The search for food and adequate nutrition determines much of an animal's behavior, as it must ingest the macronutrients, micronutrients, and water needed for growth, reproduction and body maintenance. These macro- and micronutrients are found in varying proportions and concentrations in different foods. A generalist consumer, such as many primates, faces the challenge of choosing the right combination of foods that confers adequate and balanced nutrition. Diet selection is further complicated and constrained by antifeedants, as well as digestive morphology and physiological limitations. Nutritional ecology is the study of the connected relationships between an organism, its nutrient needs (determined by physiological state), its diet selection, and the foraging behavior it uses within a specific food environment. Additionally, these relationships are complex and changeable since the nutrient needs of a consumer change over time and food resources (including the nutritional composition) vary spatiotemporally. Published data on primate nutritional ecology are limited, with most investigations of nutritional needs stemming from captive populations and few field studies. To contribute to the body of knowledge of nutritional ecology in natural populations, I examined the nutritional ecology of wild adult female blue monkeys, Cercopithecus mitis. I used the geometric framework (GF) to quantify nutritional patterns, as it allows simultaneous examination of multiple nutrients that may be driving foraging behavior and patterns of food intake. Blue monkeys are known to be generalist feeders, with flexible feeding behavior. The population I studied inhabits the Kakamega Forest, western Kenya. This forest has a history of variable human modification on a small scale, and offered a unique opportunity to examine environmental factors (e.g. degree of human-modification of forest type, food availability), social factors (dominance rank), and physiological factors (reproductive demand) that may alter blue monkey nutritional strategies. From January and September 2015, a team of field assistants and I collected behavioral data from 3 study groups, intensively sampling 24 adult females that varied in dominance rank and reproductive condition. I used all-day focal follows to quantify feeding behavior, which allowed me to assess diet selection and nutrient intake on a daily basis. I also monitored subjects' daily movement. To assess food availability, I quantified vegetative differences among major habitat types within each group's home range and monitored biweekly changes in plant production of fruits and young leaves, which were major constituents of the plant-based diet. I collected >300 food samples, as well as fecal samples, and analyzed them for macro-nutritional content using wet chemistry and near-infrared spectroscopy techniques. I combined data to examine patterns in diet and nutritional strategy on different scales: patterns across subjects, between groups and within the population as a whole, patterns in the diet on the food composition level versus nutrient intake level, and patterns in nutrient intake on a daily basis versus a long term basis (i.e. over the course of the study period). Additionally, I evaluated factors that might affect variation in nutritional strategies, including a female's reproductive condition, dominance rank, habitat use, and degree of frugivory or folivory in daily intake, as well as food availability in the environment. Kakamega blue monkeys ate a broad diet of over 445 food items (species-specific plant parts and insect morphotypes). Fruit was preferred food, and particular species-specific fruits constituted the majority of important food items (i.e., those contributing >1% of total caloric intake by group); many fruits were highly selected (i.e. eaten more than expected based on availability). Many species-specific young leaves also were important food items, though they were eaten in proportion to their availability, or even less often. Regardless of whether group diet was characterized by time spent feeding or by calories, fruit remained the largest constituent and young leaves the second largest. A subject's daily path length was negatively related to proportion of fruit in the diet (by kcal) because females focused feeding in particular trees when important fruits ripened and thus traveled less. Daily path length was not related to group size, probably because females spread out when foraging to avoid within-group scramble competition over food. Group differences in the food composition of diets likely reflected habitat differences in food distribution. Comparison of the population's diet to data from previous studies showed that as study groups moved into new areas and habitats, they capitalized on new food resources, reinforcing the idea that blue monkey are flexible feeders. During this study, subjects adjusted their diet in response to food availability in the environment, consuming more fruit (by percentage of diet and absolute kcal) when fruit was more available. In contrast, subjects ate fewer young leaves (by absolute kcal) when either fruit or young leaves were more available, suggesting that young leaves served as fallback food. At the level of nutrient intake, it was also true that females consumed significantly more structural carbohydrates when fruit availability was low. Despite their diverse diets and changes related to food availability, females actively regulated food intake to converge daily on a similar nutrient intake (grand mean of 637 kcal, with 108 kcal from protein, 149 kcal from lipid, 88 kcal from structural carbohydrates, and 293 kcal