Graduate Program in Neuroscience - Ph.D. / Sc.D.
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Item Open Access Behavioral and neural investigation on the effect of spatial attention on surround suppression(2023-09) Kınıklıoğlu, Merve; Boyacı, HüseyinWhen a visual stimulus is presented together with other stimuli surrounding it, behavioral sensitivity and neural responses may change, often reduce, compared to when the same stimulus is presented alone. This is commonly referred to as center-surround interaction or surround suppression, and it is one of the most fundamental mechanisms in biological vision. It is well documented that in motion perception, center-surround interaction is affected by the size and contrast of the stimulus. As the size of a drifting grating increases, motion direction discrimination performance, as well as neural activity in one of the main cortical motion processing areas, medial temporal complex (MT+), decreases if the grating has high contrast (surround suppression). Whereas, when the size increases within certain limits, both the discrimination performance and the neural activity in MT+ may increase if the grating has low contrast (surround facilitation). On the other hand, spatial attention is known to modulate surround suppression both in humans and non-human animals with static stimuli. No previous study, how-ever, has directly and systematically investigated the effect of the spatial extent of attention on surround suppression in human motion perception. The studies presented in this dissertation aim to investigate the effect of the extent of spatial attention on center-surround interaction in visual motion processing. In our experiments, we used two attention conditions and a novel stimulus design, where a ‘center’ and a ‘surround’ drifting grating were presented to the participants. Under one of the attention conditions, which we call the ‘narrow-attention’ condition, participants performed a task that limited their attention to the central part of the stimulus. Under the other attention condition, which we call the ‘wide-attention’ condition, participants performed tasks that required them to extend their attention to both the center and surround gratings. Using this experimental paradigm, we measured motion direction discrimination thresholds behaviorally and cortical activity with fMRI. Behaviorally, we found increased thresholds, that is, stronger surround suppression, under the wide attention condition. In the hu-man homolog of MT+ (hMT+), we found that increasing the spatial extent of attention leads to reduced cortical responses, that is, to stronger neural suppression. This was not the case for the activity in the primary visual cortex (V1). Finally, we show that a parsimonious computational model that incorporates spatial attention and response normalization can successfully predict the response patterns in hMT+ and V1. Furthermore, the model could provide a link between cortical responses and behavioral thresholds. Overall, our findings and analyses showed that the behavioral effect can be successfully predicted by hMT+ activity. These results reveal the critical role of spatial attention on surround suppression, namely that surround suppression in motion perception becomes stronger with a wider attention field, and reveal possible cortical mechanisms underpinning the effect.Item Open Access Effects of context and expectations on dynamics of visual processing(Bilkent University, 2023-09) Malik, Amna; Boyacı, HüseyinLiving organisms rely on the information they receive through their senses to survive and function in an environment that is constantly changing. However, incoming information from the senses is often ambiguous, noisy, or incomplete. Context, either spatial, temporal, or cognitive, is fundamental, especially in visual perception, to disambiguate and complete this noisy input and optimize behavior. The mechanisms by which contextual information influences visual perception, however, are not fully understood. Studies in the current thesis aim to add to the understanding of those mechanisms. It comprises two lines of work. The first line of work focuses on the spatial context, where using fMRI, we investigate the neural correlates of spatiotemporal properties of context-dependent lightness perception. Results show that activity in the primary visual cortex (V1) correlates with context-dependent lightness perception, providing evidence for low-level mechanisms underlying the contextual effects. The second line of work focuses on the cognitive context, where we systematically study the effect of expectations about dynamic material properties on perceptual decisions. To do so, we used behavioral methods, where we manipulated participants’ long-term and short-term expectations about material properties. Results show that expectations about material properties can impact relatively low-level perceptual decision-making processes. Furthermore, we found an interplay between long-term and newly learned expectations. In conclusion, the current thesis broadens our understanding of how context influences visual processes, particularly by pro-viding evidence that low-level processes are affected by the visual context. This knowledge has the potential to help develop more accurate models of visual perception, which in turn can have implications in clinical neuroscience, artificial intelligence, computer vision, and marketing.Item Open Access Cortical processes underlying