Neuropeptides are a class of neural signaling molecules that play a pivotal role in brain function and human health through neuromodulatory influences. There are over 100 types of neuropeptides identified and characterized, yet genomic analysis suggests that it is only the tip of the iceberg, with extra hundreds of putative neuropeptides awaiting further investigation. Neuropeptides collectively regulate a variety of developmental, physiological, and behavioral functions. While each neuropeptide is idiosyncratic in regard to its molecular structure, chemical properties, and anatomical distribution, they impinge on the nervous system in a similar fashion.
Surprisingly, despite their fundamental importance, techniques for measuring the localization, expression and release of neuropeptides, at large scale and with high spatiotemporal resolution, have lagged far behind. Microdialysis and fast-scanning cyclic voltammetry are useful primarily for measuring “volume transmission,” but are invasive, and have poor spatial resolution and limited general applicability. FP-tagged vesicle reporters are mainly tested and used in limited cell types. Little is characterized about their functional universality and specificity. GPCR-based sensors are designed to visualize the binding, instead of expression and release, of a neuropeptide.
Therefore, I aim to develop new methods for visualizing, detecting, and inhibiting NP expression and release in vivo. The long-term goal is to apply these methods to understanding the dynamics of neuromodulation of specific, behaviorally relevant neural circuits, and to providing a dynamic, high-resolution view of chemical modulation of circuit function.
In Chapter 2, I will describe the design, screening, and proof-of-concept validation of novel genetically engineered neuropeptide release reporters (NPRR) in Drosophila. I further demonstrated the idiosyncrasy of neuropeptide release dynamics, as well as cell-type specific release properties of a neuropeptide. In Chapter 3, I conceived and constructed a neuropeptide imaging platform that exploits the discoveries and strategies from Drosophila NPRRs. Besides a series of redesign of mammalian NPRRs, a collection of sister reporters to visualize localization and expression (Neuropeptide Localization and Expression Reporter, NPLER) were built in parallel. I also established a prototypical pipeline to systematically screen for appropriate cell lines for the purpose of NPRR/NPLER applications.
Malfunctioning of neuropeptide pathways can potentially result in a variety of mental illnesses triggered by stress, and metabolic disorders including obesity. Drugs targeting neuropeptide signaling have received heavy investment, but most have failed in the clinical trials. We therefore propose alternative strategies to target the processing/release of the neuropeptide from neurons, rather than blocking its receptor. In Chapter 4, I describe the ongoing process of adapting modern biotechnologies to the imaging platform to explore novel therapeutic strategies for neuropeptide- relevant disorders and abnormalities.
The Appendix includes a serendipitous finding from our attempt to generalize NPRR to Caenorhabditis elegans.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/v13r-yt57, author = {Yang, Bin}, title = {Transformations and Functions of Neural Representations in a Subcortical Social Behavior Network}, school = {California Institute of Technology}, year = {2022}, doi = {10.7907/v13r-yt57}, url = {https://resolver.caltech.edu/CaltechTHESIS:05232022-073842501}, abstract = {The brain functions by processing sensory information such as vision, smell, and touch, integrating it with internal states (hunger, fear, aggression) and memory to produce relevant motor outputs (eating, fleeing, or fighting). To understand the brain, neuroscientists study neural representations (patterns of neural activity that correlate with features of the outside world) to and perform perturbations (activate or silence groups of neurons) to determine its function. Past studies on neural representations gave us insights into how sensory regions filter complex inputs to retain relevant information and how coordinated activity in the motor regions produce complex motor actions. However, little is known about how information is processed in the inner brain (between sensory and motor) and how behaviors are controlled. Mating and aggression are innate social behaviors that are essential for animals’ survival. During social interactions, such as those preceding mating or fighting, the brain must determine the sex of a conspecific to produce sex-appropriate behaviors that are conducive to its survival. Functional studies demonstrated that they are controlled by deep subcortical circuits in the extended amygdala and hypothalamus. My thesis attempts to understand how the inner brain works by 1) showing that chemosensory cues encoding conspecific’s sex are transformed to neural representations of mating and aggression during social interactions by recording from a genetically defined group of neurons in different regions of the extended amygdala and hypothalamus. 2) Demonstrating that the neural activity representing conspecific’s sex is necessary for the emergence of behavioral representations in the hypothalamus.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/9rf6-5727, author = {Chiu, Hui}, title = {Neural Control of Male and Female Aggression in Drosophila}, school = {California Institute of Technology}, year = {2021}, doi = {10.7907/9rf6-5727}, url = {https://resolver.caltech.edu/CaltechTHESIS:05222021-190729800}, abstract = {Aggression is essential for an individual’s survival, but it can also lead to unfavorable consequences when misregulated. It is thus important to study the neural basis of this behavior not only for learning how the nervous system is constructed to generate an innate behavior but also for finding the causality of misregulation. Although many circuit and molecular mechanisms underlying aggression have been revealed, our knowledge is mostly restricted to males. Given that sexual differences in aggression are seen in most if not all species, the mechanisms that we learned in one sex may not be directly applied to the other. Therefore, studying the neural basis of aggression in both sexes is necessary for gaining a full understanding of this behavior. Drosophila serves as a unique model for such studies because males and females differ not only in the level of aggressiveness but also in the motor patterns. Interestingly, the aggression-promoting neurons that have been identified so far are mostly sex-specific, raising the possibility that males and females adopt distinct circuits for controlling aggression. However, many sexually shared features of aggression also imply the existence of common circuit elements. My thesis work investigated whether any aggression circuit modules are shared by the two sexes and how the circuit is organized to generate sexually shared and dimorphic motor patterns. Through a behavioral screen and the genetic intersection approach, we identified a pair of sexually shared neurons, CAP, that regulates aggressive approach in both sexes, as well as a pair of male-specific neurons, MAP, whose activation promotes the transition from approach to male-specific attack. We subsequently identified the female homologue, fpC1 neurons, whose activation induces female aggression. Supported by the in vivo imaging and the behavioral epistasis results, we confirmed the functional connectivity between CAP and MAP/fpC1 in males and females, respectively. Lastly, we showed that the connectivity between CAP and MAP/fpC1 is strengthened in socially isolated flies, which exemplifies how circuits can be modified by social isolation to enhance aggression in both sexes. The connectivity between CAP and MAP/fpC1 provides a circuit logic for the control of sexually shared and dimorphic aggressive behaviors. It can be used as an entry point for circuit mapping as well as for further investigation of mechanisms underlying sexual differences in aggression.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/RGVK-9962, author = {Kim, Dong-Wook}, title = {Multimodal Analysis of Cell Types in a Hypothalamic Node Controlling Social Behavior in Mice}, school = {California Institute of Technology}, year = {2020}, doi = {10.7907/RGVK-9962}, url = {https://resolver.caltech.edu/CaltechTHESIS:12162019-183140887}, abstract = {The advent and recent advances of single-cell RNA sequencing (scRNA-seq) have yielded transformative insights into our understanding of cellular diversity in the central nervous system (CNS) with unprecedented detail. However, due to current experimental and computational limitations on defining transcriptomic cell types (T-types) and the multiple phenotypic features of cell types in the CNS, an integrative and multimodal approach should be required for the comprehensive classification of cell types.