from non-structural carbohydrates, N=24). Thus, considering a multidimensional nutritional niche, I characterized their feeding behavior at two levels: they were both food composition generalists and nutrient intake specialists. Blue monkeys showed a nutritional strategy on two different temporal scales: 1) daily protein prioritization and 2) long term non-protein energy (NPE; i.e. lipid + carbohydrate energy) to available protein (P) balancing. On a daily basis, protein intake (by kcal) showed the least amount of variation (by coefficient of variation) and subjects consumed similar amounts of protein, regardless of potential influences from environmental, social or physiological factors. Females allowed more variation in daily ratio of non-protein energy to protein (NPE:P), taking advantage of high NPE foods like fruit. They allowed higher NPE:P ratios when fruit was a larger proportion of their diet and when they spent less time in near-natural forest. There was no evidence that reproductive demand or dominance rank affected protein intake or NPE:P balance. Dominance rank also did not predict deviation (absolute or directional) from mean protein intake or mean NPE:P ratio. On a long term basis (i.e. over the 8 months of data collection), all subjects tightly balanced cumulative NPE:P intake, regardless of dominance rank. This long-term pattern in all 24 subjects suggests that it a species-typical strategy. However, lower ranking females ate more unique food items per day than higher ranking females. Varying daily dietary breadth may allow females to cope with social constraints while feeding, such that dominance rank had no effect on nutritional strategies. Further, the prevalence of NPE:P balancing in most nutritional ecology studies of primates suggests that the diversity of feeding strategies within this order of mammals may have evolved to allow them to adhere to that particular nutrient balance, though the rule of compromise (e.g. protein versus NPE prioritization) and the exact ratio balanced may differ by population or species. Blue monkeys regularly used human-modified habitats and ate considerable amounts of the non-natural foods found there (and elsewhere in the forest). Non-natural foods were directly derived from humans or human activity (e.g. via scavenging from trash) and exotic (non-native) plants, generally introduced inadvertently or for silviculture. Subjects incorporated a substantial amount of non-natural foods into their diets, with approximately a third of their daily calories derived from non-natural foods. Subjects in the group with the most access to human-modified habitat used non-natural foods the most extensively. Further, subjects in two groups showed clear preference for human-modified habitat while members of the third group used habitat types in proportion to their occurrence in the home range. Human-modified habitat, and the non-natural foods found within, may have been readily used because many non-natural foods provided similar access to nutritional space as natural foods. Some non-natural foods, like oil palm fruit and ugali (cooked maize flour), represented energetically dense food resources, which also proved attractive. Regardless of whether subjects fed primarily on natural or non-natural foods, they consumed similar amounts of daily protein. This prioritization of protein, coupled with the fact that females had higher NPE:P ratios when feeding mostly on non-natural foods, indicated that blue monkeys capitalized on non-natural resources to increase NPE intake as long as they were able to consume a threshold amount of protein. What remains unclear though, is whether there are adaptive advantages associated with the ability to consume diets of variable NPE:P ratios. Overall, blue monkeys in Kakamega Forest are very flexible feeders, perhaps to a greater degree than previously acknowledged. Subjects were able to consume a diverse diet of hundreds of species-specific food items, to shift their diet in response to changes in food availability, to capitalize on food resources found in different habitat types, to take advantage of non-natural food resources, and to tolerate a wide range of NPE:P ratios in daily diets. Further, on a nutritional level, they successfully navigated potential stressors from the physiological demands of reproduction and dominance rank to adhere to a particular nutritional strategy. Flexible behavior, such as spreading out during feeding or varying dietary breadth, indicates how blue monkeys may use particular feeding strategies to arrive at a common nutrient intake target. Despite daily fluctuations in NPE:P ratio that varied with environmental and dietary factors, all subjects were able to consume a consistent daily amount of protein and prioritized its intake above all other nutritional components. Finally, their tight adherence to long term NPE:P balancing suggested that they followed a nutritional strategy that operated on both daily and longer timescales. Primates are increasingly threatened from habitat loss, degradation and other human-disturbances. There is growing awareness that some species, like blue monkeys, may be able to persist in regenerating human-modified landscapes, where they regularly and readily use non-natural food resources. More species- and habitat-specific nutritional studies are needed to predict population-level responses to varying degrees of habitat alteration. The data generated may help us assess the potential value of human-modified habitats that may require protection, as these habitats may contribute to the persistence of primate populations around the globe, especially in novel ecosystems.