attentional modulations of dynamic vision(Bilkent University, 2022-09) Çatak, Esra Nur; Kafalıgönül, Hacı HulusiVisual attention is one of the most fundamental cognitive functions guiding and influencing a various number of processes. However, how different neural mechanisms are modulated by selective attention to process information is still subject to debate. Utilizing electroencephalography (EEG), the current thesis focused on understanding the time course of visual information processing and its neural underpinnings with paradigms that operate in different attentional modes, such as visual masking, attentional load, and transparent motion design. First, we aimed to understand the role of spatial attention in information processing and its possible interactions with metacontrast masking mechanisms. The behavioral results revealed an interaction effect that suggests differential effects of spatial attention on metacontrast masking. The following EEG analyses revealed significant activation due to masking and attentional load on early negative components located over occipital and parieto-occipital scalp sites, followed by a late positive component centered over centro-parietal electrodes. These findings suggest that the effect of spatial attention may have distinct characteristics at different stages of sensory and perceptual processing regarding its relationship with metacontrast masking. Secondly, by employing a novel variant of transparent motion design with color and motion swapping, we aimed to isolate the object-based cueing effect from a possible feature-based explanation in both psychophysical measures and neural activities. Our results demonstrate that the behavioral effects of attentional cueing survived feature swaps, providing evidence for an object-based attention mechanism. We also observed event-related potential correlates of these object-based selection effects in the late N1 component range, over occipital and parieto-occipital scalp sites, significantly associated with the variation in behavioral performance. Our findings provide the first evidence of the role of the N1 component in object-based attention in this transparent-motion design under conditions that rule out possible feature-based explanations. Taken together, the present results highlight the substantial effects of selective attention on the processing of visual information after the initial entry of information into the visual system and before the completion of its processing.Item Open Access Transcriptomic alterations in the aged brain with and without dietary and dietary- mimetic manipulations(Bilkent University, 2021-12) Erbaba, Begün; M. Adams, MichelleAside from many genetic and environmental influences on the brain, aging itself is a significant risk factor for accelerated cognitive decline, making aging research crucial due to the increasing population age in our era. We aimed to discover gene expression differences in the aging zebrafish brain using three age groups in the first aim. We identified gjc2 (CX47) and alcamb (ALCAM) cell adhesion genes showing consistent downregulation with age across all experiments. ALCAM is also known to be associated with neuroinflammation, which has been implicated to be lowered using anti-aging, non-genetic nutrient interventions. In the second aim, we applied 12 weeks of two opposing nutrient interventions, caloric restriction (CR) and overfeeding (OF) in aging zebrafish, in order to be able to propose a reliable therapeutic approach for reversing age-related neurobiological changes. We measured protein and expression level differences of selected genes related to proliferation to inflammation with these diets. The results showed that sox2 gene expression was significantly upregulated following OF treatment than CR diet, and myca and tp53 mRNA levels were significantly downregulated with advanced age. Alcamb and tfdp1 expression levels were also marginally significantly lowered with CR compared to other groups. Meanwhile, we also conducted another transcriptomic approach using microarray to assess gene expression differences with CR compared to Ad-libitum (AL) feeding. Thus, lastly, in the third part, we found that CR causes changes in cell cycle regulation among several other functional regulatory pathways in zebrafish brains. We identified the tfdp1 gene, which showed downregulation with CR, as a possible CR regulator. Then, to create a CR mimic, we performed morpholino oligo (MO) injections to zebrafish embryos and adult brains to knock down tfdp1 gene expression levels. The injections were not successful in altering Tfdp1 protein levels in neither embryos and adults. However, 8ng tfdp1-MO injections in embryos significantly increased myca and tp53 expression levels, which are among the downstream targets of tfdp1. Our examinations shed light on healthy brain aging and possibly propose new drug targets.Item Open Access Spatially informed voxelwise modeling and dynamic scene category representation in the human brain(Bilkent University, 2021-12) Çelik, Emin; Çukur, TolgaHumans have an impressive ability to rapidly process global information in natural scenes to infer their category. Yet, it remains unclear whether and how scene categories observed dynamically in the natural world are represented in cerebral cortex beyond few canonical scene-selective areas. To address this question, here we examined the representation of dynamic visual scenes by recording whole-brain blood oxygenation level-dependent (BOLD) responses while subjects viewed natural movies. We fit voxelwise encoding models to estimate tuning for scene categories that reflect statistical ensembles of objects and actions in the natural world. Voxelwise modeling (VM) is a powerful framework to predict single voxel responses evoked by a rich set of stimulus features present in complex natural stimuli. However, because VM disregards correlations across neighboring voxels, its sensitivity in detecting functional selectivity can be diminished in the presence of high levels of measurement noise. Here, we introduce spatially-informed voxelwise modeling (SPIN-VM) to take advantage of response correlations in spa-tial neighborhoods of voxels. To optimally utilize shared information, SPIN-VM performs regularization across spatial neighborhoods in addition to model fea-tures, while still generating single-voxel response predictions. Compared to VM, SPIN-VM yields higher prediction accuracies and better capture locally congruent information representations across cortex. We find that this scene-category model explains a significant portion of the response variance broadly across cerebral cortex. Cluster analysis of scene-category tuning profiles across cortex reveals nine spatially-segregated networks of brain regions consistently across subjects. These networks show heterogeneous tuning for a diverse set of dynamic scene categories related to navigation, human activity, social interaction, civilization, natural environment, non-human animals, motion-energy, and texture, suggesting that the organization of scene category representation is quite complex.Item Open Access Identification of modulatory functions of TP53, estrogen signaling, and 14q32.31 miRNA cluster on CHRNA5 knock-down expression profile and development of syneRgy APP(Bilkent University, 2021-09) Keşküş, Ayşe Gökçe; Karakayalı, Özlen KonuCholinergic receptor subunit alpha 5 (CHRNA5) is a ligand-gated ion channel expressed in not only the nervous system but also other tissues. Differential expression and the polymorphisms of CHRNA5 have been associated with addiction, particularly nicotine and various cancer types. The tumor-suppressive properties of CHRNA5 depletion, i.e., decrease in cell proliferation, induction of DNA damage response, and drug sensitivity, have been identified in breast cancer cell lines. This thesis focuses on identifying critical factors modulating or modulated by the knock-down of CHRNA5 in breast cancer cell lines using both wet-lab and bioinformatics approaches. Here I have first found the significant correlation between CHRNA5 and DNA damage response in breast cancer tumor datasets. Moreover, I discovered that the introduction of siTP53 antagonized the actions of siCHRNA5 and reverted the siCHRNA5-mediated cell cycle inhibition and drug sensitivity in the MCF7 breast cancer cell line. Furthermore, siCHRNA5 was found to inhibit the secondary signaling of estrogen/ESR1 in time and dosage-dependent manners. CHRNA5 depletion also downregulated the conserved 14q32.31 miRNA cluster expression. Among those miRNAs, miR495-3p appeared to be the most prominent candidate, exhibiting a similar expression profile with selective estrogen degraders and partially with siCHRNA5. However, the inhibitory effect of the combinatorial treatment with siCHRNA5 and miR495-3p on the secondary targets of estrogen signaling indicated that siCHRNA5 and miR495-3p might target converging pathways, evidenced by the antagonism (rather than addictiveness) between them. In addition, the Shiny-based syneRgy app was developed to analyze the transcriptome-based synergy between treatments and/or genetic modifications. As a case study, syneRgy analysis using novel MDM2 inhibitor and/or temozolomide treated neuroblastoma cell lines revealed that although the tumor-suppressor effect of combination therapy was more than each individual treatment, it was less than additive. syneRgy was applied to understand the combinatorial treatment of siCHRNA5 with siTP53 as well as siCHRNA5 with miR495-3p mimic and enhanced our understanding of the TFs that might have a role in the crosstalk. To our knowledge, syneRgy is the first-ever online tool to perform statistical synergy analysis using RNAseq count or logFC transcriptomic data and synreg, i.e. our novel methodology allowing for statistical tests of TF target enrichment using regression models.Item Open Access Natural speech representations in the human brain during a cocktail party(Bilkent University, 2021-08) Kiretmitçi, İbrahim; Çukur, TolgaHumans are remarkably adept in selectively listening to a desired speaker in a crowded environment, while filtering out non-target speakers in the background. Attention is key to solving this difficult cocktail-party task, yet a detailed char-acterization of attentional effects on speech representations is lacking. It remains unclear across what levels of speech features and how much attentional modula-tion occurs in each brain area during the cocktail-party task. Besides, it should be clarified whether unattended speech is represented in cortex during selective listening and if so, at what feature levels its representations are maintained. To address these questions, we recorded whole-brain blood-oxygen-level-dependent (BOLD) responses while subjects either passively listened to single-speaker stories, or selectively attended to a male or a female speaker in temporally-overlaid stories in separate experiments. Spectral, articulatory, and