Though mutant proteins are broadly expressed in neurodegenerative diseases, only some neuronal subsets are vulnerable. In patients with amyotrophic lateral sclerosis (ALS), most motor neurons degenerate but those innervating extraocular muscles, pelvic sphincters and slow limb muscles exhibit selective resistance. Previous work in our lab used laser capture microdissection followed by microarray analysis of vulnerable lumbar level (L5) motor neurons and the resistant motor neuron populations that innervate the extraocular muscles (III) and the pelvic sphincter muscles (DL) to identify hundreds of molecular markers of each subset. By looking at the genes that had > 10 fold differential expression and that were selectively and strongly expressed in motor neurons into adulthood, we identified matrix metalloproteinase-9 (MMP-9) as a potential susceptibility gene. We first examined the expression of MMP-9 in wild-type mice and found that it is expressed only after postnatal day 5 in fast motor neurons, which are selectively vulnerable in ALS. Further, MMP-9 was the only secreted member of the MMP family constitutively expressed in this population. In mutant SOD1 mice, MMP-9 expression was strongly positively correlated with cell loss at end-stage of the disease and with early induction of endoplasmic reticulum stress, as measured by p-EIF2á. To test whether MMP-9 is a driver of disease, we crossed Mmp9 null mice with mSOD1 mice and found that both partial reduction and complete ablation of MMP-9 levels delayed muscle denervation, prolonged survival, and improved motor function, measured both behaviorally and electrophysiologically. Importantly, even acute knock-down in motor neurons with AAV6 gene therapy or central inhibition of MMP-9 after symptom-onset were able to protect muscle innervation of the fast hindlimb muscle, TA. Further, MMP-9 expressed by motor neurons is required for full ER stress activation, suggesting it may be a very early intermediate in the disease pathway triggered by mutant SOD1. However, virally introducing MMP-9 into resistant pools does not confer susceptibility, implying that there is an additional factor (or factors) also present in fast motor pools that is necessary to induce axonal die-back. These studies suggest that MMP-9 is a promising candidate therapeutic target for ALS. Importantly, these data support the study of neuronal diversity as a potential way to define novel therapeutic strategies for the treatment of neurodegenerative diseases.

Carbonation, or the presence of carbon dioxide (CO2) dissolved in solution, is a commonly encountered feature of beverages in the contemporary human diet. While the popularity of carbonation may be attributed to its distinct sensory qualities, the specific orosensory pathways mediating CO2 detection in mammals have not previously been delineated. This dissertation describes the identification of specific cellular and molecular mechanisms that mediate taste sensation of carbonation, using the mouse as a model system. The mammalian gustatory system is sensitive to CO2, and these responses are sensitive to inhibition of carbonic anhydrases, enzymes that catalyze the interconversion of carbon dioxide with carbonic acid. Through gene expression profiling I discovered that the gene carbonic anhydrase IV (Car4), encoding an extracellular enzyme, is specifically expressed in acid sensing taste receptor cells (TRCs). Genetic ablation of the Car4 locus resulted in a major deficit in gustatory CO2 sensation that is stimulus specific, not affecting responses to acid. Ablation or silencing of acid sensing TRCs likewise produced a profound deficit in taste responses to CO2. These studies identified a primary pathway of the gustatory carbonation response, substantiating acid sensing TRC and the Car4 enzyme as key mediators. A smaller gustatory neural response to carbonation remains even in the absence of sour-sensing TRC and/or Car4. To identify additional carbonation sensing pathways, I applied an in vivo calcium-imaging assay to define the ensemble of primary gustatory neurons activated by CO2. These studies revealed that in addition to robust activation of sour sensing neurons, a secondary gustatory pathway for CO2 detection is mediated by subpopulations of bitter and sweet responsive neurons. I identified carbonic anhydrase VII (Car7) as an intracellular carbonic anhydrase specifically expressed by sweet, bitter and umami sensing TRC. Pharmacological and gene expression data support a role for Car7 in transducing the secondary CO2 sensing pathway. These studies suggested that carbonation acts as a complex gustatory stimulus, stimulating sour, sweet and bitter taste qualities simultaneously. The rules governing peripheral encoding of multi-modal taste stimuli are not well understood. To address this issue, I examined the peripheral gustatory response to binary mixtures of taste qualities. I found that most combinations of taste qualities are represented as a superimposition of the component responses. However, neural responses to attractive stimuli, including natural sugars, artificial sweeteners and umami tastants, are selectively suppressed by simultaneous co-stimulation with a sour (acidic) stimulus. Acid-mediated suppression of sweet is cell autonomous, occurring even in the absence of gustatory acid sensing. Remarkably, carbonation stimulates sour signaling without suppressing sweet taste response. These studies suggest that cross-modal interactions at the periphery modulate the sensory response to complex taste stimuli.