One of the central questions in neuroscience is how the nervous system generates and regulates behavior. To understand the neural code for any behavior, an ideal experiment would entail (i) quantitatively defining that behavior, (ii) recording neuronal activity in relevant brain regions to identify the underlying neuronal circuits and eventually (iii) manipulating them to test their function. Novel methods in neuroscience have greatly advanced our abilities to conduct such experiments but are still insufficient. In this thesis, I developed methods for these three goals. In Chapter 2, I describe an automatic behavior identification and classification method for the cnidarian Hydra vulgaris using machine learning. In Chapter 3, I describe a fast volumetric two-photon microscope with dual-color laser excitation that can image in 3D the activity of populations of neurons from visual cortex of awake mice. In Chapter 4, I present a machine learning method that identifies cortical ensembles and pattern completion neurons in mouse visual cortex, using two-photon calcium imaging data. These methods advance current technologies, providing opportunities for new discoveries.

abstract: Human-inhabited or -disturbed areas pose many unique challenges for wildlife, including increased human exposure, novel challenges, such as finding food or nesting sites in novel structures, anthropogenic noises, and novel predators. Animals inhabiting these environments must adapt to such changes by learning to exploit new resources and avoid danger. To my knowledge no study has comprehensively assessed behavioral reactions of urban and rural populations to numerous novel environmental stimuli. I tested behavioral responses of urban, suburban, and rural house finches (Haemorhous mexicanus) to novel stimuli (e.g. objects, noises, food), to presentation of a native predator model (Accipiter striatus) and a human, and to two problem-solving challenges (escaping confinement and food-finding). Although I found few population-level differences in behavioral responses to novel objects, environment, and food, I found compelling differences in how finches from different sites responded to novel noise. When played a novel sound (whale call or ship horn), urban and suburban house finches approached their food source more quickly and spent more time on it than rural birds, and urban and suburban birds were more active during the whale-noise presentation. In addition, while there were no differences in response to the native predator, rural birds showed higher levels of stress behaviors when presented with a human. When I replicated this study in juveniles, I found that exposure to humans during development more accurately predicted behavioral differences than capture site. Finally, I found that urban birds were better at solving an escape problem, whereas rural birds were better at solving a food-finding challenge. These results indicate that not all anthropogenic changes affect animal populations equally and that determining the aversive natural-history conditions and challenges of taxa may help urban ecologists better understand the direction and degree to which animals respond to human-induced rapid environmental alterations. / Dissertation/Thesis / Doctoral Dissertation Biology 2018

Migraine is a complex neurological disorder that affects approximately 38 million Americans. For over 25 years, the neuropeptide calcitonin gene-related peptide (CGRP) has been implicated in the pathogenesis of migraine. In fact, several pharmaceutical companies are tailoring treatments to antagonize CGRP actions. However, due to the complexity of migraine, exactly how and where CGRP acts to contribute to migraine have remained controversial: whereas several studies suggest that CGRP acts in the central nervous system (CNS) in this context, others have indicated a role in the periphery. Central nervous system sites of action include the trigeminal nucleus and several higher brain regions, and peripheral sites include the vasculature and dural mast cells in the meninges. Among the sites of CGRP action, the trigeminal nerve, which is the major somatosensory structure of the face, is of particular interest because it bridges the CNS and the periphery. Migraine is generally thought to involve abnormal signaling in the trigeminovascular system, and about 50% of trigeminal neurons have CGRP immunoreactivity. Although the notion that CGRP has a central site of action in relation to migraine had gained ground over the past decade, the recent discovery that monoclonal antibodies against CGRP can prevent migraine attacks has resurrected the possibility that a peripheral site of action is involved as well. Clarification of the sites of CGRP action in migraine will be crucial to