To this end, performing multimodal analysis of scRNA-seq in hypothalamus would be very beneficial in that hypothalamus, controlling homeostatic and innate survival behaviors which known to be highly conserved across a wide range of species and encoded in hard-wired brain circuits, is likely to display the more straightforward relationship between transcriptomic identity, axonal projections, and behavioral activation, respectively. In my dissertation, I have been focused on the cell type characterizations of a hypothalamic node controlling innate social behavior in mice, the ventrolateral subdivision of the ventromedial hypothalamus (VMHvl). VMHvl only contains ~4,000 neurons per hemisphere in mice but due to its behavioral, anatomical, and molecular heterogeneity, which T-types in VMHvl are related to connectivity and behavioral function is largely unknown.
In Chapter II, I described my main thesis work to perform scRNA-seq in VMHvl using two independent platforms: SMART-seq2 (~4,500 neurons sequenced) and 10x (~78,000 neurons sequenced). Specifically, 17 joint VMHvl T-types including several sexually dimorphic clusters were identified by canonical correlation analysis (CCA) in Seurat, and the majority of them were validated by multiplexed single-molecule FISH (seqFISH). Correspondence between transcriptomic identity, and axonal projections or behavioral activation, respectively, was also investigated. Immediate early gene analysis identified T-types exhibiting preferential responses to intruder males versus females but only rare examples of behavior-specific activation. Unexpectedly, many VMHvl T-types comprise a mixed population of neurons with different projection target preferences. Overall our analysis revealed that, surprisingly, few VMHvl T-types exhibit a clear correspondence with behavior-specific activation and connectivity.
In Chapter III, I will discuss about future directions for a deeper and better understanding of VMHvl cell types. Briefly, my previous data from whole-cell patch clamp recording in VMHvl slices suggested that there were at least 4 distinct electrophysiological cell types (E-types). Additionally, two distinct neuromodulatory effects on VMHvl were observed (persistently activated by vasopressin/oxytocin vs. silenced by nitric oxide) by monitoring populational activities using two-photon Ca2+ imaging in slices. Based on the results from the first part and combined with advanced molecular techniques (e.g. Patch-seq and CRISPR-Cas9), we can further dissect out the cellular diversity in VMHvl and their functional implications.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/pard-ed88, author = {Jung, Yonil}, title = {Neurons that Control Social States in Drosophila melanogaster}, school = {California Institute of Technology}, year = {2020}, doi = {10.7907/pard-ed88}, url = {https://resolver.caltech.edu/CaltechTHESIS:03122020-231054692}, abstract = {Animal behaviors are influences not only by the immediate stimuli they are receiving but also by internal states. Internal states such as fear, hunger, and arousal can change subjective “feeling”, and result in complex behavioral outcome even if animals receive the same stimuli. In most cases, these state-dependent behavioral changes persist long after the sensory input that caused internal state change is removed, and affect future behavior, reflexing previous experience. This feature of state-control allows animals to adapt their behavior to be more suitable for their internal demands.
The influence of the internal state on animal behavior has been emphasized for decades. There are multiple studies and attempts to identify persistent neuronal mechanisms which are the important feature of the internal state. However, how persistency makes the behavioral state interact with behavioral process to induce input/output relationship has been largely unknown. In addition, it is not clear what the behavioral functions of the persistence are, and what the circuit implementation of persistent activity is. Are there neurons that are persistently activated by external stimulus? Here we approached these questions by investigating social state of fruit flies, Drosophila melanogaster.
Fruit flies exhibit complex social behaviors that are appropriate for given social cues. For example, male flies show courtship behavior toward female flies, and show aggressive behavior such as wing threat and fighting when they encounter opponent male files. Previous studies have been focused on what sensory cues induce these behaviors: detection of female specific pheromones, 7,11-HD, causes male files to court, and male specific pheromone, cVA, induces inter-male aggression. In this study, we have focused more on how these cues might affect internal state changes rather than immediate behavioral response.
Studying persistent social state change has been challenging due to the difficulty of precise, time-resolved presentation of the social cues. For instance, courtship behaviors require constant presence of female object toward which male flies show oriented behavior. The male-male aggressive behaviors such as lunging and tussling require constant interaction between two animals, and removal of opponent male fly is technically impossible. Therefore, we first developed an optogenetic tool in fly systems to study persistent feature of the social state change to mimic transient presentation of the social cue. In Chapter II, we describe an optogenetic tool that allows the manipulation of neural activity in a freely moving fly. We used Red activatable Channelrhodopsin (ReachR), which enabled us to manipulate activation of neurons in freely behaving adult flies in millisecond precision without interfering normal visual function. Using such an activation tool, we show that activation of female sensing neurons, P1 neurons, induces persistent courtship behaviors in male flies that last several minutes after the stimulation of P1 neurons.
