The AMP-activated protein kinase (AMPK) is a master metabolic regulator and energy sensor that has been extensively studied in the context of cancer and metabolic disorders. However, its role in neuronal function and morphology remains largely unexplored. This dissertation aims to bridge this gap by investigating the roles of AMPK in maintaining axon homeostasis and regulating mitochondrial morphology in neurons. By identifying novel regulators of AMPK across different cellular compartments, this thesis sheds light on the multifaceted functions of AMPK in shaping neuronal and mitochondrial architecture. The dissertation is organized into five chapters. Chapter 1 provides a brief background on AMPK, its activation mechanisms, and the downstream pathways it regulates. Chapter 2 introduces with-no-lysine kinase (WNK), a novel axon morphogenic kinase with dual roles in terminal axon branch development and maintenance in mature axons. Chapter 3 investigates the role of AMPK in mediating the loss-of-WNK axonal phenotypes, revealing a critical link between AMPK and axonal integrity. Chapter 4 shifts focus to the mitochondrial, characterizing mitochondrial fission regulator 1-like (MTFR1L) as a novel AMPK-activated protein that regulates mitochondrial morphology. Finally, Chapter 5 explores the role of AMPK in mediating activity-dependent mitochondrial morphology in hippocampal CA1 neurons, highlighting the dynamic interplay between neuronal activity and mitochondrial dynamics. Collectively, these findings provide novel insights into the multifaceted roles of AMPK in neuronal development, degeneration, and organelle regulation, underscoring the importance of this master kinase in maintaining neuronal health and homeostasis.

Segmented analysis of subcortical structures within the nonhuman primate can potentially have a profound impact on studying the relationship between volumetric characteristics and alcohol dependencies. Image segmentations have been widely used in quantifying structural information. There are a variety of methods in which users can extract desired structures from a medical image; ranging from manual segmentations to fully-automated segmentations and 2-D to 3-D. The implications of this possibility can have tremendous applicability to medical research and diagnosis. The primary goal of my thesis is to investigate different implementation methodologies for segmenting subcortical structures such as the hippocampus and striatum and then apply that knowledge towards the development of an approach to segment these two structures from a group of alcohol-dependent Rhesus Macaque monkeys. Using the Level Set Deformable Model (LSDM) with a priori structural information, a series of T1-weighted MR images of Rhesus Macaque hippocampi and striatum were segmented in an effort to compare the structural hippocampal and striatal volumes between early and late stages of alcohol dependency. The results suggest that the volumes of both subcortical structures are affected negatively by alcoholism. Volume deficits of as much as 5% for the hippocampus and 8% for the caudate were found. / Master of Science

