Animals have evolved sensory systems that afford innate and adaptive responses to stimuli in the environment. Innate behaviors are likely to be mediated by hardwired circuits that respond to invariant predictive cues over long periods of evolutionary time. However, most stimuli do not have innate value. Over the lifetime of an animal, learning provides a mechanism for animals to update the predictive value of cues through experience. Sensory systems must therefore generate neuronal representations that are able to acquire value through learning. A fundamental challenge in neuroscience is to understand how and where value is imposed in brain during learning. The olfactory system is an attractive sensory modality to study learning because the anatomical organization is concise in that there are relatively few synapses separating the sense organ from brain areas implicated in learning. Thus, the circuits for learned olfactory behaviors appear to be relatively shallow and therefore more experimentally accessible than other sensory systems. The goal of this thesis is to characterize the representation and function of neural circuits involved in olfactory associative learning. Odor perception is initiated by the binding of odors onto olfactory receptors expressed in the sensory epithelium. Each olfactory receptor neuron (ORN) expresses one of 1500 different receptor genes, the expression of which pushes the ORN to project with spatial specificity onto a defined loci within the olfactory bulb, the olfactory glomeruli. Therefore, each and every odor evokes a stereotyped map of glomerular activity in the bulb. The projection neurons of the olfactory bulb, mitral and tufted (M/T) cells, send axons to higher brain areas, including a significant input to the primary olfactory cortex, the piriform cortex. Axons from M/T cells project diffusely to the piriform without apparent spatial preference; as a consequence, the spatial order of the bulb is discarded in the piriform. In agreement with anatomical data, electrophysiological and optical imaging studies also demonstrate that individual odorants activate sparse subsets of neurons across the piriform without any spatial order. Moreover, individual piriform neurons exhibit discontinuous receptive fields that defy chemical or perceptual categorization. These observations suggests that piriform neurons receive random subsets of glomerular input. Therefore, odor representations in piriform are unlikely to be hardwired to drive specific behaviors. Rather, this model suggests that value must be imposed upon the piriform through learning. Indeed, the piriform has been shown to be both sufficient and necessary for aversive olfactory learning without affecting innate odor responses. However, how value is imposed on odor representations in the piriform and downstream associational areas remain largely unknown. We first developed a strategy to track neural activity in a population of neurons across multiple days in deep brain areas using 2-photon endoscopic imaging. This allowed us to assay changes in neural responses to odors during learning in piriform and in downstream associative areas. Using this technique, we first observe that piriform odor responses are unaffected by learning, so learning must therefore impose discernable changes in neural activity downstream of piriform. Piriform projects to multiple downstream areas that are implicated in appetitive associative learning, such as the orbitofrontal cortex (OFC). Imaging of neural activity in the OFC reveal that OFC neurons acquire strong responses to conditioned odors (CS+) during learning. Moreover, multiple and distinct CS+ odors activatethe same population of OFC neurons, and these responses are gated by context and internal state. Together, our imaging data shows that an external and sensory representation in the piriform is transformed into an internal and cognitive representation of value in the OFC. Moreover, we found that optogenetic silencing of the OFC impaired the ability of mice to acquire learned associations. Therefore, the robust representation of expected value of the odor cues is necessary for the formation of appetitive associations. We made an important observation: once the task has been learned with a set of odors, the OFC representation decays after learning has plateaued and remains silent even when mice encounter novel odors they haven’t previously experienced. Moreover, silencing the OFC when it was not actively engaged during the subsequent learning of new odors had no effect on learning. These sets of imaging and silencing experiments reveal that the OFC is only important during initial learning; once task structure has been acquired, it is no longer needed. Task performance after initial task acquisition must therefore be accommodated by other brain regions that can store the learned association for long durations. We therefore searched for other brain regions that held learned associations long-term. In the medial prefrontal cortex (mPFC), we observe that the learned representation persists throughout the entire course of training. Unlike the OFC, not only does this representation encode the positive expected value of CS+ odors, it also encodes the negative expected value of CS- odors in a non-overlapping ensemble of neurons. We further show through optogenetic silencing that this representation is necessary for task performance after the task structure has already been acquired. Therefore, while the OFC representation is required for initial task acquisition, the mPFC representation is required for subsequent appetitive learning and performance. Why would a learned representation vanish in the OFC and betransfered elsewhere? We hypothesize that the brain may allocate a portion of its real estate to be a cognitive playground where experimentation and hypothesis testing takes place. Once this area solves a task, it may unload what it has learned to storage units located elsewhere to free up space to learn new tasks. We further imaged another associative area, the basolateral amygdala (BLA), and found a representation of positive value that appears to be generated from a Hebbian learning mechanism. However, the silencing of this representation during learning had no effect. This suggests that while multiple and distributed brain areas encode cues that predict the reward, not all may be necessary for the learning process or for task performance. In summary, we have described a series of experiments that map the representation and function of different associational areas that underlie learning. The data and the techniques employed have the potential to significantly advance the understanding of learned behavior.