Although we show that persistent internal state change can be induced by transient stimulation of the sensory cues in Chapter II, the circuit implementation of such a persistency is not clear. In Chapter III, we show that activation of P1 neurons triggers persistent activity in its downstream neurons, pCd neurons, that is necessary for the persistent social behavior induced by transient social behaviors. Interestingly, manipulation of the pCd neurons do not affect immediate behavioral response that are shown during the presentation of social cues (P1 stimulation), implying that there are parallel and dissociable pathways for the immediate response and enduring response derived from persistent internal state change, although these responses are caused by common cue. Although the neural mechanism to encode persistent activity is still unclear, this finding shows how internal state and command pathway interact with each other to affect behavioral outcome.
Altogether, these findings described in this dissertation offer new insights for future researchers to understand behavioral state control.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/Z9R78C4T, author = {Lim, Rod S.}, title = {How Resources Control Aggression in Drosophila}, school = {California Institute of Technology}, year = {2015}, doi = {10.7907/Z9R78C4T}, url = {https://resolver.caltech.edu/CaltechTHESIS:11222014-220142839}, abstract = {
How animals use sensory information to weigh the risks vs. benefits of behavioral decisions remains poorly understood. Inter-male aggression is triggered when animals perceive both the presence of an appetitive resource, such as food or females, and of competing conspecific males. How such signals are detected and integrated to control the decision to fight is not clear. Here we use the vinegar fly, Drosophila melanogaster, to investigate the manner in which food and females promotes aggression.
In the first chapter, we explore how food controls aggression. As in many other species, food promotes aggression in flies, but it is not clear whether food increases aggression per se, or whether aggression is a secondary consequence of increased social interactions caused by aggregation of flies on food. Furthermore, nothing is known about how animals evaluate the quality and quantity of food in the context of competition. We show that food promotes aggression independently of any effect to increase the frequency of contact between males. Food increases aggression but not courtship between males, suggesting that the effect of food on aggression is specific. Next, we show that flies tune the level of aggression according to absolute amount of food rather than other parameters, such as area or concentration of food. Sucrose, a sugar molecule present in many fruits, is sufficient to promote aggression, and detection of sugar via gustatory receptor neurons is necessary for food-promoted aggression. Furthermore, we show that while food is necessary for aggression, too much food decreases aggression. Finally, we show that flies exhibit strategies consistent with a territorial strategy. These data suggest that flies use sweet-sensing gustatory information to guide their decision to fight over a limited quantity of a food resource.
Following up on the findings of the first chapter, we asked how the presence of a conspecific female resource promotes male-male aggression. In the absence of food, group-housed male flies, who normally do not fight even in the presence of food, fight in the presence of females. Unlike food, the presence of females strongly influences proximity between flies. Nevertheless, as group-housed flies do not fight even when they are in small chambers, it is unlikely that the presence of female indirectly increases aggression by first increasing proximity. Unlike food, the presence of females also leads to large increases in locomotion and in male-female courtship behaviors, suggesting that females may influence aggression as well as general arousal. Female cuticular hydrocarbons are required for this effect, as females that do not produce CH pheromones are unable to promote male-male aggression. In particular, 7,11-HD––a female-specific cuticular hydrocarbon pheromone critical for male-female courtship––is sufficient to mediate this effect when it is perfumed onto pheromone-deficient females or males. Recent studies showed that ppk23+ GRNs label two population of GRNs, one of which detects male cuticular hydrocarbons and another labeled by ppk23 and ppk25, which detects female cuticular hydrocarbons. I show that in particular, both of these GRNs control aggression, presumably via detection of female or male pheromones. To further investigate the ways in which these two classes of GRNs control aggression, I developed new genetic tools to independently test the male- and female-sensing GRNs. I show that ppk25-LexA and ppk25-GAL80 faithfully recapitulate the expression pattern of ppk25-GAL4 and label a subset of ppk23+ GRNs. These tools can be used in future studies to dissect the respective functions of male-sensing and female-sensing GRNs in male social behaviors.
Finally, in the last chapter, I discuss quantitative approaches to describe how varying quantities of food and females could control the level of aggression. Flies show an inverse-U shaped aggressive response to varying quantities of food and a flat aggressive response to varying quantities of females. I show how two simple game theoretic models, “prisoner’s dilemma” and “coordination game” could be used to describe the level of aggression we observe. These results suggest that flies may use strategic decision-making, using simple comparisons of costs and benefits.
In conclusion, male-male aggression in Drosophila is controlled by simple gustatory cues from food and females, which are detected by gustatory receptor neurons. Different quantities of resource cues lead to different levels of aggression, and flies show putative territorial behavior, suggesting that fly aggression is a highly strategic adaptive behavior. How these resource cues are integrated with male pheromone cues and give rise to this complex behavior is an interesting subject, which should keep researchers busy in the coming years.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/MPZX-TN59, author = {Inagaki, Hidehiko K.}, title = {Neuronal Mechanism of State Control in Drosophila melanogaster}, school = {California Institute of Technology}, year = {2014}, doi = {10.7907/MPZX-TN59}, url = {https://resolver.caltech.edu/CaltechTHESIS:02112014-133121063}, abstract = {The changes in internal states, such as fear, hunger and sleep affect behavioral responses in animals. In most of the cases, these state-dependent influences are “pleiotropic”: one state affects multiple sensory modalities and behaviors; “scalable”: the strengths and choices of such modulations differ depending on the imminence of demands; and “persistent”: once the state is switched on the effects last even after the internal demands are off. These prominent features of state-control enable animals to adjust their behavioral responses depending on their internal demands. Here, we studied the neuronal mechanisms of state-controls by investigating energy-deprived state (hunger state) and social-deprived state of fruit flies, Drosophila melanogaster, as prototypic models. To approach these questions, we developed two novel methods: a genetically based method to map sites of neuromodulation in the brain and optogenetic tools in Drosophila.
These methods, and genetic perturbations, reveal that the effect of hunger to alter behavioral sensitivity to gustatory cues is mediate by two distinct neuromodulatory pathways. The neuropeptide F (NPF) – dopamine (DA) pathway increases sugar sensitivity under mild starvation, while the adipokinetic hormone (AKH)- short neuropeptide F (sNPF) pathway decreases bitter sensitivity under severe starvation. These two pathways are recruited under different levels of energy demands without any cross interaction. Effects of both of the pathways are mediated by modulation of the gustatory sensory neurons, which reinforce the concept that sensory neurons constitute an important locus for state-dependent control of behaviors. Our data suggests that multiple independent neuromodulatory pathways are underlying pleiotropic and scalable effects of the hunger state.