semantic models of the natural stories were constructed to enable comprehensive assessments on the hierarchy of speech features. Intrinsic selectivity profiles were identified via vox-elwise models fit to passive listening responses. Attentional modulations were then quantified based on model predictions for attended and unattended stories in the cocktail-party task. We find that acoustic representations are confined to the early auditory cortex whereas linguistic representations are broadly distributed across cortex, that attention causes broad modulations at multiple levels of speech representations (articulatory and semantic) while growing stronger towards later stages of processing, and that unattended speech is represented up to the semantic level in parabelt auditory cortex. These results provide insights on speech perception and attentional mechanisms that underlie the ability to selectively listen to a desired speaker in noisy multi-speaker environments.Item Open Access Investigating motion detection of aging zebrafish with optomotor response(Bilkent University, 2021-07) Karaduman, Ayşenur; Kafalıgönül, Hacı HulusiVarious aspects of visual functioning including motion perception have been shown to be significantly altered throughout aging. Contrary to the traditional view focusing on structural changes in the eye and retina, accumulating evidence suggests that subtle alterations in neural circuitry and functioning are respon-sible for the age-related changes in visual sensitivity and perception. However, the mechanisms underlying age-related changes in motion perception are still poorly understood. This thesis was aimed to investigate the detection of first-and second-order motion direction during aging by using zebrafish optomotor re-sponses (OMR). Furthermore, exposure-based visual learning was investigated by repeated presentation of first-order motion. The studies included both wild-type and achesb55/+ zebrafish with decreased levels of acetylcholinesterase which has been previously shown to delay age-related cognitive decline. In this way, it was also aimed to explore the possible functional links between cholinergic functioning and age-related changes in visual motion processing. The results indicated that adult zebrafish mainly exhibit negative OMR (i.e., position shift in the opposite direction of visual motion) to drifting first-order gratings which is significantly dependent on spatial frequency and contrast level of the motion. Rather than an overall effect of aging, the results revealed a three-way interaction between the contrast level of first-order motion, genotype, and age. Therefore, the findings pointed out a complex relationship between the physical characteristics of first-order motion stimulation and the cholinergic system during neural aging. Contrary to the first-order motion, the second-order motion did not induce strong optomotor responses in adult zebrafish. Although young and old zebrafish exhibited OMR with different polarities (negative and positive OMR, respectively), future work revealing robust responses will be informative to better understand and characterize age-related changes. Lastly, passive exposure of repeated first-order motion induced significant improvements in negative OMR of young and old zebrafish, suggesting that adult zebrafish can be used as a model organism to study passive forms of visual perceptual learning. Overall, these behavioral results pave the way for a detailed investigation of the functional links between the physical motion characteristics and the cholinergic system in the zebrafish aging model which will ultimately have important implications for developing interventions to improve human visual performance during aging.Item Open Access Genetic and environmental interventions altering the course of brain aging: evidence from the zebrafish (Danio Rerio) model(Bilkent University, 2021-04) Eravşar, Elif Tuğce Karoğlu; Adams, Michelle MarieAge-related cognitive decline occurs during normal aging, although there is no prominent neural loss in the brain. Subtle molecular alterations in synaptic and cellular dynamics are likely underlying these cognitive alterations. One challenge is the widely heterogeneous profile regarding age-related behavioral changes and neurobiological underpinnings. Therefore, it is crucial to characterize how individual factors can contribute to successful or unsuccessful aging and whether these factors can induce shared patterns of alterations in the cellular and synaptic dynamics. Three different intervention approaches were utilized in the current study. The first intervention was a genetic manipulation in the cholinergic system component acetylcholinesterase (AChE), which results in reduced levels of this enzyme in the achesb55/+ mutants. Previous studies have characterized this model as a delayed aging model because of its preserved cognitive abilities at an older age. The current study was the first study analyzing the neurobiological changes in this mutant model within the context of aging. It was shown that reduced brain AChE activity levels persist in different age groups, including old age in the mutant animals. This reduction was accompanied by subtle decreases in the other elements of the cholinergic system, including acetylcholine and nicotinic acetylcholine receptor subunit alpha-7. Genotype significantly altered key glutamatergic receptor subunits such as N-methyl D-aspartate-type receptor subunit 2B (NR2B) and