Dendrites develop in highly complex environments and their interactions with neighboring neurons and the substrate are thought to be important for the establishment of their dendritic territories. Mechanisms required for the establishment of dendritic territories remain largely elusive. This thesis investigates the role of both dendrite-dendrite interactions and dendrite-substrate interactions in determining dendritic boundaries. As a part of this study I have uncovered novel mechanisms that neurons utilize to define dendritic borders and how those borders may be important determinants of neuronal function. The experiments described in this thesis have taken advantage of genetic tools in Drosophila that allow manipulation and visualization of individual neurons.

Regulating emotions effectively is an indispensable human task, essential for maintaining proper health and well-being. While the investigation of emotions and strategies for regulating them has been a timeless and irresistible activity, pursued by artists and philosophers throughout human history, recent decades have given rise to the controlled examination of emotion and emotion regulation by psychologists in the laboratory. While substantial progress has been made in describing, categorizing, and understanding the effectiveness of multiple strategies to regulate emotion in the laboratory, and while several long-term cognitive treatment modalities incorporating numerous regulation strategies are in practice in clinical psychology, there has been substantially less basic investigation into two overarching questions that form the basis of this dissertation: (1) how we can effectively prepare to regulate emotion using specific strategies? and (2) how can emotion regulation efficacy using particular strategies can change over time through repeated training? In this dissertation, I will focus on one promising type of cognitive change-based emotion regulation strategy, that of cognitive reappraisal. Cognitive reappraisal refers to reevaluating the meaning of an affective stimulus in a way that alters its emotional impact. In a series of four studies, I will address the two above questions using a combination of dependent measures, including questionnaire and task-based self-reported behavior, psychophysiology, and functional neuroimaging. In Study 1, I will provide evidence for the neural mechanisms that are conducive to reappraisal success and failure (measured via behavioral self-report) during anticipation of emotion regulation using whole-brain mediation and pattern expression analyses. Anticipatory activity in an area of rostrolateral prefrontal cortex (RLPFC) commonly associated with stimulus-independent mind-wandering was associated with poorer regulation outcomes, while anticipatory anterior insula activity implicated in internal affective integration was associated with better regulation outcomes. In Study 2, I will examine whether a short course of reappraisal training (in one of two reappraisal modalities: reinterpretation and psychological distancing, or a no-regulation control group) yields improvements in self-reported levels of negative affect during a laboratory task and in questionnaire reports of perceived stress in daily life. Results indicated that distancing shows promise as a trainable emotion regulation strategy, yielding decreasing reports of negative affect over time that were not attributable to habituation. Study 3 used the same experimental paradigm, adding psychophysiological data collection during the laboratory task (mean changes in heart rate). The combined results of Studies 2 and 3 indicated that while there was evidence of longitudinal decreases in negative affect for both distancing and reinterpretation, in distancing these effects were not attributable to habituation, and distancing was further uniquely associated with decreases in perceived stress in daily life among participants. Further, Study 3 indicated that mean changes in heart rate for distancing training yielded a pattern of increasing differentiability between regulated and unregulated trials over time, but this pattern was absent for reinterpretation training and the no-regulation control group. Finally, in Study 4, I examined the effects of a short course of reappraisal massed practice, where one engages in repeated distancing episodes using the same stimuli. Specifically, I examined the behavioral and neural sustainability of responses to stimuli for which one has engaged in massed distancing practice versus simple repeated viewing, versus stimuli regulated but not practiced, and versus novel negative stimuli. Results indicated that distancing massed practice resulted in a sustained adaptive response pattern in a key subcortical appraisal region (amygdala) over time relative to other conditions. Overall, these studies elucidate the temporal dynamics involved in reappraisal response patterns, including evidence for adaptive anticipation mindsets, as well as evidence for the effectiveness of short courses of reappraisal training, particularly using psychological distancing.