In addition, using optogenetic tool, we show that the neural control of male courtship song can be separated into probabilistic/biasing, and deterministic/command-like components. The former, but not the latter, neurons are subject to functional modulation by social experience, supporting the idea that they constitute a locus of state-dependent influence. Interestingly, moreover, brief activation of the former, but not the latter, neurons trigger persistent behavioral response for more than 10 min. Altogether, these findings and new tools described in this dissertation offer new entry points for future researchers to understand the neuronal mechanism of state control.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/T6WZ-BV58, author = {Wang, Liming}, title = {Genetic and Neural Regulation of Aggressive Behavior in Drosophila melanogaster}, school = {California Institute of Technology}, year = {2011}, doi = {10.7907/T6WZ-BV58}, url = {https://resolver.caltech.edu/CaltechTHESIS:05152011-122043232}, abstract = {
Aggression is an evolutionarily conserved behavior across the animal kingdom. Aggressive behavior among conspecifics is critical for the acquisition and defense of important resources including food, mates, and shelter, hence contributing to the survival and reproduction of animals. Therefore, it is of particular interest to understand how this behavior is regulated.
We use the fruit fly Drosophila melanogaster as a model system to understand the regulation of aggression. We identify Cyp6a20, a cytochrome P450, as a gene mediating the suppressive effect of social experience on the intensity of male-male aggression. Notably, Cyp6a20 has been previously identified by profiling Drosophila strains subjected to genetic selection for differences in aggressiveness. Therefore our findings reveal a common genetic target for environmental and heritable influences on aggressiveness. Interestingly, Cyp6a20 is expressed in a subset of non-neuronal support cells associated with pheromone-sensing olfactory sensilla, suggesting that olfactory pheromone(s) may contribute to the regulation of aggression. Consistent with this idea, we find that cis-11-vaccenyl acetate (cVA), a previously identified olfactory pheromone, promotes male-male aggression via a group of olfactory receptor neurons expressing Or67d.
Despite its robust behavioral effect, cVA is not required for baseline male-male aggression, and exogenous cVA does not induce male-female aggression, suggesting that sex specificity of male aggression is independent of cVA. Our subsequent studies show that the sex specificity of male social behaviors is determined by a different class of pheromones, named male cuticular hydrocarbons. Male flies perform significantly less aggression and more courtship towards male flies lacking male CHs, both of which can be rescued by synthetic (Z)-7-tricosene (7-T), the most abundant male cuticular hydrocarbon. The opposite influences of 7-T on aggression and courtship are independent, but both require the gustatory receptor Gr32a. Surprisingly, sensitivity to 7-T is required for the aggression-promoting effect of cVA, but not vice versa. Furthermore, the increased courtship in the absence of male cuticular hydrocarbons is induced by pheromone(s) detected by an olfactory receptor Or47b. Thus, male social behaviors are controlled by gustatory pheromones that promote and suppress aggression and courtship, respectively, and whose influences are dominant to olfactory pheromones that enhance these behaviors.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, month = {July}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/BSHW-AP17, author = {Hergarden, Anne Christina}, title = {The Role of Peptidergic Neurons in the Regulation of Satiety in Drosophila}, school = {California Institute of Technology}, year = {2011}, doi = {10.7907/BSHW-AP17}, url = {https://resolver.caltech.edu/CaltechTHESIS:02272011-173157140}, abstract = {Understanding the neural mechanisms that motivate us to eat is important because of the increasing rates of obesity and the consequential increasing rates of diabetes and cardiovascular disease in our society. The aim of this dissertation is to gain insight into the neuromodulators and neural mechanisms that regulate satiety. To do this, we turned to Drosophila melanogaster, which has been a powerful model organism to study the molecular mechanisms underlying innate animal behaviors and which exhibits many conserved elements of feeding regulation and energy homeostasis found in mammals. A common theme in animal behavior is that food deprivation modifies behavioral responses, e.g., the likelihood that an animal will accept a low-nutrient food. I manipulated the parameters of a feeding assay to screen for animals that lacked several starvation-induced feeding behaviors: increased foraging for food, increased acceptance of low-nutrient food, and increased ingestion of low-quality food. Using this feeding assay, I identified a neuronal circuit manipulation that inhibits several starvation-induced behaviors. Activation of a subset of Allatostatin-A-expressing neurons, using a novel transgenic tool that we generated, inhibits starvation-induced changes in both the acceptance and the ingestion of low-quality foods. In contrast, this circuit manipulation did not affect starvation-induced metabolic changes or foraging behavior. This suggests that we tapped into a mechanism that regulates a specific subset of starvation-induced changes in feeding behavior that is independent from general starvation-induced behavioral responses and energy metabolism. Studies in blowflies have revealed that the primary mechanism that promotes satiety is inhibitory proprioceptive feedback from the gut, but whether such a mechanism operates in Drosophila is unclear. While Allatostatin A has been implicated as a satiety factor and as a myoinhibitor in several other insects, it has no known function in Drosophila. A mechanism that promotes satiety but that does not alter energy metabolism has not previously been identified in Drosophila. I have used this circuit manipulation to better understand how a state of satiety is achieved in Drosophila, by integrating the knowledge acquired from studies in other insects with the knowledge acquired from molecular genetic manipulations in Drosophila.