glutamate receptor subunits 2 and 3 (GluR2/3), with these markers significantly reduced in the achesb55/+ mutants and likely maintaining homeostatic synaptic scaling. At old age, a significant age-related elevation was observed in the synaptophysin levels (SYP) of the old achesb55/+ mutants, and this mutation prevented an age-related decline in the gephyrin (GEP) levels which was evident in the wild-type controls. This mutation also altered the cellular dynamics; an immature neuronal marker, embryonic lethal abnormal-vision (ELAV Drosophila) like-3 (HuC) was significantly upregulated in the achesb55/+ mutants at all ages. In contrast, the levels of inflammation-related markers, glial fibrillary acidic protein (GFAP) and reactive oxygen species (ROS), were downregulated subtly in the mutants. It can be concluded that reduced levels of brain AChE can be associated with altered excitatory homeostasis and preserved levels of GEP and SYP through aging. At the same time, the neuronal marker was upregulated, and inflammation-related markers were downregulated. The second intervention was applying short-term environmental enrichment using the sensory cues to young and old zebrafish to induce successful aging. It was shown that environmental enrichment increases the brain weight in old zebrafish, prevents age-related decrements in the levels of synaptic proteins, including SYP and NR2B, and doublecortin-like kinase (DCAMKL1). Additionally, environmentally enriched old zebrafish had elevated levels of GEP while applying this environmental intervention did not modulate the age-related increases in oxidative stress indicators. The third intervention was also a non-genetic approach. Two short-term opposing dietary treatments, such as caloric restriction (CR) and over-feeding (OF), were applied to young and old zebrafish and an ad-libitum diet. It was demonstrated that a short-term CR regimen upregulated the glutamatergic components of neurotransmission such as GluR2/3 and post-synaptic density 95 (PSD95). Significant age-related decline in GEP levels was observed in old zebrafish in the OF dietary condition. Expression levels of synaptic and regulatory genes were relatively stable, while inflammation-related gene tnfa was altered in an age-dependent manner. Additionally, in the young zebrafish, a significant elevation of trunk cortisol was demonstrated in the OF group compared to CR-fed young zebrafish. Taken together, evaluating different components such as the cholinergic system, diet, and environment can provide us insights into the neurobiological underpinning of successful aging and possible determinants of unhealthy aging.Item Open Access Behavioral and computational investigation of the effect of prior knowledge on visual perception(Bilkent University, 2021-01) Ürgen, Buse Merve; Boyacı, HüseyinVisual perception results from the dynamic interaction of bottom-up and topdown processes. Top-down prior knowledge and expectations can guide us to predict upcoming events and even determine what we see in an ambiguous or noisy sensory stimulus. Despite the well-established facilitating effects of expectations on recognition or decision-making, whether and how early sensory processes are affected by expectations remain unclear. This dissertation attempts to investigate the effect of expectations on early visual processes. To this end, we used behavioral experiments to examine the effects of expectation on visual perception at the threshold level and implemented a recursive Bayesian model and a recurrent cortical model to unravel the computational mechanisms underlying those effects. In the behavioral experiments, we systematically manipulated expectation’s validity in separate sessions and measured duration thresholds, which is the shortest presentation time sufficient to achieve a certain success level. Our behavioral findings showed that valid expectations do not reduce the thresholds, rather unmet expectations lead them to increase. Next, using a recursive Bayesian updating scheme, we modeled the empirical data obtained in the behavioral experiments. Model fitting showed that higher thresholds observed in the unmet expectations are not due to a change in the internal parameters of the system. Instead, additional computations are required by the system to complete the sensory process. Finally, within the predictive processing framework, we implemented a recurrent cortical model to explain the behavioral findings and discuss possible neural mechanisms underlying the observed effects. The cortical model findings were in agreement with the Bayesian model results, revealing that longer processing is needed when expectations are not met. Overall, the computational models that are proposed in this study provide a parsimonious explanation for the observed behavioral effects. The proposed experimental paradigm and the computational models offer a novel framework that can be extended and used in other stimuli, tasks, and sensory modalities.Item Open Access Identification of preclinical implications for novel indole-benzimidazoles and phenothiazines using in vitro cancer cell line and in vivo zebrafish models(Bilkent University, 2020-09) Yaman, Murat; Karakayalı, Özlen KonuBreast cancer (BC) and hepatocellular carcinoma (HCC) are two major health problems with significant mortality rates. Although drug therapies are available, therapeutic success remains limited. Because of low bioavailability, high toxicity and recurring drug resistance, novel