New neurons continue to be added to the dentate gyrus (DG) throughout adulthood and enhancing neurogenesis in this region holds therapeutic potential. However, the molecular mechanisms underlying DG neurogenesis remain elusive. Since developmental and adult neurogenesis often share the same signaling pathways, understanding how the DG develops is crucial to understanding adult neurogenesis. This study aims to determine the role of the retinoblastoma (Rb) protein in DG development and to determine if modulation of this pathway holds potential for enhancing neurogenesis in an adult system. A FoxG1 driven Cre is used to delete Rb in the developing forebrain and the resulting effects are analyzed in in vitro and in vivo mouse models. We show that Rb deletion enhances DG neurogenesis by specifically increasing proliferation of immature neurons. Overall this study suggests that Rb pathway modulation could hold potential for enhancing neurogenesis in the adult.

Developmental ethanol exposure causes both structural and functional changes in the brain that can result in cognitive and behavioral abnormalities. The hippocampal formation, an area of the brain strongly linked with learning and memory, is particularly vulnerable to the teratogenic effects of ethanol. Research in this thesis focused on uncovering the effects of developmental ethanol exposure on hippocampal function in adulthood, particularly synaptic plasticity (a putative neurobiological mechanism of learning and memory). The first experiment sought to determine the temporal vulnerability of hippocampal synaptic plasticity as a function of exposure to ethanol during a single trimester. Ethanol exposure during the 1st or 3rd trimester equivalent resulted in minor changes in synaptic plasticity in adult offspring. In contrast, ethanol exposure during the 2nd trimester equivalent resulted in a pronounced decrease in long-term potentiation (LTP), indicating that the timing of exposure determines the severity of the deficit. The second experiment was aimed at determining the effects of prenatal ethanol exposure (1st and 2nd trimester equivalent combined) on bidirectional synaptic plasticity. Prenatal ethanol exposure resulted in a profound reduction in LTP but did not affect long-term depression. These findings show that prenatal ethanol exposure creates an imbalance in bidirectional synaptic plasticity. The third experiment sought to determine if prenatal ethanol exposure alters the affect of acute ethanol exposure in adulthood on synaptic plasticity. Acute exposure to ethanol in adulthood attenuated LTP in control offspring. Conversely, the magnitude of LTP was not affected by acute ethanol application in prenatal ethanol offspring. These results suggest that prenatal ethanol exposure alters the physiological response to ethanol in adulthood. Together, the results from the experiments undertaken in this thesis demonstrate long-lasting alterations in synaptic plasticity as the result of developmental ethanol exposure. Furthermore, these results allude to a malfunction of neural circuits within the hippocampal formation, perhaps relating to the learning and memory deficits observed in individuals with fetal alcohol spectrum disorders. / Graduate

Cognitive aging is a biological process characterized by physical changes in the brain and subsequent alterations in cognitive function. While neurodegenerative diseases result in extensive neuronal death and anatomical abnormalities, normal aging has subtle changes resulting in a range of cognitive abilities. Early studies of cognitive aging focused on changes in the neuronal population, but evidence has demonstrated that forebrain neurons are largely preserved with age. Furthermore, the proliferation of new neurons in the adult brain has generated great speculation regarding the role and contribution of new neurons to cognitive function. Conversely, both imaging and ultrastructural analyses have shown that age-related alterations in white matter and myelin are good predictors of cognitive impairment, suggesting that alterations in connectivity between brain regions may result in cognitive decline. In this dissertation, a rhesus monkey model of normal aging was used to assess the contribution of adult-neurogenesis and oligodendrogenesis to cognitive function. First, cell proliferation and adult neurogenesis were assessed in the subgranular zone of the hippocampal dentate gyrus. Aged animals demonstrated a decline in proliferating cells and neurogenesis but only limited correlations with behavioral impairment. Immature neurons were also identified in temporal lobe cortices, but results indicate these immature cortical neurons are most likely not adult-generated. Moreover, despite an age-related decline in numbers, they persist throughout the lifespan and many differentiate into Calretinin neurons. Further investigation of white matter alterations used immunohistochemistry and diffusion spectrum imaging to correlate oligodendrocyte numbers with white matter connectivity. In the corpus callosum and cingulum bundle, there were no correlations with age, but cognitive impairment was associated with increased oligodendrocyte number and decreased white matter connectivity. These correlations were only present in the anterior aspect of the cingulum bundle, not the posterior cingulum suggesting differential oligodendrocyte responses along the anterior-posterior axis of the brain. Together, these data demonstrate an age-related decline in adult neurogenesis may be only a small contributor to cognitive impairment. Additionally, a reserve pool of immature neurons continues to differentiate in the temporal cortex potentially contributing to local plasticity. Furthermore, cognitive impairment rather than aging has a stronger correlation with oligodendrocytes alterations and connectivity.

