel environment activated the hippocampus in binocular vision conditions. However, if either one of the eyes was occluded, the hippocampal c-Fos expression did not rise above what observed in the baseline condition (chicks maintained in a familiar environment). Thus, successful hippocampal response to a novel environment requires input from both eyes. Our results also suggest that monocular occlusion equally affects the left and the right hippocampus. Overall, access to information from both eyes plays a crucial role for the acquisition of a spatial map of a novel environment, in line with the behavioral results of the first study. Moreover, a task independent lateralization effect, with higher c-Fos expression in the left compared to the right hippocampus, could be observed in all the experimental conditions. This confirms the presence of neuroanatomical lateralization in dark-incubated chicks. The last study investigated whether chicks’ hippocampus would also respond to novel social stimuli, in line with the activation observed in this structure after exposure to a novel environment. Only few studies have directly investigated the involvement of birds’ hippocampal formation in social functions. Here, the hippocampal activation was compared between chicks exposed to an unfamiliar conspecific vs. chicks exposed to a familiar one. We found that the ventral and dorsomedial portion of the right hippocampus of dark-incubated chicks responds to an unfamiliar individual. This provides the first demonstration of hippocampal sensitivity to social novelty in birds. Overall the studies performed in this thesis indicate a selective lateralized involvement of domestic chicks’ hippocampal formation not only in spatial, but also in social functions.