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This project analyzed gender and color-specific differences in event-related potentials (ERPs). Previous studies have shown that males process color differently than females. In a recent study, sex differences were found in ERPs during a visual object recognition task. There were higher EEG amplitudes in females (especially P300) than males. Significant sex and color-specific differences have been found in diseases involving altered dopamine (DA) machinery. Thus, we analyzed differences between ERPs in males vs females during a color task. We also compared the color-specific differences in ERPs between males and females. Males and females participated in EEG recording sessions for 2 color studies during a color-go-no-go task, where two studies examined the gender and color-specific differences in ERPs, respectively. Data from 32 males and 24 females and 21 females and 31 males, respectively, in two color studies demonstrated significant sex-specific differences in ERPs during a color-go-no-go task. Males consistently showed higher EEG amplitudes (particularly P300) than females, which is contradictory to what we demonstrated previously in the object recognition task, indicating different color processing systems in males and females. Regarding color-specific differences, no significant differences were found in P300s between the three colors red, green and blue in males and females when each color was the relevant stimulus, suggesting that color is not a marker for inducing ERPs in normal subjects. These studies will provide the impetus to compare patients having altered DA mechanisms such as in attention-deficit hyperactivity disorder (ADHD), Parkinson's, or chemical addiction.

Much can be gained by applying knowledge and insight gleaned from the field of neuropsychology to the field of education. Diagnosis and treatment of learning disabilities (LD) could be enhanced through an increased understanding of neurolinguistic functioning. The present study examined the effect of five instructional techniques aimed at stimulating the cognitive functioning of students with diagnosed learning disabilities. The defining characteristic of each of the five techniques is the use of interactive dialogue to stimulate oral language production leading to greater cognitive efficiency. Evidence is presented for the need for interhemispheric collaboration in complex linguistic tasks such as reading, writing, spelling, and arithmetic. Students with learning disabilities could be viewed as having a breakdown in dynamic functioning impacting neurological systems.;The intervention model developed by the National Institute for Learning Disabilities (NILD) assessed in the present study is based upon the theoretical foundations of Feuerstein (1980), Luria (1981), Piaget (1959), and Vygotsky (1962/1975). The interrelatedness of thought and language, the creation of the zone of proximal development, the recognition of the plasticity of intelligence and the belief in the importance of a human mediator in the learning process, each contributes to the design of techniques used in the NILD program.;The statistical analysis showed significant group-by-time interaction effects in the areas of general and verbal cognitive functioning for the experimental group (n = 47), as assessed by the Detroit Tests of Learning Aptitude - Second Edition (DTLA-2) when compared to the control group (n = 25). Significant gains over time were evidenced by the experimental group in reading, spelling, and arithmetic scores as measured by the Wide Range Achievement Test - Revised (WRAT-R), and in nonverbal cognitive functioning as measured by the DTLA-2.;Overall results indicated that students with diagnosed learning disabilities benefited from an intensive individualized program over a three-year period in a modified pull-out approach involving 160 minutes of instruction per week. Specifically, the interactive effects of five core instructional techniques appeared to significantly impact neurolinguistic functioning for the experimental group.

Polyglutamine (polyQ) diseases are progressive fatal neurodegenerative movement disorders. Although many cellular processes are perturbed in polyQ disease, recent studies highlight the importance of protein misfolding as a central event in polyQ toxicity. Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, is a particularly interesting polyQ disease because of the special qualities of the disease protein ataxin-3, which normally participates in cellular protein quality control. Here I use multiple mouse models of disease to explore toxic protein species and the role of protein homeostasis in SCA3 pathogenesis. In Chapter 1, I review the key features of polyQ disease, and outline the background and rationale behind our strategy for identifying toxic protein species in SCA3. In Chapter 2, I examine the role of the protein quality control ubiquitin ligase, CHIP (C-terminus of Hsp70 interacting protein), in regulating the toxicity of expanded ataxin-3 in vivo. Genetic reduction or removal of CHIP increases formation of detergent-resistant ataxin-3 microaggregates specifically in the brain. Concomitant with this, reduction or removal of CHIP exacerbates the phenotype of SCA3 mice, revealing a correlation between high levels of microaggregates and phenotypic severity. Additional cell-based studies confirm that CHIP may not directly mediate ataxin-3 degradation, suggesting that CHIP reduces expanded ataxin-3 toxicity in the brain primarily by enhancing ataxin-3 solubility. In Chapter 3, I use various biochemical techniques to reveal the presence of brain-specific ataxin-3 microaggregates in two genetically distinct mouse models of SCA3. Selective neuropathological evaluation of SCA3 mice reveals that major neuronal loss and reactive glial proliferation are not shared features of phenotypically-manifesting SCA3 mice. Additional studies fail to provide evidence for loss-of-function of endogenous ataxin-3 in SCA3 mice. Our results suggest that neuronal dysfunction in SCA3 is mediated through a toxic gain-of-function mechanism by ataxin-3 microaggregates in the CNS. In Chapter 4, I discuss important areas for future research in polyQ disease. I describe studies that would help elucidate the structural nature of toxic soluble microaggregates, and their effects on other cellular proteins. I also consider how the results described in this thesis inform potential treatment strategies.

