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Learned motor patterns such as speaking, playing musical instruments and dancing require a defined sequence of movements. The mechanism of acquiring and perfecting these types of learned behaviors involve a highly complex neurological process not exclusive to humans. In fact, vocal learning in songbirds is a well-known model to study the neural basis of motor learning, particularly human speech acquisition. In this dissertation, I explored differences in the intrinsic physiology of vocal cortex neurons – which underlie song acquisition and production in the zebra finch (Taeniopygia guttata) – as a function of age, sex, and experience using a combination of electrophysiology and mathematical modeling. Using three developmental time points in male zebra finches, Chapter 3 presents evidence of intrinsic plasticity in vocal cortex neurons during vocal learning. The experimental results in this chapter revealed age- and possibly learning-related changes in the physiology of these neurons, while the mathematical models suggest possible variations in both the expression and kinetics of several ion channels that cause the physiological changes. Exploiting the fact that male zebra finches exhibit auditory and vocal song learning, while females exhibit auditory song learning only, in Chapter 4 I compared the physiology of vocal cortex neurons between sexes. This comparison reveals aspects of the neurons’ physiology specialized for singing (males only) vs. auditory learning of song (both males and females). Finally, in Chapter 4 I explored the effect of auditory learning in the physiology of vocal cortex neurons in females. Experimental results and mathematical models revealed regulation in ion channel expression due to auditory learning. In summary, this dissertation describes the effect of three new variables – age, sex, and experience – now known to influence the physiology of key neurons in vocal learning. / A Dissertation submitted to the Department of Mathematics in partial fulfillment of the Doctor of Philosophy. / Summer Semester 2017. / July 20, 2017. / Includes bibliographical references. / Richard Bertram, Professor Directing Dissertation; Debra Ann Fadool, University Representative; Richard Hyson, Committee Member; Harsh Jain, Committee Member; Frank Johnson, Committee Member; Washington Mio, Committee Member.

Despite intense therapeutic and diagnostic focus on dyshomeostasis of amyloid \(\beta\)-peptide \((A\beta)\) in Alzheimer’s disease (AD), we still lack insight into the in vivo economy of \(A\beta\) in the normal and diseased brain. Thus, my thesis research focused on understanding the dynamics of \(A\beta\) in the living brain during the development of AD-type pathology. Using in vivo microdialysis, I showed that the steady-state level of \(A\beta\) that remains diffusible in the hippocampal interstitial fluid (ISF) of awake, behaving hAPP transgenic mice falls as \(A\beta\) steadily accumulates in the brain parenchyma. In accord, I observed distinct dispositions of microinjected radiolabeled \(A\beta\) in plaque-rich versus plaque-free mice, suggesting that cerebral amyloid deposits rapidly sequester newly released \(A\beta\). This provides the first in vivo evidence from controlled animal experiments for the hypothesis that soluble \(A\beta42\) in human cerebrospinal fluid (CSF) falls in AD because it is sequestered into insoluble parenchymal deposits as the disease develops. My data further show that the association of \(A\beta\) with insoluble parenchymal deposits is not irreversible, as acute inhibition of \(\gamma\)-secretase in plaque-rich mice failed to lower ISF \(A\beta42\), whereas it did in plaque-free mice. Hence, the ISF in plaque-rich mice seems to be a reservoir for both newly produced \(A\beta\) and \(A\beta\) that diffuse off of cell membrane- and plaque-bound deposits. Finally, I showed that \(A\beta\) dimers, which are known to be potent synaptic neurotoxins, are undetectable in the aqueous compartments of the central nervous system, i.e., the brain ISF and CSF, in hAPP transgenic mice. Acute injection of \(A\beta\) dimers into living wild-type mice showed a rapid sequestration of the dimers away from the hippocampal ISF pool and a higher recovery in the membrane-bound pool than in the cytosolic pool of the brain homogenates. Interestingly, I found that the \(A\beta\) recovered in the membrane-bound pool was tightly associated with endogenous GM1 ganglioside. Taken together, my results suggest that \(A\beta\) dimers, and probably higher oligomers, are rapidly sequestered away from the ISF and bind to GM1 ganglioside-enriched lipid membranes, such as raft-like microdomains of secreted vesicles or on the plasma membranes of neurons and other cells.