developing an understanding of mechanisms that underlie migraine so that future treatments can be rationally designed. One diagnostic criterion for migraine is photophobia, a painful and often debilitating response to non-noxious levels of light. Our laboratory previously developed a preclinical model of migraine in which the light-aversive behavior of mice is used as a surrogate of photophobia. Specifically, mice were sensitized to CGRP by introducing a nestin/hRAMP1 transgene. In these mice versus control littermates, light aversion in response to central (intracerebroventricular, ICV) injection of CGRP was enhanced in dim light. In wild-type mice, CGRP (ICV) also elicited aversion to very bright light; this did not occur in vehicle-treated mice. Additionally, I have shown that CGRP injected peripherally (intraperitoneal, IP) can induce significant light aversion in wild-type mice. I have begun to identify the sites of action outside of the central nervous system, using four lines of transgenic mice with different patterns of overexpression of CGRP receptors: global hRAMP1 mice (expression in all tissues), nestin/hRAMP1 mice (expression only in nervous tissue), tagln/hRAMP1 (expression only in smooth muscle cells), and tek2/hRAMP1 (expression in endothelial cells). As predicted, in the global hRAMP mice light aversion, in response, to IP-injected CGRP was enhanced. However, in nestin/hRAMP1 mice, only ICV-injected, and not IP-injected, CGRP induced enhanced light aversion. This finding suggests that peripheral CGRP activates neural pathways involved in light aversion, but by an indirect mechanism. To determine where in the periphery CGRP is acting, a pharmacological and genetic approach was taken. Since CGRP is one of the most potent vasodilators in the body, it is well positioned to have vascular effects that induce light aversive behavior. This hypothesis was based on findings that 1) intravenous administration of CGRP in human subjects can cause migraine pain, and 2) perivascular CGRP can sensitize the trigeminal nerve, which could alter synaptic transmission to the central nervous system and 3) CGRP monoclonal antibodies are effective in clinical trial and likely do not cross the blood brain barrier. Thus, there is a mechanism by which CGRP in the periphery can sensitize the trigeminal nerve and alter sensory perception, leading to photophobia. The role of the vasculature in migraine, specifically vasodilation, has been controversial and now the consensus is that it is neither necessary nor sufficient. First, I wanted to test the role of vasodilation in this model. I pharmacologically inhibited CGRP-induced vasodilation using two vasoconstrictors, phenylephrine and endothelin-1. Blocking CGRP-induced vasodilation partially attenuates the light aversive response. Moreover, mice that overexpress the CGRP receptor in smooth muscle, but not endothelial, cells exhibit enhanced light aversion indicating a role for vascular actions of CGRP in this preclinical model of migraine. These results present clear evidence that CGRP has actions on the vasculature to induce light aversion. Additionally, the inability of blocking vasodilation to completely rescue the light aversion suggests that the vasculature may not be the only peripheral target of CGRP in migraine pathophysiology. This work improves the understanding of peripheral CGRP actions in migraine and raises awareness that contribution of the vasculature in migraine should not be ignored. The identification of sites of CGRP action in regions inside and outside of the CNS could lead to improved and more successful therapeutics for migraine.

Eight experiments examined the role of the intertrial interval in the extinction of conditioned fear to a context. Rats were shocked in one context (A) but not in another (B) and freezing responses to Context A were extinguished. The interval between extinction trials was spent in the home cages. Experiments 1a and 1b showed that massed extinction trials produced better response loss but worse learning than spaced trials. Experiment 2 demonstrated that the interval between the final extinction trial and test mediated the level of responding on a test exposure. Experiments 3 and 4 showed that the duration of the extinction trial affected long term response loss, whereby long durations facilitate response loss compared to shorter durations. Subsequent experiments (Experiments 5 to 8) demonstrated that the first in the series of massed extinction trials reduced the associability of subsequent trials. Associability was restored by alternating extinction trials between Context A and Context B. The results are discussed in terms of the role accorded to self-generated priming in the models developed by A. R. Wagner (1978; 1981).