Current models suggest that perception is a decision process: given noisy perceptual signals, the brain has to decide what they represent. While attention is known to enhance the perceptual signal, it has been unclear how it modulates the decision mechanism itself. Here we explored this issue in a series of studies. We used a spatial cuing paradigm to manipulate the attentional focus of observers, and found that attention leads to a conservative detection criterion such that attended stimuli are reported less often than unattended ones (Chapter 1). We investigated whether this effect would generalize to situations that do not involve detection tasks by using the same cuing paradigm, but instead asking observers to discriminate between two stimulus categories. We found that attention leads to low subjective ratings of visibility (Chapter 2). In both sets of experiments, the results were strongest when detection or discrimination capacity d' was equated between different levels of attention, or when stimuli had low contrast. To account for these results, we developed a variance reduction (VR) model of attention in which attention is postulated to reduce the variability of the perceptual signal, while keeping the decision criteria constant (Chapter 3). The VR model provided a good fit to the data observed in Chapters 1 and 2. We tested critical assumptions of the model using functional magnetic resonance imaging (Chapter 4). We found that high activity in the dorsal attention network (DAN) in the brain, which is indicative of a high attentional state, led to lower variability in the evoked signal in motion sensitive area MT+, thus supporting the idea that attention reduces perceptual variability. Further, high DAN activity resulted in lower confidence ratings, which confirmed that the findings from Chapter 2 generalize to exogenous attentional fluctuations and are not limited to spatial cuing. We tested the VR model further by extending it beyond the realm of attention (Chapter 5). We used transcranial magnetic stimulation (TMS) to directly increase the variability of the perceptual signal. The effects mirrored the effect of lack of attention: TMS led to decreased performance but increased subjective ratings. Finally, we explored the influence of attention on the amount of information carried by one's subjective ratings. We found that attention made subjective ratings more predictive of accuracy (i.e., attention improved metacognitive sensitivity) despite the fact that it decreased the overall magnitude of the subjective ratings (Chapter 6). To account for this finding, we developed a simple extension to the VR model - the "variance and criterion jitter reduction" (VCJR) model of attention which postulates that attention reduces the amount of trial-to-trial criterion jitter. Computational modeling shows that this reduction of criterion jitter leads to improved metacognitive sensitivity. We discuss these findings in relation to current debates related to attention and subjective perception, and speculate how they may account for our impression that we clearly see everything in our visual fields, including unattended objects that receive little processing.

Animals can quickly learn to make appropriate decisions according to their environment that can change over a wide range of timescales. Yet the neural computation underling the adaptive decision making is not well understood. To investigate basic computational principles and neural mechanisms, here we study simple neural network models for decision making with learning on multiple timescales, and we test our model's predictions in experimental data. We provide basic network models for value-based decision making under uncertainty.

L-type voltage-gated calcium channels are involved in many important physiological processes, including muscle contraction, hormone secretion and neuronal gene expression. These channels are regulated by many different mechanisms to tightly control calcium influx. Our lab has uncovered a new form of L-type channel regulation that involves the proteolysis of the channel in the main body of the alpha1 subunit in response to increased intracellular calcium, channel activity and age. I investigated the immediate and long-term functional impact of mid-channel proteolysis on the CaV1.2 channel. Mid-channel proteolysis causes an acute change in gating and a decrease in channel activity over a longer time scale. Fragment channels result from proteolysis, and these fragments associate on the plasma membrane to form functional channels. These L-type fragment channels exhibit different biophysical properties than full-length CaV1.2. While fragment channels must combine so that all four domains are present to be functional, non-complimentary pairs containing more than four domains still produce discernible current. L-type fragment channels co-immunoprecipitate with the full-length CaV1.2, indicating that fragments bind to either the alpha1 subunit or the channel complex. Some of these fragments cause a shift in inactivation and in the I-V curve of the channel, and one fragment comprising Domain IV and the C-terminus (fragment C2) inhibits full-length CaV1.2 in a dominant negative manner. These results demonstrate the functional effects of mid-channel proteolysis. L-type mid-channel proteolysis increases with animal age. Therefore, to identify the protease responsible for mid-channel proteolysis, I turned to proteases involved in aging diseases. Amyloid-β precursor protein (APP), a protein implicated in Alzheimer's disease (AD), modulates L-type channels and is itself extensively proteolyzed. One of those proteases is presenilin, the catalytic component to gamma-secretase. I found that APP dramatically reduced human CaV1.2 current in Xenopus oocytes. The current-voltage relationship and inactivation profiles of CaV1.2 in the presence of APP mirrored those of the fragment channels. Moreover, a gamma-secretase inhibitor, DAPT, completely reversed this effect. When an AD APP mutant was co-expressed with CaV1.2, currents were further diminished. Astonishingly, an APP mutant that protects against AD had the opposite effect, allowing larger CaV1.2 currents than wild-type APP. Western blots stained with an antibody against CaV1.2 revealed a ~100 kD band when APP was coexpressed with the channel, which was absent in oocytes solely expressing CaV1.2. DAPT application reversed this effect, indicating the band was a product of presenilin proteolysis. A putative cut site was found on the alpha1 subunit that would produce a band similar in size to the one observed in Western blots. When this site was mutated, the ~100 kD band no longer appeared when CaV1.2 was coexpressed with APP. Unfortunately, the CaV1.2 II-III loop antibody was later found to cross-react with APP. Therefore, additional experiments are necessary to determine whether the ~100 kD band is CaV1.2 Interestingly, APP induced mid-channel proteolysis was detected in primary neurons using imaging techniques. While the mechanism for APP-induced inhibition of the channel is still unresolved, my data clearly shows this effect is mediated by presenilin. Whether or not presenilin is responsible for cutting CaV1.2 remains to be resolved.