Numerous memory studies have demonstrated that epigenetic-mediated transcriptional regulation, such as post-translational histone modifications, is essential to memory formation and maintenance. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, which helps to understand post-traumatic stress disorder (PTSD). However, these mainly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited. The trimethylation of histone 3 lysine 4 (H3K4me3) is an essential regulator of active gene transcription in cells and has been shown to be critical for memory formation in the hippocampus, a major site of memory storage. However, it is unknown how H3K4me3 is coordinated to target genes during memory formation. Monoubiquitination of histone H2B (H2Bubi) is critical for recruiting H3K4me3 to DNA in a gene-specific manner during memory formation in the hippocampus. Furthermore, there is a great overlap between H3K4me3 and phosphorylation of histone H2A.X at serine 139 (H2A.XpS139), a marker to study DNA double-strand break (DSB) loci. DSB is a critical mechanism for solving DNA-related topological issues during transcription and replication, which could be triggered in some immediate early genes (IEGs) by neuronal activity, such as memory consolidation.Here, we used rat fear conditioning paradigms in combination with quantitative molecular assays, such as chromatin immunoprecipitation (ChIP), and gene editing techniques, like siRNAs and CRISPR-dCas9 manipulations, to study the role of hippocampal 1) H2Bubi and 2) DSBs in contextual fear memory consolidation and reconsolidation, respectively. Additionally, we behaviorally and molecularly characterized IFL and compared it to directly acquired fear subjects. We found that contextual fear conditioning changed the expression of 86 genes in the hippocampus one hour after training. Remarkably, siRNA knockdown of the H2Bubi ligase, Rnf20, abolished changes in all but one of these genes, Per1. Additionally, we report that the loss of Rnf20 in neurons, but not astrocytes, of the hippocampus impaired long-term memory formation. We next found an increase in H2A.XpS139 and H3K4me3 levels in the Npas4, an IEG important for contextual fear memory, promoter region 5 minutes after retrieval. In vivo siRNAmediated knockdown of the enzyme responsible for DSB, topoisomerase II β, prior to retrieval, decreased Npas4 promoter-specific H3K4me3 and H2A.XpS139 levels and impaired long-term memory. Lastly, our data show that both sexes can indirectly acquire fear from either sex using the auditory-cued IFL model. Moreover, our data show that molecular profiles in the amygdala are largely unique to direct or indirect fear learning and vary by sex. Collectively, this data reveals novel roles for histone phosphorylation and ubiquitination in regulating H3K4me3 and memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear. / Doctor of Philosophy / Changes in epigenetic mechanisms, processes that control the expression of genes without changing the original sequences, play a crucial role in the formation and maintenance of memory. Moreover, many studies on the mechanisms of memory have focused on fear memories underlying traumatic events, helping to understand post-traumatic stress disorder (PTSD). However, these majorly focus on individuals directly experiencing the event, while different species have shown the ability to learn fear indirectly by observing a conspecific experiencing a trauma. Thus, our understanding of indirect fear learning (IFL)'s characteristics is very limited. In the present study, we investigated some of these epigenetic mechanisms called histone modifications. In the brain, histone 3 lysine 4 trimethylation (H3K4me3), a histone modification, is critical for memory formation in the hippocampus, a key area for memory storage. However, it is still not fully understood how H3K4me3 is coordinated during memory formation. Another histone modification called H2B monoubiquitination (H2Bubi) helps recruit H3K4me3 to DNA and so is also crucial for memory formation. Here, using rat models, we found that the expression of 86 genes is changed during memory formation in the hippocampus and that this result is almost entirely dependent on the presence of H2Bubi. We also discovered that H2Bubi is critical for longterm memory formation only in neurons of the hippocampus, and not astrocytes (another type of brain cells). Additionally, there is a connection between H3K4me3 and the phosphorylation of histone H2A.X, another epigenetic mechanism that co-occurs with DNA breaks and may serve as a markerfor studying these breaks. DNA breaks play a vital role during gene expression and could be triggered by neuronal activity during memory formation. We observed an increase in H2A.X phosphorylation and H3K4me3 levels in a memory-permissive gene five minutes after memory retrieval. Inhibition of DSBs, prior to retrieval abolished these changes, and impaired long-term memory. This suggests a critical role for DSBs in memory maintenance and that H2A.X phosphorylation is necessary for the recruitment of H3K4me3 to DNA. Lastly, our data demonstrated that both males and females could learn fear indirectly from either sex by observing them undergoing auditory-cued fear conditioning. Additionally, we found distinct molecular patterns in the amygdala, a brain region involved in fear processing, depending on whether fear was directly or indirectly acquired, and it varied between sexes. Collectively, data from this dissertation reveals novel roles for histone modifications in memory formation and shows behavioral and molecular differences in each sex based on the way they acquire fear.

The memory system is adaptive in so far as it is able to provide the most robust predictions of what will happen next in our environment. Three means through which it can do this are: arbitrating between temporally embedded recollections and generalized knowledge; rescuing remote memories that are learned to be behaviorally relevant; and updating existing memories if they provide invalid predictions of the outside world. Here, I cover three studies that probe these functions in behavior and in the brain. In Chapter 1, I show how a data-driven parcellation reveals non-linear gradients in measures of signal heterogeneity across the body of the hippocampus, suggesting novel areas of investigation into how the memory system flexibly constructs fine- and coarse-grained memories. In Chapter 2, I then explore how memories might be rescued by later aversive experiences, finding novel evidence that the online inferences participants make as to what current stimuli are relevant to their arousal subtly shapes what previous stimuli they retroactively maintain in memory. Finally, in Chapter 3, I show that signals from the ventral tegmental area modulate the effect of replaying memories on the eventual updating of those memories. Each of these provides novel pieces of evidence into the neural and behavioral markers of how memories are constructed, strengthened, or updated in the brain.

In dieser Arbeit wurden die Auswirkungen der Herzinsuffizienz und ihrer Komorbiditäten Hypertonie und Diabetes mellitus auf Morphologie und Histologie des Hippocampus am Mausmodell untersucht. / In this paper we studied the effects of heart failure and its comorbidities hypertension and diabetes mellitus on morphology and histology of the hippocampus in the mouse model.