The activity of sensory neurons can be modulated by both external stimuli and an animal’s internal state. Characterizing the role of these bottom-up and top-down factors as well as the way in which they interact is critical for an understanding of how the activity of sensory neurons contributes to perception. To this end, we recorded from the middle temporal area (MT) in awake-behaving primates in order to measure the joint tuning properties of these neurons for two commonly studied feature dimensions, direction of motion and binocular disparity. Additionally, we set out to determine whether attention directed to these two features can modulate the responses of MT neurons. We showed that MT neurons have fixed tuning preferences for direction of motion and binocular disparity and thus represent these features in a separable manner. Further, we have demonstrated that MT neurons can be modulated by feature attention for both direction of motion and binocular disparity and that the amount of this modulation depends on a neuron’s tuning strength. These results further our understanding of how stimulus features are jointly represented in the brain and how the attentional system interacts with these representations in order to facilitate perception.

We completed two separate studies examining chemosensation in Drosophila. The first study investigated taste processing. It was our aim in this study to identify and characterize higher-order gustatory neurons. Our strategy for tackling this problem involved complementary functional and anatomical approaches. First, we used calcium imaging to screen for cells responding to stimulation of gustatory receptor neurons. Second, we used photo-activatable GFP to localize the cell bodies of neurons innervating the gustatory neuropil. Third, based on the information we gained from these imaging experiments, we were able to identify some promising Gal4 lines that labeled candidate gustatory neurons. Fourth and finally, we made whole-cell patch clamp recordings from these candidate gustatory neurons while stimulating the proboscis with tastants. Unfortunately, none of these candidates turned out to be gustatory neurons. However, this study illustrates a flexible and powerful general approach to identifying and characterizing sensory neurons in the Drosophila brain. The second study investigated olfactory transduction. Specifically, we examined the effect of air speed on olfactory receptor neuron responses (ORNs) in Drosophila. We constructed an odor delivery device that allowed us to independently vary concentration and air speed, and we used a fast photoionization detector to precisely measure the actual odor concentration at the antenna while simultaneously recording spikes from ORNs in vivo. Our results demonstrate that Drosophila ORN odor responses are invariant to air speed, as long as odor concentration is kept constant. This finding was true across a >100-fold range of air speeds. Because odor hydrophobicity has been proposed to affect the air speed dependence of olfactory transduction, we tested a >1,000-fold range of hydrophobicity values, and found that ORN responses are invariant to air speed across this full range. These results have implications for the mechanisms of odor delivery to Drosophila ORNs. Our findings are also significant because flies have a limited ability to control air flow across their antennae, unlike terrestrial vertebrates which can control air flow within their nasal cavity. Thus, for the fly, invariance to air speed may be adaptive because it confers robustness to changing wind conditions.

The development of anxiety and mood disorders often coincides with exposure to stress. Accumulating evidence indicates that both corticotropin-releasing factor (CRF) and dynorphin, the endogenous ligand for the kappa-opioid receptor (KOR), can mediate the effects of stress. My dissertation research utilized laboratory animals to investigate the role of KORs in stress-induced increases in the acoustic startle response, a metric often used to study stress effects in humans. Using wild-type mice, I first demonstrated that systemic administration of a KOR antagonist produced an anxiolytic-like effect on acoustic startle following central (intracerebroventricular) infusion of CRF. Immunohistochemical analysis revealed that KOR blockade decreased c-Fos cell counts in the dentate gyrus of the hippocampus in both vehicle- and CRF-treated mice, and reduced CRF-induced increases in the ventral tegmental area (VTA). Within the VTA, reductions were predominantly in dopaminergic neurons. KOR antagonist pretreatment also produced anxiolytic-like effects on footshock-potentiated startle, a model that quantifies context-specific fear conditioning. To complement the antagonist studies, we developed constitutive knockout mice that lack KORs throughout the brain (KOR-/-), and conditional KOs that lack KORs only within dopaminergic neurons (DAT-KORlox/lox). Initial characterization demonstrated that these two mutant lines did not differ from controls in hearing, vision, weight gain, and locomotor activity. KOR-/- mice were similar to controls in unconditioned anxiety-like behavior, but DAT-KORlox/lox mice displayed nominal decreases in anxiety-like behavior in the open field and light/dark box. Unexpectedly, KOR ablation did not affect CRF-induced increases in startle in either mutant line. Importantly, however, KOR antagonist treatment did not alter CRF-induced increases in startle in KOR-/- mice, suggesting that KOR antagonist effects in wild-type mice are due to blockade of KORs. These findings raise the possibility that differences in KOR antagonist and KOR-/- studies may be related to brief KOR blockade during adulthood versus a lack of KORs during the entire lifespan. In the footshock-potentiated startle paradigm, KOR-/- mice were comparable to littermate controls, whereas DAT-KORlox/lox mice showed attenuated effects of footshock. My findings confirm a role for KORs in fear and anxiety-like behavior in rodents, and implicate KORs expressed on dopaminergic neurons in modulating important aspects of stress-related behavior.