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/8H1W-RW51, author = {Yorozu, Suzuko}, title = {Distinct Sensory Representations of Wind and Near-Field Sound in the Drosophila Brain}, school = {California Institute of Technology}, year = {2010}, doi = {10.7907/8H1W-RW51}, url = {https://resolver.caltech.edu/CaltechTHESIS:05282010-134450366}, abstract = {Behavioral responses to wind are thought to play a critical role in controlling the dispersal and population genetics of wild Drosophila species, as well as their navigation in flight, but their underlying neurobiological basis is unknown. I show that Drosophila melanogaster, like wild-caught Drosophila strains, exhibits robust wind-induced suppression of locomotion (WISL), in response to air currents delivered at speeds normally encountered in nature. Furthermore, I identify wind-sensitive neurons in the Johnston’s organ (JO), an antennal mechanosensory structure previously implicated in near-field sound detection. Using Gal4 lines targeted to different subsets of JO neurons, and a genetically encoded calcium indicator, I show that wind and near-field sound (courtship song) activate distinct JO populations, which project to different regions of the antennal and mechanosensory motor center (AMMC) in the central brain. Selective genetic ablation of wind-sensitive JO neurons in the antenna abolishes WISL behavior, without impairing hearing. Different neuronal sub-populations within the wind-sensitive population, moreover, respond to different directions of arista deflection caused by airflow and project to different regions of the AMMC, providing a rudimentary map of wind direction in the brain. Importantly, sound- and wind-sensitive JO neurons exhibit different intrinsic response properties: the former are phasically activated by small, bidirectional displacements of the aristae, while the latter are tonically activated by unidirectional, static deflections of larger magnitude. These different intrinsic properties are well suited to the detection of oscillatory pulses of near-field sound and laminar airflow, respectively. These data identify wind-sensitive neurons in JO, a structure that has been primarily associated with hearing, and reveal how the brain can distinguish different types of air particle movements, using a common sensory organ.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/NMN1-N566, author = {Carvalho, Gil Bastos de}, title = {Drosophila Feeding Behavior and Demographic Mechanisms of Lifespan Extension}, school = {California Institute of Technology}, year = {2010}, doi = {10.7907/NMN1-N566}, url = {https://resolver.caltech.edu/CaltechTHESIS:06022010-183440587}, abstract = {Why do we age? Most organisms undergo senescence, a process involving progressive functional decline culminating in death, yet this widespread phenomenon remains largely mysterious. A number of genetic and environmental factors affect longevity, the best conserved and most widely studied of which is dietary restriction (DR), a reduction of nutrient intake short of malnutrition. Since nutrient ingestion determines lifespan, any factor affecting longevity—particular food components, genetic pathways or drugs—may do so indirectly, by altering feeding behavior. This is particularly true in Drosophila, which is normally kept in conditions where food is present in excess. Moreover, since DR is applied by aging flies on two different food concentrations—diluted media are associated with an extended lifespan—animals may alter their intake in response to the change in nutrient content. Since the medium is also the only water source, this compensatory feeding would result in changes in hydration, introducing a second experimental variable. Despite these issues, Drosophila feeding behavior has classically been ignored or superficially characterized in the context of aging research, partly due to the absence of appropriate methodology. We developed two complementary assays allowing long-term measurement of food intake. Using these techniques, we present the first characterization of real-time Drosophila feeding behavior. Our results reveal gender-specific feeding trends and show that mating stimulates female appetite via the seminal Sex Peptide. Additionally, we show that ingestion is dramatically affected by food dilution or dietary additives. Animals fed concentrated media restrict their intake and are chronically thirsty. We have found that lifespan extension by classical DR paradigms is abolished in the presence of ad libitum water, challenging the long-held assumption that DR affects longevity by altering nutrient intake. We characterize a new regime that robustly prolongs lifespan irrespective of water availability, and thus likely represents a more relevant model for mammalian DR. In contrast to previous claims, demographic analysis using this paradigm indicates that DR acts not by reducing the immediate risk of death, but by slowing the accumulation of age-related damage. Our findings directly challenge current views on the mechanistic basis of DR and have broad implications for the study of aging and nutrition in model organisms.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J. and Benzer, Seymour}, } @phdthesis{10.7907/KSPD-J220, author = {Hochstim, Christian John}, title = {Pax6 Controls Astrocyte Positional Identity in the Spinal Cord}, school = {California Institute of Technology}, year = {2007}, doi = {10.7907/KSPD-J220}, url = {https://resolver.caltech.edu/CaltechETD:etd-07262008-115538}, abstract = {Astrocytes constitute the most abundant cell type in the CNS, and play diverse functional roles, but the ontogenetic origins of this phenotypic diversity are poorly understood. We have investigated whether positional identity, a fundamental organizing principle governing the generation of neuronal subtype diversity, is also relevant to astrocyte diversification. We identified three positionally distinct subtypes of white matter astrocytes in the spinal cord, which can be distinguished by the combinatorial expression of Reelin and Slit1. These astrocyte subtypes derive from progenitor domains expressing the homeodomain transcription factors Pax6 and Nkx6.1, respectively. Loss- and gain-of-function experiments indicate that the positional identity of these astrocyte subtypes is controlled by Pax6 and Nkx6.1, in a combinatorial manner. Thus, positional identity is an organizing principle underlying astrocyte, as well as neuronal, subtype diversification, and is controlled by a homeodomain transcriptional code whose elements are re-utilized following the specification of neuronal identity earlier in development.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/w6ht-9m14, author = {Choi, Gloria Bohyun}, title = {Characterization of the Circuits Mediating Innate Reproductive and Defensive Behaviors from the Amygdala to the Hypothalamus}, school = {California Institute of Technology}, year = {2005}, doi = {10.7907/w6ht-9m14}, url = {https://resolver.caltech.edu/CaltechETD:etd-05212005-123104}, abstract = {All metazoan organisms must reproduce and defend themselves in order to survive as individuals and as a species. These innate behaviors are so crucial that they are “hard-wired” into the brain during the animal’s development. They are also released primarily by the olfactory stimuli detected by the AOB, which synapses into the MEA. The MEA in turn projects to the medial hypothalamic behavior control column, which contains a series of nuclei orchestrating either reproductive or defensive behaviors. These amygdalar-hypothalamic projections are topographically organized, and the sub-circuitries controlling reproduction and defense are segregated both functionally and anatomically.
The topographically organized projections suggest that these neural pathways for reproduction and defense are likely genetically determined, but genes that might control their wiring have not yet been identified. Such a parallel circuit organization with very few cross-talks between the two sub-circuits also poses the problem of how rapid decisions between competing reproductive and defensive behaviors are made by organisms faced with conflicting cues.