No / In addition to their role in cellular metabolic activity, thyroid hormones (THs), also regulate neural development; the central nervous system is particularly dependent on TH for normal maturation and function. Specifically, there appears to be extensive inter-reliance between TH and acetylcholine (Ach), nerve growth factor and hippocampal function. These associations led us to investigate the possible effects of thyroxine (L-T4) on performance of a spatial learning task, where cholinergic activity and hippocampal function are known to be important. Groups of rats (n=20) received saline (controls) or L-T4 at 2.5 or 5 mg/kg daily for 4 days as a sub-chronic treatment, or 0, 5 or 10 mg/kg doses administered every third day for 28 days prior to testing as a chronic regimen. Rats were assessed in a watermaze for their ability to find a submerged or visible platform. Forty minutes prior to watermaze testing, half the animals in each group received 1 mg/kg scopolamine to elicit a cognitive deficit. Following testing, rats were decapitated, blood samples taken, and the frontal cortex and hippocampus were dissected out for acetylcholinesterase (AChE) assay. The results showed that L-T4 treatment, administered both sub-chronically and chronically, significantly enhanced the ability of rats to learn a spatial memory task, compared with controls. Moreover, both short-term and long-term L-T4 treatment reduced the cognitive-impairing effects of scopolamine. Improvements in performance were shown to occur alongside significantly increased cholinergic activity in frontal cortex and in the hippocampus of treated animals.These findings demonstrate an augmentative effect of L-T4 upon cognitive function, possibly mediated by an enhancement of cholinergic activity. The results support previous findings of a relationship between L-T4 and acetylcholine, and underscore possible mechanisms by which disorders of thyroid function may be associated with cognitive decline.

No / Acute administration of the psychotomimetic phencyclidine (PCP) can mimic some features of schizophrenia, while a repeated treatment regimen of PCP may provide a more effective way to model in animals the enduring cognitive dysfunction observed in many schizophrenic patients. The present study aims to investigate behavioural and neuropathological effects of sub-chronic PCP administration. The cognitive deficit induced by sub-chronic PCP was examined using a previously established operant reversal-learning paradigm. Subsequently, the effect of sub-chronic PCP on parvalbumin-immunoreactive (parvalbumin-IR) neurons was assessed using immunohistochemical techniques. Rats were trained to respond for food in an operant reversal-learning paradigm for approximately 6 weeks, followed by sub-chronic administration of PCP (2mg/kg) or vehicle twice daily for 7 days followed 7 days later by behavioural testing. Six weeks post PCP, brains were analysed using immunohistochemical techniques to determine the size and density of parvalbumin-IR in the frontal cortex and hippocampus. Sub-chronic PCP significantly reduced (p <0.001) percentage correct responding in the reversal phase relative to the initial phase, an effect that persisted throughout the experimental period (4 weeks). The density of parvalbumin-IR neurons was reduced in the hippocampus, with significant reductions in the dentate gyrus and CA2/3 regions (p <0.001). There were significant changes in the frontal cortex, with a reduction (p <0.01) in the M1 (motor area 1) region and increases in the M2 (motor area 2) region and cingulate cortex (p <0.01-p <0.001). These results parallel findings of profound hippocampal and more subtle cortical deficits of parvalbumin-IR neurons in schizophrenia, and provide evidence to suggest that sub-chronic PCP can induce a lasting cognitive deficit, an effect that may be related to the observed neuronal deficits.