When confronted with changing environments, animals can generally adjust their behavior to optimize reward and minimize punishment. The process of modifying one's behavior based on its consequences is referred to as operant or instrumental learning. Operant learning makes specific demands on the animal. The animal must exhibit some flexibility in its behavior, switching from unsuccessful motor responses to potentially successful ones. The animal must represent the consequence of its actions. Finally, the animal must select the correct response based on its past history of reinforcement. Studies in mammalian systems have found competing and complementary circuits in the cortex and striatum that mediate different aspects of this learning process. The larval zebrafish is an ideal system to extend the study of operant learning due to its genetic and optical properties. We have developed a behavioral paradigm and imaging system that have allowed us to comprehensively image neural activity throughout the zebrafish brain during the process of operant conditioning. Our analysis of the neural network activity underlying this learning process reveals several classes of neurons whose activity correlates with learning and decision making. The distribution of these learning-related neurons is highly localized to regions of the habenula and forebrain. We describe, in particular, a lateralized habenula circuit that may encode prediction and relief prediction error.

Animals undergo periods of behavioral quiescence and arousal in response to environmental, circadian, or developmental cues. During larval molts, C. elegans undergoes a period of profound behavioral quiescence termed lethargus. Locomotion quiescence during lethargus was abolished in mutants lacking a neuropeptide receptor (NPR-1), and was reduced in mutants lacking NPR-1 ligands (FLP-18 and -21). Wild type strains are polymorphic for the npr-1 gene, and their lethargus behavior varies correspondingly. Locomotion quiescence and arousal were mediated by decreased and increased secretion of an arousal neuropeptide (PDF-1) from central neurons. PDF receptors (PDFR-1) expressed in peripheral mechanosensory neurons enhanced touch-evoked calcium transients. Thus, a central circuit stimulates arousal from lethargus by enhancing the sensitivity of peripheral mechanosensory neurons in the body. These results define a circuit mechanism controlling a developmentally programmed form of quiescence.

Autism spectrum disorder (ASD) and intellectual disability (ID) are neurodevelopmental disorders of cognition that remain incompletely understood. Here, using a computation-assisted interaction proteomics approach in neural cells including primary neurons, we isolate high-confidence binding partners of proteins linked to ASD and ID. As part of these studies, we uncover the brain-enriched, coiled-coil domain protein huntingtin-associated protein 1 (Hap1) as a novel functional binding partner of the tuberous sclerosis complex (TSC) protein Tsc1. We validate and map the Hap1-Tsc1 interaction, and find that Hap1 and Tsc1 form a complex endogenously in the brain. Hap1 knockdown in primary hippocampal neurons triggers the specification of supernumerary axons, and in utero knockdown of Hap1 in mice profoundly impairs the positioning of pyramidal neurons in the hippocampus in vivo. Importantly, the Hap1 knockdown-induced phenotypes in primary neurons and in vivo recapitulate the phenotypes induced by Tsc1 knockdown. We also define a mechanism by which Hap1 regulates Tsc1 function. We observed that exogenous Hap1 promotes the abundance of soluble, stable Tsc1 expressed in cells. Hap1 knockdown in neurons reduces Tsc1 abundance and accordingly stimulates the activity of mTORC1, as reflected by phosphorylation of the ribosomal protein S6. Importantly, inhibition of mTORC1 signaling suppresses the Hap1 knockdown-induced axon phenotype in hippocampal neurons. Collectively, these findings define a novel relationship between Hap1 and Tsc1 that regulates neuronal Tsc1 abundance, pyramidal neuron development, and neuronal mTORC1 signaling, with important mechanistic implications for our understanding of neurodevelopmental disorders of cognition.