Drug use and abuse remains an important public health problem in the United States. In particular, there has been considerable recent concern regarding the illicit use of marijuana, methamphetamine, and prescription pain relievers. However, several important gaps remain in our knowledge of these drugs. The current studies aim to address three of these gaps. Further, the present studies utilized a translational, multilevel approach in order to better understand substance use as a whole. First, although there have been a number of studies examining marijuana use in college students, there is a lack of information regarding the consequences of marijuana and alcohol co-use, as well as the relationship between marijuana use and mental health and stress. Study 1 examined the relationship between frequency of marijuana use and other substance use, binge drinking, negative consequences associated with drinking, mental health problems, and stress. Results show that students who reported more frequent marijuana use were more likely to use all other substances, binge drink, and have drinking-related encounters with the police. Frequency of use was also related to diagnosis and/or treatment for major depression and substance use disorders. On the other hand, any marijuana use was associated with greater likelihood of experiencing a number of negative consequences from drinking, and diagnosis or treatment for anxiety disorders. Marijuana was not related to stress. This data contributes to the field by indicating that marijuana use is indeed related to mental health among this population. This has important implications for university administrators and health professionals. Study 2 was the first empirical investigation of the acute effects of marijuana during simulated night shift work in marijuana users. Night shift workers are particularly susceptible to performance impairments and often use drugs in order to manage their sleep-wake cycles. The results indicated that smoked marijuana attenuated performance and mood disruptions during simulated night shift work. This data furthers the database by indicating that marijuana has cognitive-enhancing effects under certain conditions. Additional research will be needed in order to determine whether these effects were caused by circadian modulation of the stimulant-related effects of marijuana, or by residual effects on improved sleep. Another important gap addressed by the present studies is the dearth of empirical information regarding the effects of methamphetamine when combined with oxycodone. Study 3 addressed this gap by beginning a systematic investigation of this drug combination utilizing a pre-clinical model with a wide range of cognitive and behavioral measures. Results indicated that the drug combination produced stronger effects than either drug alone, although this was observed in a limited number of measures. These data importantly provide the first empirical determination of the effects of the methamphetamine-oxycodone combination.