Using oligonucleotide microarrays and laser-capture microdissection, I identified that several LIM homeodomain transcription factors mark different regions of the MEA involved in either reproductive or defensive behaviors. I have characterized the projections of these neurons to the hypothalamus, using both genetically encoded anterograde and traditional retrograde tracers. I have also carried out behavioral experiments to assess their differential activations by reproductive and defensive stimuli.
My results indicate that Lhx6 delineates a reproductive pathway, which involves neurons in both MEApd and BSTpr, and their projections to the three reproductive nuclei in the hypothalamic medial behavioral control column (MPN, VMHvl and PMv). Further analysis reveals, counter-intuitively, that VMHvl receives inhibitory projections from this reproductive pathway, and a convergent excitatory projection from neurons in MEApv that are activated by a predator odor. The results suggest that this point-of-convergence may serve to “gate” the expression of reproductive behavior, under conditions where animals are exposed to threatening stimuli. Thus, my data identifies a potential neural substrate within the hypothalamus for controlling behavioral decisions in the face of conflicting cues and a transcription factor family that may contribute to the development of this substrate.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/XV0S-9D71, author = {Shin, Donghun}, title = {Identification and Characterization of Endothelial Specific Genes}, school = {California Institute of Technology}, year = {2005}, doi = {10.7907/XV0S-9D71}, url = {https://resolver.caltech.edu/CaltechETD:etd-05232005-173743}, abstract = {Cardiovascular development and its proper function are essential for the development and survival of animals, while malformation of vasculature leads to a variety of diseases. The significance of vasculature during development and in adulthood has been delineated by investigating the functions of genes expressed in the vasculature. Endothelial cells lining the lumen of vessel tubes with a single layer, had long been considered inert, homogeneous cells. However, molecular and genetic studies have provided numerous pieces of evidence, which indicate that endothelial cells are active, dynamic, heterogeneous cells. Among these studies, molecular differences between arterial and venous endothelial cells were first revealed by the observation that ephrin-B2 and its cognate receptor EphB4 are restrictively expressed in arterial and venous endothelial cells, respectively. These genes are not only molecular markers of arteries and veins, but they also play essential roles in cardiovascular development.
To investigate whether the molecular difference between arteries and veins persists into adulthood, I analyzed ephrin-B2 expression in adult tissues including pathological settings. These data indicate that the molecular distinction is maintained in adults, and ephrin-B2 further distinguishes arterial smooth muscle cells from venous smooth muscle cells in adults.
Ephrin-B2 was serendipitously identified as an arterial marker; therefore, I performed a systematic screen to isolate novel arterial- and venous-specific genes, whose identification and characterization might improve current understanding of vascular biology. Through this screen, I isolated several novel arterial-restricted genes, and one of these genes, Depp (decidual protein induced by progesterone), was characterized in detail by generating a knockout of the Depp locus. Although the homozygous mutant mice appear phenotypically normal, the detailed analysis of Depp expression reveals the heterogeneity of arterial endothelial cells from the early stage of vascular development.
I identified another novel gene, D1.1, through the screen; however, D1.1 is expressed in both arterial and venous endothelial cells. The fact that D1.1 is specifically expressed in endothelial cells and encodes a predicted transmembrane protein, prompted me to characterize D1.1 in detail using a tau-LacZ knock-in to the D1.1 locus. The data from the expression analysis suggest D1.1 as a novel marker of adult neovasculature. In addition, the data using a soluble D1.1-Fc fusion protein in several different acute assays suggest that D1.1 may play a functional role in angiogenesis that is compensated in vivo by other, structurally distinct proteins.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/SV83-BY17, author = {Zhou, Qiao}, title = {Glial Cell Development in the Vertebrate Central Nervous System}, school = {California Institute of Technology}, year = {2003}, doi = {10.7907/SV83-BY17}, url = {https://resolver.caltech.edu/CaltechETD:etd-04092003-155305}, abstract = {Neurons and glial cells are the two most fundamental cell types of the vertebrate central nervous system (CNS). While neurons are directly responsible for information processing via their electrical activities, glial cells play essential supportive roles. For example, oligodendroglia insulates axons, microglia performs immune functions, and astroglia maintains homeostasis of the entire CNS. Malfunction of glial cells causes numerous debilitating diseases directly (such as glial tumors), or indirectly by disrupting the normal functions of neurons that they support (as in multiple sclerosis).
Despite their functional importance, relatively little is known about the development of vertebrate CNS glial cells. Focusing on the possibility that members of the basic helix-loop-helix (bHLH) transcription factors may play important roles in the development of vertebrate glial cells, similar to their functions in neurons, I searched for novel bHLH factors expressed in glial cells. A new family of bHLH factors was found and named Olig. Intriguingly, one member of this family, Olig2, is sequentially expressed first in motoneuron progenitors and later in the oligodendroglia. The sequence and expression pattern of Olig2 is highly conserved among different vertebrate species including fish, birds and mammals.
To understand the role of Olig2 in oligodendroglia development, I ectopically expressed Olig2 singly or in combination with other factors in chick embryos. My result suggests that Olig2 can promote oligodendrocyte formation in the absence of neurogenic bHLH factors, which are negative regulators of glial fate. Other groups of researchers reported that in the presence of neurogenic factors, Olig2 promotes motoneuron development instead. Olig2 gene is therefore sufficient to specify the fate of either a neuronal subtype or a glial subtype, together with neurogenic factors.
To further assess whether Olig genes are required for motoneuron and oligodendroglia development, I knocked out both Olig2 and Olig1 genes in mouse. In double null mutants, spinal motoneurons and oligodendroglia precursors from the entire CNS fail to develop, demonstrating that Olig genes are absolutely necessary for the generation of these cell types. Unexpectedly, in the absence of both Olig1 and Olig2, spinal motoneurons are transformed into V2 interneurons whereas oligodendroglial cells are respecified as astroglial cells. These results suggest that Olig genes are not involved in neuron-glia decision, but rather in specifying subtype identities of neuron and glia. Given that motoneurons and oligodendrocytes likely derive from common precursors, the expression of Olig may serve to couple the subtype identities of both neurons and glial cells sequentially generated from the same stem cells.
