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.

Thesis (Ph. D.)--College of Computing, Georgia Institute of Technology, 2004. Directed by Larry F. Hodges. / Vita. Includes bibliographical references (leaves 112-115).