Proper control of movements is critical for an animal’s survival, and requires the robust function of a number of genetic, molecular, neuronal and biomechanical processes. This dissertation describes a body of inter-related studies utilizing a diverse collection of Drosophila mutants to probe the roles individual genes play in shaping motor pattern generation. A particular emphasis is placed on describing the consequences of genetic perturbations of voltage-gated sodium, calcium and potassium ion channels (NaV, CaV, and KV respectively) on the function of neuronal circuits that drive motor behavior. Here, I describe the development of several quantitative protocols to study alterations in of walking (IowaFLI Tracker) and flight motor program activity and behavior in Drosophila mutants. These approaches were utilized to analyze the highly-stereotypic aberrant motor program associated with electroconvulsive stimulation (ECS)-induced seizure discharge activity in each hyperexcitable mutant. Several quantitative and mechanistic similarities between flight motor program activity and ECS-evoked discharges were identified, and the distinct aberrant ECS-evoked activity disclosed an electrophysiological signature of each mutation. Ion channel mutants display a diverse spectrum of neuronal excitability phenotypes that was highlighted in a novel hyperexcitable mutant, Shaker wings down (Swd), characterized by ether-induced leg shaking reminiscent of certain KV channel mutants (e.g. Shaker, KV1) is presented. Detailed analyses revealed disrupted walking and flight, correlated with neuronal hyperexcitability and aberrant action potential generation. Surprisingly, the Swd mutation site was mapped to a single amino acid in the voltage sensor region in paralytic (para, encoding the only NaV gene in Drosophila). Genetic analysis of intra-genic heteroallelic interactions amongst Swd and other identified para alleles further revealed a number of complex mechanisms underlying a wide phenotypic spectrum of altered neuronal excitability and motor pattern generation. The effects of perturbed ion channel function on motor program generation are compared with progressive alterations associated normal aging as well as neurodegeneration. A number of age-resilient and age-vulnerable circuits were identified along with circuit-function biomarkers of aging. Throughout this study, an integrative framework utilizing non-linear dimensional reduction approaches unraveled a broader perspective to visualize and quantify similarities and distinctions between discharge phenotypes across a large collection of Drosophila mutants.

To experience the world, we depend on the ability of our brains to process information. Problems can occur when communication between neurons is not regulated, and a significant enough loss of stability could lead to conditions such as migraine and epilepsy. Homeostatic plasticity is thought to constrain activity within physiologically useful ranges. Our lab uses the fruit fly neuromuscular junction as a model synapse to study homeostatic plasticity. Homeostatic potentiation and homeostatic depression are two forms of homeostatic synaptic plasticity. Expression of a dominant negative glutamate receptor subunit in the muscle impairs its sensitivity to glutamate and triggers an increase in the number of vesicles released per evoked potential, or quantal content. This increase in quantal content is called homeostatic potentiation. We found that homeostatic potentiation is a reversible process: quantal content returns to normal levels when expression of the dominant negative ceases. We additionally found that homeostatic potentiation can be ablated at high temperature. Overexpression of the Vesicular Glutamate transporter (VGlut) causes an increase in the amplitude of spontaneous events, leading to a corresponding decrease in quantal content, called homeostatic depression. It is unknown to what degree homeostatic potentiation and homeostatic depression may share regulatory machinery. We screened genes required for homeostatic potentiation in the neuron for additional roles in homeostatic depression. We found that certain genes involved in calcium regulation, such as the IP3 receptor and ryanodine receptor, showed a substantial decrease in evoked potential amplitude in a VGlut overexpression background.