In dieser Arbeit wurde der Einfluss sozialer Stresserfahrung sowie des 5-Htt-Genotyps auf die neuronale Morphologie bestimmter Hirnregionen anhand eines Mausmodells untersucht. Es wurde in mit Golgi-Cox gefärbten Gehirnen der 5-HTT-KO-Linie in der lateralen Amygdala (LA) die Apikal- und Basaldendriten pyramidenzellähnlicher Neurone und die Apikaldendriten der Pyramidenzellen der Cornu ammonis (CA)3-Region des Hippocampus mithilfe des Neurolucidasystems rekonstruiert und die so gewonnenen Daten anschließend statistisch ausgewertet. Die erzielten Ergebnisse belegen, dass vor allem die Erfahrung von sozialem Verteidigungsstress aber auch der 5-Htt-Genotyp (WT, HET, KO) im Mausmodell signifikanten Einfluss auf die Morphologie der Neurone der LA und der CA3-Region besitzen. Um die in dieser Arbeit mit allen drei 5-Htt-Genotypen erzielten Ergebnisse der LA-Neurone besser mit den Ergebnissen von Nietzer und Bonn (nur WT, KO) vergleichen zu können (Nietzer et al., 2011), wurden die von mir erhobenen Daten nicht nur in einem 3er-Vergleich, sondern auch einem 2er-Vergleich (WT vs. KO) statistisch analysiert. Untersuchungen der LA-Neurone aller drei 5-Htt-Genotypen zeigen, dass sozialer Stress zu einer Zunahme der Komplexität der Dendritenbäume durch längere und auch stärker verzweigte Dendriten vor allem in der Gruppe der WT-Mäuse führt. HET- und KO-Mäuse zeigten keinen entsprechenden Stress-Effekt. Darüber hinaus zeigten sich deutliche Genotypeffekte. Unabhängig vom Stresserleben besitzen HET-Mäuse längere Dendriten als WT-Mäuse sowie eine höhere Spinedichte als WT- und KO-Mäuse. Die Hypothese, die in der Arbeit von Nietzer et al. aufgestellt wurde, dass eine vollständige 5-HTT-Defizienz zu mehr Spines führt, ließ sich hier weder durch den 3er- noch durch den 2er-Vergleich replizieren. Die Pyramidenzellen der CA3-Region, die in dieser Studie zum ersten Mal analysiert wurden, zeigen in Bezug auf die durch den Stress ausgelösten Veränderungen ein im Vergleich zu den LA-Neuronen entgegengesetzten Effekt. Der soziale Stress führt hier zu einer Dendritenatrophie in der WT-Gruppe mit kürzeren und weniger komplexen Dendriten. Außerdem führte er zu einer geringeren Spinedichte bei den HET-Mäusen. Es zeigten sich klare Genotypeffekte, unabhängig von der Stresserfahrung, mit einer reduzierten Spinedichte der KO-Mäuse gegenüber den WT-Mäusen und einer nur in den Kontrollen detektierten, reduzierten Spinedichte der KO-Mäuse im Vergleich zu den WT- und HET-Mäusen. Sowohl in der LA als auch in der CA3-Region lassen sich Kompensationsmechanismen des 5-HTT-Defizits der HET-Tiere vermuten, über die die KO-Tiere nicht verfügen. Die in LA und CA3 gezeigten gegensätzlichen Auswirkungen des sozialen Stresses weisen auf die unterschiedlichen Funktionen dieser beiden Regionen im Furchtkreislauf und/oder bei der Verarbeitung von Stress hin. Darüber hinaus deutet diese Arbeit darauf hin, dass Arbeiten mit ähnlichen Untersuchungsmethoden und sogar gleichem Untersuchungsmaterial unterschiedliche Ergebnisse liefern können. / In this study, the influence of social stress experience and the 5-Htt genotype on the neuronal morphology of certain brain regions was investigated using a mouse model. The apical and basal dendrites of pyramidal cell-like neurons and the apical dendrites of the pyramidal cells of the cornu ammonis (CA)3 region of the hippocampus were reconstructed in Golgi-Cox-stained brains of the 5-HTT-KO line in the lateral amygdala (LA) using the neurolucida system and the data obtained was then statistically analyzed. The results obtained show that especially the experience of social defense stress but also the 5-Htt genotype (WT, HET, KO) have a significant influence on the morphology of the neurons of the LA and the CA3 region in the mouse model. In order to better compare the results of the LA neurons obtained in this study with all three 5-Htt genotypes with the results of Nietzer and Bonn (WT, KO only) (Nietzer et al., 2011), the data collected by me were statistically analyzed not only in a 3-way comparison, but also in a 2-way comparison (WT vs. KO). Investigations of the LA neurons of all three 5-Htt genotypes show that social stress leads to an increase in the complexity of the dendrite trees due to longer and also more branched dendrites, especially in the group of WT mice. HET and KO mice showed no corresponding stress effect. In addition, there were clear genotype effects. Regardless of the stress experience, HET mice have longer dendrites than WT mice and a higher spin density than WT and KO mice. The hypothesis put forward in the work of Nietzer et al. that complete 5-HTT deficiency leads to more spines could not be replicated here by either the 3-way or 2-way comparison. The pyramidal cells of the CA3 region, which were analyzed for the first time in this study, show an opposite effect compared to the LA neurons with regard to the changes triggered by stress. Here, social stress leads to dendrite atrophy in the WT group with shorter and less complex dendrites. It also led to a lower spin density in the HET mice. There were clear genotype effects, independent of the stress experience, with a reduced spin density in the KO mice compared to the WT mice and a reduced spin density in the KO mice compared to the WT and HET mice, which was only detected in the controls. Compensatory mechanisms for the 5-HTT deficit in the HET animals, which the KO animals do not have, can be assumed in both the LA and the CA3 region. The contrasting effects of social stress shown in LA and CA3 indicate the different functions of these two regions in the fear circuit and/or in the processing of stress. Furthermore, this work suggests that studies using similar research methods and even the same research material may yield different results.