therapeutic options are essential. In the present thesis, a multitude of in vitro, in silico and in vivo approaches were executed to test anti-cancer effects and preclinical potentials of novel indole-benzimidazoles and phenothiazines in BC and HCC, respectively. In the first component of the thesis, I evaluated BC cell line toxicity and estrogen receptor (ER) relationship of novel indole-benzimidazole derivatives using in vitro cancer lines, in vivo zebrafish embryos/larvae, and in silico comparative transcriptomics analyses. In the second part, antipsychotic compounds phenothiazines (PTZ) were repurposed for HCC therapy. Therefore, generic PTZ derivatives alone or in combination with sorafenib (SFB) were tested using in vitro cancer lines followed by zebrafish developmental assays and embryonic stage xenografts. In addition, RNAseq analyses were performed on trifluoperazine (TFP), SFB, and TFP+SFB combination treated Hep3B cells to understand synergistic/antagonistic effects of the drugs at gene expression level. Lastly, anti-HCC potential of novel PTZ derivatives were explored by in vitro and in vivo screenings. Moreover, effects of the novel and generic derivatives on neural pathways were evaluated by cholinesterase assays and motor response measurements. The findings of the dissertation present potential leads for conducting further preclinical studies tailored towards novel BC and HCC therapies.Item Open Access Probing sensory plasticity with rapid forms of motion adaptation(Bilkent University, 2020-09) Akyüz, Sibel; Kafalıgönül, Hacı HulusiPerception is shaped by both immediate pattern of sensory inputs and previous experience with the external environment. Visual adaptation, a temporary change in perception following exposure to a stimulus, has been widely employed to understand how previous sensory experience on different timescales shapes perception. Visual motion adaptation is a powerful investigative tool to understand sensory plasticity and neural adaptation. However, the neural mechanisms underlying adaptation induced changes by visual motion are still subject to debate. In the present thesis, spatiotemporal dynamics, neural substrates, and functional role of sensory plasticity in the human visual system was examined using rapid forms of motion adaptation paradigm combined with EEG. Specifically, how motion adaption-induced short-term sensory plasticity is reflected at the neural level and parallel with perceptual performance were explored. Participants were adapted to directional drifting gratings for either short (640 ms in Experiment 1; 188 ms in Experiments 2 and 3) or long (6.4 s in Experiment 1; 752 ms in Experiments 2 and 3) durations and used a counter-phase flickering (with constant polarity in experiments 1 and 2; polarity inverting within every step in Experiment 3) grating as a test pattern. Sinusoidal gratings of phi motion were employed in Experiment 1 whereas; square wave gratings were used for phi and reverse-phi adaptations in Experiments 2 and 3 to examine how ON and OFF pathways operate in the visual processing stream. Based on the EEG analyses in Experiment 1, the scalp sites relevant to motion adaptation were identified. Experiment 1 showed that both adapting durations led to significant motion aftereffects and EEG results showed that long adaptation produced stronger aftereffects than the short adaptation condition within 64-112 ms time range over occipital and parieto-occipital sites. Taken together, these findings provide important electrophysiological evidence that motion aftereffects reflect changes in cortical areas mediating low- and mid-level visual motion processing. They also suggest that adaptation is an active process that involves neural mechanisms operating at different time scales. In Experiments 2 and 3, the short-term adaptation induced changes over these identified scalp sites were further examined based on Experiment 1. Given that the phi and reverse-phi motion mainly engage within (ON or OFF) and across (ON and OFF) pathway mechanisms, the comparisons of adaptation induced changes across these motion types provided further insights into the nature of corresponding mechanisms over visual cortex. The behavioral and EEG findings pointed to efficient convergence of information provided by these pathways and some distinct characteristics of across pathway mechanisms.Item Open Access Effects of aging, diet and potential genetic interventions on the levels of Smurf2 and its interacting partners in Zebrafish (Danio Rerio) brain(Bilkent University, 2020-09) Şaşik, Melek Umay Tüz; Adams, Michelle MarieAging is a natural process that is ultimate combination of numerous intrinsic and extrinsic changes in an organism. Contrary the common belief, brain aging is not a loss of neurons while it has been shown that subtle cellular and synaptic alterations have contribution to brain aging. Therefore, the molecular and cellular alterations may give more insight into the brain aging process. There are some hallmarks of aging that are common features in different organisms including genomic instability, telomere attrition, cellular senescence. There are some common factors with the ability to regulate more than one of the hallmarks of aging such as Smurf2. HECTdomain E3 ubiquitin ligase Smurf2 has several roles in the cellular processes for example, telomere attrition and cellular senescence. Moreover, its gene expression is higher in the aged brain. Although there are several