New neurons continue to be added to the dentate gyrus (DG) throughout adulthood and enhancing neurogenesis in this region holds therapeutic potential. However, the molecular mechanisms underlying DG neurogenesis remain elusive. Since developmental and adult neurogenesis often share the same signaling pathways, understanding how the DG develops is crucial to understanding adult neurogenesis. This study aims to determine the role of the retinoblastoma (Rb) protein in DG development and to determine if modulation of this pathway holds potential for enhancing neurogenesis in an adult system. A FoxG1 driven Cre is used to delete Rb in the developing forebrain and the resulting effects are analyzed in in vitro and in vivo mouse models. We show that Rb deletion enhances DG neurogenesis by specifically increasing proliferation of immature neurons. Overall this study suggests that Rb pathway modulation could hold potential for enhancing neurogenesis in the adult.

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.

No description available.

Animals utilize a repertoire of defensive behaviors to avoid predators and other noxious stimuli. Successful implementation of these behaviors depends on both the external environment and the internal state of the animal as the brain makes a series of computations to integrate and use such information. Memory systems in particular play a highly influential role in mediating defensive strategies based on previous experiences. These events are encoded in the brain as a form of episodic memory and recruit the hippocampus. Yet, how hippocampal cell populations (i.e., engram ensembles) that drive memory expression modulate downstream neural systems to properly gate defensive behaviors is unknown. To address this, we use activity-dependent labeling strategies to leverage optical control over a hippocampal engram ensemble that encodes the information of a fearful experience (i.e., hippocampal CFC [Contextual Fear Conditioning] engram). Our first experiment aimed to test for the behavioral flexibility of a hippocampal CFC engram, where we use optogenetics to artificially reactivate this fear memory-bearing ensemble across environments that differed in size. We quantified freezing behavior, which is a passive defensive behavior that is commonly associated with negative affective states in rodents, such as fear, and found that environment size influenced the amount of light-induced freezing. From there, our second experiment utilized whole-organ immunohistochemistry, light-sheet microscopy, and graph theoretical analyses to identify regions of interest that were preferentially engaged during hippocampal CFC engram reactivation. Our manipulations conferred positive correlations in brain-wide endogenous cFos expression, induced alterations in network topology, and recruited regions spanning putative memory and defense systems as hubs in respective networks. Lastly, our third experiment aimed to test for the necessity of a hub region for generating light-induced freezing when a hippocampal CFC engram was reactivated in a small arena. Our preliminary results suggest that the lateral hypothalamic area could be one of many regions important for integrating information to generate light-induced freezing, but future work is required to further tease out its role. Overall, by identifying and manipulating the circuits supporting memory function, as well as their corresponding brain-wide activity patterns, it is thereby possible to resolve systems-level biological mechanisms mediating memory’s capacity to modulate behavioral states.

The dorsal dentate gyrus (DDG) of the hippocampus plays a role in the expression of different forms of flexible behaviour mainly due to its ability to sustain neurogenesis throughout life. In the present thesis, we examined the role that the DDG and its adjacent areas, both collectively referred to as dorsal medial hippocampus (DMH), play in flexible, adaptive behaviour and cognitive processing. We used the neurotoxin, colchicine, to induce lesions of the DDG, which were found to affect neighbouring areas. Thus these lesions will be referred to as lesions of the DMH. In the first experiment, rats were tested for (1) perseverative behaviour before and after receiving chronic methamphetamine (METH) treatment, (2) METH-induced locomotor activity and stereotypy in an open field, and (3) working memory in a T-maze. The results showed that rats with lesions of the DMH exhibited perseveration and supersensitivity to the locomotor- and stereotypy-inducing effects of METH (0, 0.1, 0.3, 1 mg/kg i.p.) as well as increased long-term METH sensitization. Rats with DMH lesions also showed significant working memory deficits. Taken together, these results reveal specific forms of behavioural inflexibility in rats with lesions of the DMH that are mainly associated with perseveration, drug-related behaviours, including stimulant motor supersensitivity and drug sensitization, and impaired working memory functions.