Cross-linguistic studies have demonstrated that learning of a second language (L2) is influenced by the phonological system of the native language (L1), with L2 learners forming mental representations of new, non-native sounds by a process of assimilation to familiar native sounds (Best, 1995; Flege, 1995). Adult sequential successive bilingual Spanish-English speakers may be specifically challenged in perceiving and acquiring American English (AE) vowel contrasts that are signaled by multiple cues not phonemically relevant in their native language. Much of the existing research on vowel perception in L1-Spanish adults has focused on the AE vowel contrast /i/ vs. /ɪ/, as in sheep vs. ship, because discrimination errors between these two vowels are common (Escudero, 2000; Morrison, 2006; 2008; 2009). However, other vowel contrasts /ʌ/-/ɑ/ (as in hut vs. hot) have also been reported to present perceptual challenges for native Spanish-speaking learners of English (Flege, Munro & Mackay, 1995; Escudero & Chládková, 2010). It is assumed that such perceptual issues contribute to poor performance in second language acquisition and processing, and have implications for access to employment and academic opportunities for a large and growing immigrant population in the United States (Labor Employment and Training Administration Report, 2005). The aim of this study is to implement electrophysiological and behavioral methods to further elucidate the perceptual and processing abilities of L1-Spanish adult learners of English, while examining less-studied AE vowel contrasts /ʌ/-/ɑ/, and to evaluate whether specific properties of these speech sounds, such as spectral and duration differences, contribute directly to difficulties encountered in L2 acquisition. More specifically, in this study we will examine response accuracy and reaction time, as well as Mismatch Negativity (MMN) and P300 Event-Related Potentials in two listening conditions: natural vowel duration, where target vowel sounds are presented naturalistically, and neutral vowel duration, in which speech sound discrimination is possible based on spectral cues alone. Event Related Potentials (ERPs - MMN and P300) are neurophysiological indices that can reflect native and non-native mental phonological representations. Findings from the pilot study that utilized natural and neutralized duration speech sounds revealed behavioral and neurophysiological differences between Spanish-English bilingual listeners and native English speakers responses to natural AE vowel contrasts. This raised a question of whether adult Spanish-English bilinguals relied on speech cues in a similar fashion to native English speakers when perceiving these AE vowel contrasts. It is understood that language-specific use of speech cues (e.g. spectral and durational) helps to distinguish between perceptually similar speech sounds. Therefore, it was assumed that removal of duration distinctions between the target vowels would reveal any underlying differences in the processing mechanism and how much L1-Spanish listeners rely on durational cues to perceive subtle differences between vowel pairs. Findings from this dissertation study indicate that adult sequential Spanish-English bilingual listeners (Study group) showed indices of discrimination and identification of AE vowel /ɑ/ but not /ʌ/ at the attentional level, when both spectral and durational information about the vowels was perceptually available in the natural vowel duration condition, but also when duration was neutralized leaving only spectral cues available to distinguish the vowels. The current findings show that Spanish-English bilinguals may use spectral and durational cues, like native English speakers, to perceive the English vowel contrast /ɑ/-/ʌ/. However, this cannot be described as an "end state" in the sense of Escudero (2005), since the neurophysiological evidence shows that these L2 learners are able to reach native-like discrimination only when they recruit attentional and cognitive resources to facilitate the perceptual process.

Learning and affect are considered empirically separable, but these constructs bidirectionally interact. While it has been demonstrated that dopamine supports the informational component of reward learning, the term "reward" inherently infers that a subjective positive experience is necessary to drive appetitive behavior. In this dissertation, I will first review the ways in which dopamine operates on the levels of physiology and systems neuroscience to support learning from both positive and negative outcomes, as well as how this framework may be employed to study mechanism and disease. I will then review the ways in which learning may interact with or be supported by other brain systems, starting with affective networks and extending into systems that support memory and other types of broader decision making processes. Finally, my introduction will discuss a disease model, schizophrenia, and how applying questions pertaining to learning theory may contribute to understanding symptom-related mechanisms. The first study (Chapter 2) will address the way in which affective and sensory mechanisms may alter pain-related decisions. I will demonstrate that subjects will choose to experience a stimulus that incorporates a moment of pain relief over a shorter stimulus that encompasses less net pain, and will suggest that the positive prediction error associated with the pain relief may modulate explicit memory in such a way that impacts later decision making. In the second study (Chapter 3), I will examine reward learning in patients with schizophrenia, and demonstrate selective learning deficits from gains as opposed to losses, as well as relationships in performance to affective and motivational symptoms. The third study (Chapter 4) will extend this disease model to a novel cohort of subjects who perform the same reward learning task while undergoing functional MRI. The data from this chapter will reveal deficits in the patient group during choice in orbitofrontal cortex, as well as an abnormal pattern of learning signal responses during feedback versus outcome, particularly in orbitofrontal cortex, a finding that correlates with affective symptoms in medial PFC. Taken together, these data demonstrate that learning is comprised of both informational and affective processes that incorporate input from dopaminergic midbrain neurons and its targets, as well as integration from other affective, mnemonic, and sensory regions to support healthy learning, emotion, and adaptive behavior.