publications about Smurf2, most of them focused on its role in cancer. We believed that Smurf2 levels should be examined in terms of brain aging. The first aim of the study was to examine the levels of Smurf2 and its interacting partners across lifespan. Although the Smurf2 protein level was not increased significantly in the whole zebrafish brain, its protein level was upregulated significantly in telencephalon and cerebellum. Also, subcellular protein fractionation demonstrated an enriched Smurf2 level in the cytosolic part. In the case of gene expression levels, smurf2 level was significantly higher in aged whole brain although its expression was downregulated during aging in telencephalon and cerebellum. In addition, the levels of mdm2, ep300a and sirt1 were lower in the aged telencephalon. According to multivariate analysis there is a potential balance between Smurf2-mediated ubiquitination, ep300a-mediated acetylation and Sirt1- mediated deacetylation but with advancing age, this balance may disrupt and other regulatory genes should also take a role to sustain cellular stability. The second aim was to investigate the roles of Smurf2 on brain aging with the help of genetic interventions including inducible knockin, stable knockout or transient knockdown. Since stable knockin and knockout models should be genotyped before further investigations, the genotyping and phenotyping methods were employed to find an efficient and reliable way. Also, transient knockdown via Vivo-morpholino was applied to adult brain and efficient post injection times of two different morpholinos were identified in order to examine the effects of Smurf2 knockdown in both young and old zebrafish. Lastly, it was aimed to examine the effects of non-genetic interventions including dietary regimens and pharmacological compounds on the gene expression of smurf2 and its interacting partners and the levels of the neuronal proteins and proliferation/senescence proteins. The opposing short-term dietary regimens, overfeeding and caloric restriction, were altered the levels of neuronal proteins, HuC and DCAMKL1, and their relation with proliferation and senescence proteins during aging. Also, the gene expression levels of smurf2 and interacting partners except tp53 was not influenced by dietary regimens and aging in terms of whole brain. Also, multivariate analysis indicated that the correlations among smurf2, mdm2, ep300a and sirt1 were conserved in both young and old ages independent to dietary regimen which may imply that the balance between ubiquitination, acetylation and deacetylation is maintained in order to provide cellular stability during aging. Heclin, an inhibitor of HECT E3 ligases, were employed to inhibit Smurf2 activity. Before using in adult zebrafish, heclin was applied to embryos to see its effects. The higher dose of heclin decreased the survival ratio and altered the gene expression levels of downstream gene drastically. So, moderate dose of heclin should be applied to the adult brain and neuronal markers should be examined to observe target effects rather than off-target, unspecific impacts. Taken together, Smurf2 has potential roles during aging and it could be a promising target to delay the brain aging process and probably the onset of age-related cognitive decline.Item Open Access Age-dependent effects of short-term intermittent fasting and rapamycin treatment in Zebrafish (Danio Rerio) brain(Bilkent University, 2020-05) Birand, Ergül Dilan Çelebi; Adams, Michelle MarieWorld populations are rapidly aging, and there is an urgent need to develop interventions that prevent or reverse age-related deterioration of health. To date, several approaches have been developed to extend health span. Among these, non genetic interventions have a higher potential to be utilized in translational studies. Caloric restriction (CR) and its pharmacological mimetic rapamycin, are two applications that have been shown to reliably extend life and health span across species. Despite a growing body of knowledge on how CR and rapamycin show their beneficial effects, their molecular mechanisms in the brain are not completely understood. Furthermore, most studies applied life-long CR, which is not suitable for translational research. To fill this gap, we investigated whether short-term durations of a CR approach intermittent fasting (IF) or rapamycin altered cellular and molecular markers of critical processes in the brain as well as metabolic parameters in the body. To assess how the age of the subjects affect the outcome of the treatments, we included young (6-10 months old) and old (26-31 months) zebrafish, which has recently emerged as a suitable model for gerontological research. Our results demonstrated that IF decreased whole-body glucose and cortisol levels, and increased neural progenitor marker DCAMKL1 in young and old animals. While this proliferation-promoting effect was preceded by suppression of mTOR activity in young, the upregulation of foxm1 and reduced autophagic flux as measured by LC3 II/LC3-I ratio were observed in old animals. Rapamycin, on the other hand, did not alter the metabolic parameters and induced entirely different molecular profiles at young and old ages. The most notable changes in young animals were reduced mTOR activity, LC3-II/LC3-I ratio and expression levels of a global proliferation marker PCNA. In old animals, the marker of activated astrocytes (i.e. GFAP) was decreased, indicating lower neuroinflammation, whereas