Adult hippocampal neurogenesis—a unique form of plasticity in the dentate gyrus (DG)—is regulated by experience, and when manipulated can have specific effects on behavior. Different methods have been used over the years to study new neurons’ functional role in the hippocampus, many of which focus on ablating neurogenesis. While ablation methods can test the necessity of adult-born granule cells (abGCs) for behavior, these techniques remove all abGCs from the circuit and thus do not allow one to determine which properties of abGCs are required for behavior. Such information is required to understand the mechanism of their action. Thus, new strategies are needed to determine what properties of young abGCs allow them to distinguish themselves from their mature counterparts and uniquely impact behavior. Recent hypotheses have suggested that the enhanced synaptic plasticity exhibited by 4–6-week-old abGCs allows them to uniquely contribute to hippocampal circuit function, and thus behavior. The primary goal of this thesis was to explore the contribution young abGCs’ heightened synaptic plasticity makes to hippocampal function. This was achieved using a transgenic mouse approach that allowed for the conditional deletion of NR2B from abGCs. Overall, iNR2BNes mice generated the same number of new neurons in adulthood as control mice at baseline. These neurons survived and matured with only a slight reduction in dendritic complexity. However, a potentially important electrophysiological property of these neurons—their enhanced synaptic plasticity—had been eliminated. From an electrophysiological standpoint, iNR2BNes mice resemble mice with ablated neurogenesis, while from all other neurogenic standpoints examined they most closely resemble wild-type mice. Consequently, these mice provided a novel model to test the extent to which young abGCs’ enhanced plasticity contributes to hippocampal-dependent behaviors. The results reveal that eliminating NR2B-containing NMDA receptors from abGCs does not alter baseline anxiety or antidepressant (AD)-like behavior. However, iNR2BNes mice differed from controls in measures of cognitive function. These mice were able to learn in the contextual fear conditioning test, but were impaired in the more difficult contextual fear discrimination test. Mice also exhibited a decreased novelty exploration phenotype that impaired their performance in the novel object recognition test. Together, these results indicate that the NR2B-dependent heightened plasticity exhibited by 4–6-week-old abGCs is necessary for responses to novelty and fine contextual discrimination, but does not contribute to baseline anxiety or emotionality. AD treatment increases levels of adult neurogenesis in the hippocampus, and these newborn neurons have been shown to be necessary for some of the behavioral effects of ADs seen in rodents. In addition, the maturation timeline of adult neurogenesis correlates with the onset of behavioral responses to ADs. ADs also enhance a neurogenesis-dependent form of long-term potentiation (LTP) in the DG evoked by medial perforant path stimulation under intact GABAergic tone called ACSF-LTP. Thus, a potential mechanism by which abGCs may contribute to AD behavioral efficacy is by providing extra plastic units to the DG circuit. This theory was tested by once again using the mouse line in which NR2B can be conditionally deleted from abGCs in the DG. Here, we found that deletion of the NR2B subunit significantly attenuated a neurogenesis-dependent behavioral response to fluoxetine in the novelty suppressed feeding test, and additionally blocked fluoxetine’s ability to enhance young abGCs’ maturation and subsequent integration into the hippocampal network. This suggests that eliminating abGCs’ enhanced plasticity decreases their ability to influence DG output resulting in an AD response that is less robust than seen in control mice. Control experiments revealed the specificity of this effect, as NR2B deletion did not impact the effect of fluoxetine in a neurogenesis-independent behavioral assay (tail suspension test) or in an assay that was insensitive to fluoxetine in this strain of mice (elevated plus maze). Our efforts to isolate the contribution of abGCs’ unique physiology from the neurogenic effects of fluoxetine were not entirely successful as the results presented here also revealed slight group differences in neurogenesis between control mice and mice lacking NR2B in young neurons. Yet, this data still supports the idea that fluoxetine increases the ability of abGCs to participate in DG output by increasing the chance that new neurons will be activated during DG stimulation. This may be achieved either by increasing their overall number, increasing their potential to make synaptic connections, or increasing their ability to strengthen their connections. However, due to the close link between activity and maturation that appears to be enhanced with fluoxetine treatment, a different approach with greater temporal resolution is needed to separate the neurogenic effects of fluoxetine from the physiological contribution abGCs make to hippocampal output. With this in mind, a mouse line in which abGCs could be temporally inhibited was also generated. Cellular and behavioral characterization of mice conditionally expressing hM4Di—a mutated muscarinic acetylcholine receptor that is insensitive to endogenous acetylcholine, but can be activated by the biologically inert, highly bioavailable compound, clozapine N-oxide (CNO)—has begun. Results show that acute CNO treatment in mice expressing this designer receptor exclusively activated by a designer drug (DREADD) in DG granule cells can impair encoding of contextual fear memory. Chronically treating these mice had an anxiogenic effect in the open field test, but otherwise anxiety and emotionality in these mice were comparable to controls. Chronic CNO treatment in mice expressing hM4Di in young abGCs effectively decreased these cells’ dendritic complexity, but did not alter proliferation or early survival. Thus, hM4Di DREADDs represent a novel tool that can be used to modulate activity of neurons in a temporally restricted manner, allowing for both acute and chronic manipulations of hippocampal granule cells. The experiments put forth in this thesis will highlight the importance of abGCs enhanced plasticity. The utility as well as potential pitfalls of the mouse models used here to test theories of abGC function will also be explored. Hopefully this analysis will provide an improved framework in which future experiments can be developed with the aim of uncovering novel insights into the hippocampal circuitry that underlies learning and memory and discovering new strategies for the treatment of neurological and psychiatric disorders.

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.