Very little is known about red-legged seriema (Cariama cristata) vocalizations and behavior. The focus of my research was to analyze the structure of red-legged seriema primary calls and to begin to test hypotheses related to their function. Captive red-legged seriema behavior, both vocal and non-vocal, was recorded at 7 institutions located in the United States from May 2007 through November 2008. To test whether calls serve a predator alarm function, seriemas were presented with a large stuffed dog, representing a predator stimulus, and 2 control stimuli, a large, stuffed penguin and a large, flowering potted plant. Baseline recordings with no manipulations were also made throughout the day, during the course of the study. All recordings of seriema vocalizations were assessed using sound analysis software to generate sound spectrograms for comparison. I identified three different note types within the seriema primary call: upsweep notes (U), ladder notes (L), and two-part notes (T). Each call begins with a set of U notes that increase in rate and lead into the main part of the call, which is made of L and T notes. Each note type was assessed for duration, frequency, energy, and entropy measurements. These measurements were reduced using factor analysis into two relevant factors. There were substantial differences across individuals for note type acoustic structure. Group differences in note types, including sex, location, and age, were not found. When presented with the dog model, vocal responses were given by only 3 individuals. Overall, it may be possible to identify individual seriemas by analyzing the structure of their primary call, which may allow researchers to identify individuals without physical markers.

Thesis (Ph.D.)--McMaster University, 2005. / Advisor: Bennett Galef. Includes bibliographical references. Also available online.

<p>Adolescence is the period of transition between childhood and adulthood, and is characterized across mammalian species by changes in behavior that include increases in risk taking, novelty/sensation seeking, and social behavior. Immaturity of the central serotonergic system during adolescence could contribute to risk taking behavior by resulting in lower avoidance of aversive stimuli in adolescents than adults. The purpose of this dissertation was to investigate whether immature serotonergic function could contribute to adolescent risk taking. We studied pre- and postsynaptic serotonergic function and circuit-level mechanisms relevant to risk taking behavior using behavioral and neurochemical approaches.</p><p>Serotonergic modulation of behavior was assessed in adult (67-74 day old) and adolescent (28-34 day old) male rats in the novelty induced hypophagia (NIH), elevated plus maze, (EPM), and light/dark (LD) tests for anxiety-like behavior. Serotonin depletion with the synthesis inhibitor p-chlorophenylalanine (PCPA) produced anxiolytic effects only in adult rats in the NIH test and in both age groups in the EPM. These data showed that some serotonin-mediated behavioral inhibition is present during adolescence. However, adolescent rats were less sensitive than adults to the anxiogenic effects of the serotonin releasing drugs fenfluramine and methylenedioxymethamphetamine (MDMA) and the serotonin uptake inhibitor fluoxetine in the LD test, suggesting that serotonin is not as effective at inhibiting behavior in adolescents as it is in adults.</p><p>Microdialysis conducted in medial prefrontal cortex (mPFC) showed that adolescent rats exhibited lower increases in extracellular serotonin after treatment with the releasing drug fenfluramine, but not the uptake inhibitor fluoxetine. Further investigation of presynaptic serotonin function in adults and adolescents revealed that adolescent rats have lower tissue serotonin content than adults in several forebrain regions, but similar rates of serotonin synthesis, density of serotonin transporter (SERT)-immunoreactive innervation, and SERT radioligand binding. These data suggest that adolescents may have a lower increase in extracellular serotonin than adults after a releasing drug, but not an uptake inhibitor, due to lower tissue serotonin stores. Lower serotonin stores may limit the ability of a releasing drug to increase extracellular serotonin, but are unlikely to affect response to an uptake inhibitor. These findings also indicate that extracellular serotonin does not completely account for lower serotonin-mediated behavioral inhibition in adolescents. </p><p>Since presynaptic serotonin function did not explain age differences in the anxiogenic effects of indirect serotonin agonists, we investigated postsynaptic serotonin signaling by testing the behavioral effects of serotonin receptor agonists in the LD test. Adolescent rats were less sensitive than adults to the anxiogenic effects of the 5-HT_1A agonist 8-hydroxy-2-(dipropylamino)tetralin (8-OH DPAT) in the LD test, but not to the 5-HT₂ agonist meta-chlorophenylpiperazine (mCPP). No age differences were observed in <super>3</super>H-8-OH DPAT binding in prefrontal cortex, amygdala, or hippocampus between adolescents and adults, and infusion of 8-OH DPAT into mPFC (prelimbic cortex), ventral hippocampus, or basolateral amygdala was unable to replicate the systemic effects of 8-OH DPAT. These data suggest that lower adolescent sensitivity to the anxiogenic effects of 8-OH DPAT is not due to age differences in receptor expression, and show that 5-HT_1A stimulation in mPFC, ventral hippocampus, and basolateral amygdala alone is not sufficient to mimic the effects of systemic 8-OH DPAT. </p><p>We tested the circuit-level effects of fluoxetine and 8-OH DPAT, since stimulating 5-HT_1A receptors in single brain regions failed to reproduce age differences in systemic 8-OH DPAT administration. Both drugs activated regions of the amygdala more in adults than adolescents, and 8-OH DPAT also produced greater prefrontal cortical activation in adults. Fluoxetine produced greater expression of the immediate early gene c-Fos in regions of the extended amygdala in adult rats, and 8-OH DPAT produced greater activation of the lateral orbital cortex and central amygdala in adult rats. Lower activation of cortical and amygdala brain regions could underlie the lower behavioral effects of these drugs in adolescents, as these brain regions are important in mediating behavioral inhibition and anxiety-like behavior. These data are also consistent with human studies showing immature cortical and amygdala function during adolescence. </p><p>This dissertation shows that adolescents are less sensitive than adults to serotonin mediated behavioral inhibition, and that this may be due to immature activation of neural circuits modulated by the 5-HT_1A receptor between the prefrontal cortex and amygdala. This immature serotonin mediated behavioral inhibition could contribute to adolescent risk taking, drug abuse, and increased risk for suicidality during SSRI therapy for depression and mood disorders.</p> / Dissertation

Video playback is a technique used to study the visual communication and behaviors of animals. While video playback is a useful tool, most experiments lack the ability for the visual stimulus to interact with the live animal. The limited number of experiments involving interactive video playback can be attributed partially to the lack of software available to conduct instructive interactive video playback experiments. To facilitate such interactive experiments, I have created a method that combines real-time animations with video tracking software. This method may be used to conduct interactive playback experiments. To demonstrate this method, a prototype was created and used to conduct automated mating choice trials on female swordtail fish. The results of the mating choice trials show that this prototype is able to create effectively interactive visual stimulus automatically. In addition, the results show that the interactive video playback has a measurable effect on the female swordtail fish, Xiphophorus birchmanni.

Very little is known about red-legged seriema (Cariama cristata) vocalizations and behavior. The focus of my research was to analyze the structure of red-legged seriema primary calls and to begin to test hypotheses related to their function. Captive red-legged seriema behavior, both vocal and non-vocal, was recorded at 7 institutions located in the United States from May 2007 through November 2008. To test whether calls serve a predator alarm function, seriemas were presented with a large stuffed dog, representing a predator stimulus, and 2 control stimuli, a large, stuffed penguin and a large, flowering potted plant. Baseline recordings with no manipulations were also made throughout the day, during the course of the study. All recordings of seriema vocalizations were assessed using sound analysis software to generate sound spectrograms for comparison. I identified three different note types within the seriema primary call: upsweep notes (U), ladder notes (L), and two-part notes (T). Each call begins with a set of U notes that increase in rate and lead into the main part of the call, which is made of L and T notes. Each note type was assessed for duration, frequency, energy, and entropy measurements. These measurements were reduced using factor analysis into two relevant factors. There were substantial differences across individuals for note type acoustic structure. Group differences in note types, including sex, location, and age, were not found. When presented with the dog model, vocal responses were given by only 3 individuals. Overall, it may be possible to identify individual seriemas by analyzing the structure of their primary call, which may allow researchers to identify individuals without physical markers.