The series of studies on Olig genes contributed on two areas of neural development. First, they shed important light on the specification of oligodendrocyte and astrocyte, the two major glial types in the vertebrate CNS. Second, they revealed that cell fate determinations of neuron and glia are not two unrelated events as often believed, on the contrary, they are deeply intertwined.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/c701-pk41, author = {Zirlinger, Mariela}, title = {Application of Microarray, Laser Capture and Transgenic Technologies to the Study of Neural Diversity}, school = {California Institute of Technology}, year = {2002}, doi = {10.7907/c701-pk41}, url = {https://resolver.caltech.edu/CaltechTHESIS:04242012-143624169}, abstract = {A major quest in modem neurobiology is to understand how the brain controls behavior. To this end, the convergence of two traditionally separate fields, systems neuroscience and molecular neuroscience, is required. The delineation of brain regions responsible for different behaviors, and in particular, their underlying neural circuits should be accompanied by the appreciation of the molecules that compose such circuits.
I have taken two approaches toward unraveling the molecular signatures of specific neural structures. First, I conducted microarray-based RNA expression analyses to search, in a large scale and with no a priori constraints, for differentially expressed gene products in several brain regions, including the amygdala, cerebellum, hippocampus, olfactory bulb and periaqueductal gray. Interestingly, only 0.3% of the genes characterized to date showed restricted expression in distinct brain areas. Further characterization by in situ hybridization was performed for genes enriched in the amygdala, a structure that modulates emotional behavior. Remarkably, this revealed that most region-specific genes possessed expression domains whose limits respected subnuclear boundaries defined by classical cytoarchitectonic criteria. These analyses were not only informative about the molecular composition of distinct brain areas, but also provided tools to genetically dissect the role of different brain nuclei in specific behaviors.
Second, I have used a genetic strategy to label all cellular derivatives of neural crest precursor cells expressing a particular gene, Ngn2. Such lineage tracing study uncovered a segregated cellular subpopulation in the developing peripheral nervous system, which was strongly biased for the generation of sensory rather than autonomic neurons. Despite this fate bias, Ngn2-derived cells in the dorsal root ganglion were equally likely to give rise to neurons or glia. This suggests that some neural crest cells become restricted to sensory or autonomic sub lineages before becoming committed to neuronal or glial fates. In general, visualization of the behavior of neural progenitors during the formation of the nervous system may further our understanding of the generation of specific neuronal subtypes and, eventually, neuronal connections that shape the functioning brain.
The combination of strategies here described will enable the characterization of brain regions at the molecular level on a broad, systems-based approach.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/SJM2-8882, author = {Gerety, Sebastian de la Soudière}, title = {Eph Signaling in Vascular Development}, school = {California Institute of Technology}, year = {2002}, doi = {10.7907/SJM2-8882}, url = {https://resolver.caltech.edu/CaltechTHESIS:04262012-140534350}, abstract = {
One of the most striking features of developmental biology is the dramatic morphological changes that an embryo must undergo to achieve its final form. Arguably, the most stunning example of this is found in the embryonic vasculature: not only does the vasculature undergo morphological changes, it must continue to do so adaptively as the size and nutritional needs of the embryo change during gestation. As embryonic blood flow starts long before the end of vessel morphogenesis, the vessels must maintain the integrity of their cell-cell contacts while at the same time remodeling into their final state. Receptor tyrosine kinases and their ligands have been implicated in the regulation of blood vessel growth and remodeling during development. Recently, the Eph receptors and their ephrin ligands were found to be expressed in the developing vasculature. While one Eph receptor, EphB4, is restricted to veins, its specific ligand, ephrinB2, is restricted to arteries. Furthermore, the ephrinB2 knockout mice exhibit defects in blood vessel remodeling, angiogenesis. Although the reciprocal expression of ephrinB2 and EphB4 suggested that Eph signaling from arteries to veins was important for blood vessel development, the presence of additional Eph receptors suggested EphB4 might not be required for this process. Additionally, the widespread expression of ephrinB2 outside the vasculature suggested that vascular-specific expression of this ligand might not be the tissue source necessary for angiogenic remodeling.
To determine which Eph receptor was mediating the ephrinB2 signal, I generated a knockout of the EphB4 gene in mouse. A reporter gene replacement in the EphB4 locus confirmed the vein-biased expression of this receptor. Homozygous EphB4 mutant mice exhibit angiogenesis and cardiac defects, and embryonic lethality indistinguishable from those of ephrinB2 knockout mice. This suggests that EphB4 is the main Eph receptor responsible for transducing the angiogenic ephrinB2 signal. To examine the importance of endothelial specific expression of ephrinB2 in angiogenesis, in contrast to its nonvascular expression, I generated a conditional ephrinB2 mouse. These mice carry a functional ephrinB2 gene, which can be inactivated in a tissue specific manner. Mice with endothelial-specific inactivation of ephrinB2 (and intact non-vascular ephrinB2 expression) exhibit severe angiogenesis and cardiac defects identical to those of the conventional ephrinB2 mutant mice. This suggests that vascular ephrinB2 is essential, and cannot be compensated for by non-vascular ephrinB2 from surrounding vessels.