Yes / The photoperiod is the main environmental cue that drives seasonal adaptive responses in reproduction, behavior, and metabolism in seasonal animals. Increasing evidence suggests that (poly)phenols contained in fruits can also modulate seasonal rhythms. (Poly)phenol-rich diets are associated with an improvement in cognitive function and neuroprotection due to their anti-inflammatory and antioxidative properties. However, it is unknown whether cherries affect neuroprotection in a photoperiod-dependent manner. To test this, F344 rats were exposed to L6 (6 h light/day), L12 (12 h light/day) and L18 (18 h light/day) photoperiods and fed a standard chow diet supplemented with either a control, lyophilized cherry 1 or cherry 2 with distinctive phenolic hallmarks. Physiological parameters (body weight, eating pattern index (EPI), testosterone, T4/T3) and hypothalamic key genes (Dio2, Dio3, Raldh1 and Ghrh) were strongly regulated by the photoperiod and/or fruit consumption. Importantly, we show for the first time that neurotrophs (Bdnf, Sod1 and Gpx1) in the hippocampus are also regulated by the photoperiod. Furthermore, the consumption of cherry 2, which was richer in total flavonols, but not cherry 1, which was richer in total anthocyanins and flavanols, enhanced neuroprotection in the hippocampus. Our results show that the seasonal consumption of cherry with a specific phenolic composition plays an important role in the hippocampal activation of neuroprotection in a photoperiod-dependent manner. / This work was supported by grant number PID2020-113739RB-I00 funded by MCIN/AEI/10.13039/501100011033 and by Pect-Nutrisalt funded by the European Regional Development Fund of the European Commission through the Operative Program Erdf of Catalonia 2014–2020. The authors thank the British Society for Neuroendocrinology (BSN) for providing a research visit Grant to F.M (Grant number: BSN-2022-1452). F.M. is the recipient of a predoctoral fellowship from Universitat Rovira i Virgili—Martí i Franquès (Grant number: 2019PMF-PIPF-19).

The hippocampal formation has been implicated in spatial formation for many decades. The hippocampus proper has received the most attention but other regions of the hippocampal formation contribute largely to spatial cognition. This thesis concentrated on one such region, the postsubiculum. The postsubiculum is considered important because it contains head direction cells and because it thought to be a major input to the hippocampus, via the entorhinal cortex. This thesis aims to test the functional role of the rat postsubiculum under two types of situation: one where the rat must rely on idiothetic cues for navigation, and another where the rat has visual cues present and can rely on these for orientation. The thesis also investigates hippocampal place cells and their stability over time after short exposures to novel environments. Chapter 3 of this thesis aimed to test whether the postsubiculum is necessary for path integration during a homing task. Rats were trained on a homing task on a circular platform maze. Once the task was acquired, rats were given lesions of the postsubiculum or sham lesions and then re-tested on the path integration task. The homing performance of rats with lesions of the postsubiculum was as good as that of the sham rats. A series of manipulations suggests that the rats were homing by path integration, confirmed by probe tests. The rats were then tested on a forced-choice delayed alternation T-maze task that revealed a significant impairment in alternation with delays of 5, 30, and 60 seconds. This suggests that the postsubiculum is not necessary for path integration in a homing task but is necessary for avoiding previously visited locations as is necessary in an alternation task. The experiments in Chapters 4 and 5 of this thesis aimed to investigate the effects of postsubiculum pharmacological inactivation on hippocampal CA1 place cells when rats were introduced to a novel environment with visual cues. A necessary first step was to assess place cells without any manipulation of the postsubiculum (Chapter 4) and then use information gained from this in the design of experiments in Chapter 5. Rats chronically implanted with recording electrodes in the CA1 region of the hippocampus were exposed to novel cue-rich environments whilst place fields were recorded. Following delays of 3, 6, or 24 hours, the same cells were recorded again in the same environment but with the cues rotated by 90°. Pixel-by-pixel correlations of the place fields show that stability of the place fields was significantly lower at 24 hours than at 3 hours. Stability after 6 hours was not significantly different from 3 hours. In the third set of experiments, rats were implanted with drug infusion cannulae in the postsubiculum and recording electrodes in CA1. Following infusions of either the AMPA receptor antagonist CXQX, the NMDA receptor antagonist D-AP5 or a control infusion of ACSF, place field stability was assessed as rats were exposed to a cylindrical environment with a single polarising cue card for 3 x 10 minute sessions and then again 6 hours later. There were no differences in place field correlations between the 3 drug conditions, although there was evidence of larger changes in spatial information content between cells in the CNQX and AP5 drug condition, but not the ACSF condition. The results suggest that, under the present testing conditions, place fields stability did not depend upon AMPA receptor-mediated transmission nor did it depend on NMDA receptor-mediated synaptic plasticity.