excitatory-inhibitory balance as measured by PSD-95/Gephyrin ratio was shifted towards a more excitatory state. These results suggested that IF and rapamycin induced distinct metabolic profiles in young and old animals. Furthermore, there was an age dependent reciprocal relationship between proliferation and autophagy, which might be partly due to differential regulation of mTOR activity. Interestingly, rapamycin treatment was more effective in suppressing mTOR activity in young animals, and compared to IF. Nevertheless, these results suggested that rapamycin crosses the blood-brain barrier in zebrafish, and that short-term durations of IF or rapamycin were sufficient to alter the expression levels of key proteins involved in critical mechanisms in the brain.Item Open Access Genetic influences on cortical structure and function(Bilkent University, 2018-07) Demirayak, Pınar; Doerschner, KatjaStructure and function of the human central nervous system is determined by both genetic and environmental in uences. One of the fundamental quests in neuroscience studies is to determine to what degree each of these two factors in uence the development and function of the nervous system and, ultimately, human behavior. However, this is an inherently di cult problem to tackle as it is nearly impossible to tease apart the individual contributions of genes and environment, since they interact heavily throughout an organism's life. Recently, our understanding about the role that speci c genes play in the development of brain structure and function, has been greatly advanced by studies that combine genetic and neuroimaging methods to investigate congenital neurodevelopmental disorders. These studies of patients, that are homozygous for a speci c mutation, allow to single out contributions of individual genes in the neurodevelopmental process and have the potential to reveal gene-based alterations in cortical structure and function that can not be compensated by mechanisms of cortical plasticity or mitigating environmental e ects. In this thesis I pursue this promising approach further and investigate the e ects of three di erent single gene mutations on brain structure and function - namely RAD51, LAMC3 and HTRA2. Each of these genes is highly expressed during neurodevelopment, and each in uences cortical structure and function di erently. Overall, I nd that these genes are all highly associated with abnormal structural and functional connectivity patterns, however, and surprisingly, highly abnormal structure does not necessarily predict highly abnormal behavior.Item Open Access The effect of context-dependent lightness on contrast detection and identification, and its neural correlates(Bilkent University, 2017-10) Karatok, Zahide Pamir; Boyacı, HüseyinPerceived contrast of a grating varies with its background (or mean) luminance: of the two gratings with the same photometric contrast the one on higher luminance background appears to have higher contrast. On the other hand, context often causes a large perceived difference between equiluminant regions (e.g., simultaneous brightness contrast). Does perceived contrast also vary with contextdependent background lightness even when the luminance remains constant? In this study, the effect of context-dependent lightness on contrast perception was investigated using psychophysical and functional magnetic resonance imaging (fMRI) methods. First, we measured appearance judgments of participants and demonstrated that context-dependent lightness of background in uences the perceived contrast of rectified gratings. Perceived contrast of gratings superimposed on equiluminant but perceptually lighter background is higher compared to ones on perceptually darker backgrounds. However, this pattern is valid only for incremental, not for decremental contrast. Literature indicates a significant difference between visual processing near and above threshold. Also, behaviorally it has been shown that appearance and threshold tasks are mediated by different mechanisms. Therefore, here, we also measured the effect of context-dependent lightness on contrast detection and discrimination thresholds using a 2-IFC procedure. Results indicate that both detection and discrimination thresholds are lower for the gratings superimposed on perceptually lighter backgrounds. Differently from the appearance results, the effect was observed both for incremental and decremental contrast. In an fMRI study, we investigated whether activity in any brain region correlates with background-lightness-dependent contrast perception. Although our stimulus was physically identical, we observed difference in BOLD response within pre-defined region of interests (ROIs) in different visual areas. Both for incremental and decremental contrast, activation, especially in V1, was greater when the grating was superimposed on lighter background for all the contrast levels tested. Variation in V1 activity with varying contrast links better with the detection and discrimination thresholds than the appearance results. Therefore, this study might offer a neural evidence for dissociation between the mechanisms underlying detection (threshold) and identification (appearance) measures. However, the relationship between the threshold and fMRI data does not really agree with the previous findings in literature. These results indicate that the neural activation caused by the detection mechanism may change depending on the absolute or perceived value of the contrast.