These studies have clarified two important issues. The first is that the ephrinB2 signal is received by EphB4 expressing endothelial cells (of the veins), rather than by perivascular cells that also express Eph receptors. Second, cphrinB2 expression in endothelial cells of the vessels is an essential tissue source of angiogenic ephrin signals. Together, these studies reinforce the original interpretation of the ephrinB2 mutant, that Eph signaling between arteries to veins is essential for angiogenesis in the early embryo.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J. and Fraser, Scott E.}, } @phdthesis{10.7907/G3NH-0V88, author = {Montgomery, John Michael}, title = {Cell migration domains in the chick telencephalon}, school = {California Institute of Technology}, year = {1996}, doi = {10.7907/G3NH-0V88}, url = {https://resolver.caltech.edu/CaltechTHESIS:06022011-144716111}, abstract = {Little is known about the process by which the vertebrate forebrain (the diencephalon and telencephalon) becomes regionalized during development. In studies reported here, DiI injections were used to label the embryonic day 3 (stage-16) chick telencephalon in ovo , and the migration patterns of labelled cells were analyzed in relation to various molecular markers, including the regulatory genes Cash-l and Sonic hedgehog (Shh). Cells generated in the ventral telencephalon (basal ventricular ridge, or BVR) were found to migrate widely, but were restricted from crossing into the more dorsal telencephalon (dorsal ventricular ridge, or DVR), and the more caudal diencephalon. The cell migration boundary between the BVR and DVR correlates with a Cash-l expression boundary, and the cell migration boundary between BVR and diencephalon correlates with a Shh expression boundary. In addition, cell migration patterns were dramatically different in the BVR and DVR territories. These results suggest that the BVR represents a “cell migration domain,” or a true unit of telencephalic compartmental organization, which is distinct from cell migration domains in both the DVR and diencephalon. In addition, two lines of evidence in the early embryo are shown to support the proposal that avian BVR is homologous to mammalian basal ganglia, and that avian DVR is homologous to mammalian cerebral cortex: regulatory gene expression patterns in the chick and mouse telencephalon are very similar; and the cell migration patterns in the chick telencephalon demonstrated here are found to correspond closely to those previously reported in the mouse telencephalon. Using the same DiI labelling technique, a regional fate map of the stage-16 chick telencephalon was derived. This fate map can now be used to guide transplantation or misexpression experiments, and to interpret gene expression patterns in the stage-16 telencephalon. For example, though Cash-l is expressed in the entire BVR at ES-E7 (stage 24-30), superimposition of its expression pattern onto the stage-16 fate map shows that it is only expressed in a subregion of the presumptive BVR at stage-16.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J. and Fraser, Scott E.}, } @phdthesis{10.7907/g1cd-aj32, author = {Stemple, Derek Lyle}, title = {Isolation of a mammalian neural crest stem cell and environmental control of cell fate choices}, school = {California Institute of Technology}, year = {1993}, doi = {10.7907/g1cd-aj32}, url = {https://resolver.caltech.edu/CaltechTHESIS:01082013-104637928}, abstract = {One question central to the study of developmental biology is: How does phenotypic diversity arise? The neural crest provides an excellent model system for investigations into the nature of cell-fate decisions and generation of lineage diversity. In the studies described here, I have examined two aspects of this question in cell culture. The first aspect concerns the cellular dynamics underlying the cell-fate decisions. A necessary prerequisite to the study lineage diversification is the reliable production of differentiated cells from undifferentiated precursors in a controllable environment. I have developed a system for the growth of rat neural crest cells. The system allows the serial propagation of a defined sub-population of neural crest cells at clonal density. In the system neural crest cells can differentiate into at least two identifiable cell types; peripheral neurons and Schwann cells. By sub-cloning I have been able to address specific predictions made by a stem cell model of neural crest development, and found neural crest cells to possess multipotency, self-renewal and the capacity to divide asymmetrically.
The second aspect of the question addressed in this study concerns the ability of the neural crest cell environment to control the choice of cell fate. I have examined various culture conditions for their ability to affect the choice of cell fate both in clonal cultures and in mass cultures. In the clonal cultures I found that the fate of neural crest cell clones can be altered in an instructive fashion by the composition of the substrate, or by the presence of fetal bovine serum. In mass cultures, we have examined the effect of medium composition on the expression of adrenergic traits and on the expression of a transcription factor, MASH-1, thought to participate in neural determination. Finally, in mass cultures of neo-natal adrenal chromaffin cells, we have examined the effects of basic fibroblast growth factor on neuronal differentiation, mitosis and acquisition of trophic dependence.
}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, } @phdthesis{10.7907/pbh4-ts59, author = {Michelsohn, Arie M.}, title = {Sequential Steps in the Determination of Chromaffin Cell Fate by Glucocorticoids}, school = {California Institute of Technology}, year = {1992}, doi = {10.7907/pbh4-ts59}, url = {https://resolver.caltech.edu/CaltechTHESIS:08242011-162533018}, abstract = {The development of the sympathoadrenal (SA) lineage has been studied as a model system in which to investigate the mechanisms that control the timing of environmental influences on cell fate. Glucocorticoids (GC) play a key role in the fate of the SA progenitor, causing it to differentiate to an adrenal chromaffin cell, rather than to a sympathetic neuron. Previously, it has been shown that GC exert both positive and negative effects on developing chromaffin cells: they promote cell survival and the expression of an adrenergic phenotype, and inhibit the expression of neuronal properties. However, the time at which GC first influence cell fate, and the mechanism(s) which underlie its effect(s), have remained matters of controversy. In this thesis, it is shown that the positive and negative effects of GC on SA progenitors during development are temporally separated and pharmacologically distinct. Most SA progenitors are competent to respond to GC by inhibition of process outgrowth two days before they are competent to respond by induction of PNMT, a chromaffm-specific marker. Competence to express PNMT appears to be acquired according to a cell-autonomous “clock”. The early inhibition of neuronal differentiation may be a prerequisite to subsequent PNMT expression, since sympathetic neuroblasts rapidly lose the capacity to express PNMT. The two effects of GC are both mediated via the type-II glucocorticoid receptor (GCR). However, lower concentrations of GC are required to inhibit neuronal differentiation than to promote the expression of PNMT, and the two effects show differential responsiveness towards the receptor-specific antagonist RU38486. That the two effects of GC in this system are pharmacologically separable suggests that they may be mediated via different interactions of the GCR with endogenous cellular transcription machinery. Such differential interactions may explain how the two effects of GC in this system are temporally separated. Taken together, the results presented here provide precedent for an inductive developmental event in which the timing of the effects of an instructive signal on a bipotential progenitor are controlled neither by the schedule of appearance of the signal, nor of its receptor, but rather by cell-intrinsic, developmental changes in the response properties of the cell.}, address = {1200 East California Boulevard, Pasadena, California 91125}, advisor = {Anderson, David J.}, }