During neuronal activity mitochondria alter cytosolic Ca2+ signaling by buffering then releasing Ca2+ in the cytosol. This calcium transport by mitochondria affects the amplitude, duration, and spacial profile of the Ca2+ signal in the cytosol of neurons. This buffering by mitochondria has been shown to affect a variety of neuronal functions including: neurotransmission, gene expression, cell excitability, and cell death. Recently, researchers discovered that the protein CCDC109A (mitochondrial Ca2+ uniporter) was the protein responsible for mitochondrial Ca2+ uptake. Using a genetic knockout (KO) mouse model for the mitochondrial Ca2+ uniporter (MCU) my research investigated the role of MCU in neuronal function. In cultured central and peripheral neurons, MCU-KO significantly reduced mitochondrial Ca2+ uptake while significantly increasing the amplitude of the cytosolic Ca2+ signal amplitude. Behaviorally, MCU-KO mice show a small but significant impairment in memory tasks: fear conditioning and Barnes maze. Using a maximal electroshock seizure threshold model of in vivo seizure activity my research found that MCU-KO significantly increases the threshold for maximal seizure activity in mice and significantly reduces seizure severity. In addition to mitochondrial Ca2+ uptake, my research also investigated the mechanisms involved in mitochondrial Ca2+ extrusion. The protein SLC8B1 (SLC24A6, NCLX) is the putative transporter responsible for the Na+/Ca2+ exchange, mitochondrial calcium extrusion. Using genetic NCLX-KO mice, our research found that in neurons NCLX contributes to cytosolic Ca2+ extrusion, but does seem to directly affect mitochondrial Ca2+ extrusion.

Interstitial cystitis (IC)/painful bladder syndrome (PBS) is a functional visceral disorder characterized by increased bladder activity and chronic pelvic pain in the absence of a pathobiological condition. Enhanced sensory transduction of peripheral bladder afferents is hypothesized to contribute to the pain and mechanical hypersensitivity of IC/PBS patients. The aim of this thesis is to test the hypothesis that purinergic receptors, including ionotropic P2X and metabotropic P2Y, are important for sensory transmission in bladder afferent neurons and may be involved in bladder hypersensitivity after bladder tissue insults. Electrophysiological, single cell RT-PCR and immunohistochemistry techniques were performed in bladder afferent neurons from naïve and bladder inflamed mice to test the hypothesis. In Chapter 2, I characterized the distribution and function of P2X receptors in thoracolumbar (TL) and lumbosacral (LS) dorsal root ganglia (DRG) neurons innervating the urinary bladder, and found that LS and TL bladder neurons have differential purinergic signaling and distinct membrane electrical properties. In Chapter 3, I examined the sensitization of bladder afferent neurons and the plasticity of P2X receptor function in a mouse model of chemical induced bladder inflammation. P2X-mediated signals in LS and TL bladder neurons after bladder inflammation were enhanced compared with those in saline-treated controls, suggesting the importance of P2X in bladder hypersensitivity associated with cystitis. In Chapter 4, the modulation of P2Y on P2X function and the co-localization of P2Y and P2X were examined in bladder sensory neurons. It has been found that P2Y2 receptor enhances bladder sensory neuron excitability and facilitates the response of homomeric P2X2 receptor to the purinergic agonist (ATP). The present study provides evidence that LS and TL mouse bladder sensory neurons exhibit distinct P2X signaling, and the function of P2X receptors could be facilitated during bladder inflammation and modulated by activation of P2Y2 receptor, indicating an involvement of P2X and P2Y2 receptors as mechano- and chemosensors in bladder sensory transmission under normal conditions and in bladder hypersensitivity associated with inflammation.

Emotion regulation is an ability that humans engage in throughout their lives. Disruption in this ability due to brain injury can have devastating consequences on the ability to function adaptively in complex environments. It has been observed that damage involving certain areas of the prefrontal cortex (PFC), including the ventromedial PFC (VMPFC), can result in long-lasting impairments in real-world emotional and behavioral functioning. However, the specific areas of the PFC that are critical for the ability to regulate emotion have not been identified. The primary aims of this project were to identify areas of the PFC that are important for the regulation of emotion, and to determine the degree to which impairments in emotion regulation may contribute to real-world dysfunction following damage to the PFC. To address these aims, emotional regulation and real-world functioning were examined in a sample of patients with focal PFC lesions. Damage involving the VMPFC appeared to have limited impact on the ability to voluntarily regulate emotion. It was also observed that damage to PFC regions outside the VMPFC was associated with reduced ability to overcome distraction by salient emotional stimuli, compared to VMPFC damage. However, analyses of lesion volume showed that more extensive damage involving the VMPFC was associated with greater emotional distraction, suggesting one form of emotional dysregulation that may result from damage to the VMPFC. In addition, it was found that brain damage in general was associated with impairments in real-world functioning, though PFC damage was not associated with more striking impairments compared to damage outside the PFC. These findings suggest that damage involving certain PFC regions can disrupt the ability to effectively regulate emotion. The results from this project also suggest that laboratory measures of emotion regulation may help in predicting real-world dysfunction following brain damage.