Pain signaling involves transmission of nociceptive stimuli in the spinal cord where a critical balance between excitatory and inhibitory inputs determines the response to noxious stimuli. The neuropeptide, substance P (SP), mediates transmission of pain in part by binding to the tachykinin receptor (NK-1R) in the dorsal horn (DH) of the spinal cord. One of SP’s downstream effects is to modulate N-type Ca2+(N-) channels. While phospholipid breakdown is a part of the inflammatory process that accompanies tissue damage, the role of this metabolic pathway has not been completely described with respect to N-channel modulation during pain signaling. Despite the incomplete understanding of this modulation, pharmacological antagonists of both NK-1R and N-channels have been used to treat pain. In Chapter II, using whole-cell patch clamp recording techniques, the SP signaling cascade that mediates inhibition of recombinant N-channel activity was characterized. By adopting a pharmacological approach, I show that this pathway resembles the slow pathway that was earlier described for modulation of N-current by the M1 muscarinic receptor (M1R). M1R couples to Gq to stimulate phospholipid breakdown. Together with previous observations, the data presented in this chapter provide evidence for involvement of the extracellular receptor kinase (ERK1/2), phospholipase A2 and release of phospholipid metabolites in the modulation of N-current by SP. Overall, this chapter shows that phospholipid metabolism involved in modulation of N-currents is not specific to M1Rs but that other Gq-coupled receptors may also modulate N-currents via the same signal transduction pathway. In Chapter III, enhancement of N-current by SP was studied as part of a collaborative project to understand current enhancement that occurs when a palmitoylated accessory CaVβ2a subunit is co-expressed with the pore-forming subunit CaV2.2 and the accessory subunit α2δ-1. When CaVβ3 is present, SP inhibits N-current as described in Chapter II. However, when palmitoylated CaVβ2a is co-expressed with CaV2.2 (and α2δ-1), current enhancement is observed at negative test potentials, demonstrating that both M1Rs and NK-1Rs exhibit the same profile of N-current modulation. This change in modulation by muscarinic agonists is not observed in the presence of a depalmitoylated CaVβ2a. However a chimeric CaVβ2aβ1b subunit that contains the palmitoylated N-terminus from CaVβ2a confers enhancement. Normally expression of the β1b subunit resulted in current inhibition. These findings indicated that the palmitoylated CaVβ2a participates in enhancement of current. Our data support a model where inhibition dominates over enhancement; when inhibition is blocked, enhancement may be observed. Lastly, we show that N-current inhibition by SP is minimized when exogenous palmitic acid is applied to cells co-expressing CaVβ3 subunits with N-channels. These results indicate that the presence of palmitic acid can prevent N-current inhibition when SP is applied most likely by interacting with CaV2.2. We propose a model where palmitic acid occupies the inhibitory site and serves to antagonize inhibition by a lipid metabolite, which is most likely arachidonic acid. The CaVβ2a protein seems to have a role in positioning the palmitoyl groups near CaV2.2. This chapter provides a new role for protein palmitoylation where the palmitoyl groups of CaVβ2a are both necessary and sufficient to block inhibition of another protein: CaV2.2. In Chapter IV, I probe the role of the relative orientation of CaVβ2a and the pore-forming subunit of the N-channel in N-current modulation. Evidence is presented that shows that not just the presence of a palmitoylated CaVβ2a is necessary, but the relative orientation of CaVβ2a to CaV2.2 is critical for blocking inhibition. Using N-channel mutants that cause a change in the orientation of CaVβ2a relative to CaV2.2, I show that the block of inhibition is disrupted; inhibition by the slow pathway is rescued. These findings further support my model that the palmitoyl groups of CaVβ2a normally reside in a specific location that overlaps with the slow pathway inhibitory site on CaV2.2. Lastly I present data showing that the enhancement of N-current, observed when palmitoylated CaVβ2a is present, occurs via the slow pathway. In Chapter V the effect of CaVβ’s orientation on N-channel modulation by the dopamine D2 receptor is tested. In this form of modulation, inhibition is rapid and voltage-dependent. The signaling pathway is membrane-delimited since Gβγ, released after receptor stimulation, directly interacts with the N-channel at a site that overlaps with a high affinity binding site for CaVβs. While N-currents are modulated by this pathway, the deletion mutants show aberrant membrane-delimited modulation. The findings in this chapter further underscore the importance of proper positioning of CaVβ to CaV2.2 for eliciting proper N-current modulation after GPCR stimulation. Overall, the data presented in this dissertation provides a mechanistic approach into examining modulation of N-current by different GPCRs via two different signaling pathways as well as the role CaVβ subunits serve in each modulatory pathway.

High temporal resolution of vision relies on the rapid kinetics of the photoresponse in the light-sensing photoreceptor neurons. It is well known that the rapid recovery of photoreceptor membrane potential at the end of light stimulation depends on timely deactivation of the visual transduction cascade within photoreceptors. Whether any extrinsic factor contributes to the termination speed of the photoresponse is unknown. In this thesis, using Drosophilaas a model system, I show that a feedback circuit mediated by both neurons and glia in the visual neuropile lamina is required for rapid repolarization of the photoreceptor at the end of the light response. In the first part of my thesis work, I provide evidence that lamina epithelial glia, the major glia in the visual neuropile, is involved in a retrograde regulation that is critical for rapid repolarization of the photoreceptor at the end of light stimulation. I identified the gene affected in a slrp (slow receptor potential) mutant that is defective in photoreceptor response termination, and found it needs to be expressed in both neurons and epithelial glia to rescue the mutant phenotype. The gene product SLRP, an ADAM (a disintegrin and metalloprotease) protein, is localized in a special structure of epithelial glia, gnarl, and is required for gnarl formation. This glial function of SLRP is independent of the metalloprotease activity. In the second part of my thesis work, I demonstrate that glutamatergic transmission from lamina intrinsic interneurons, the amacrine cells, to the epithelial glia is required for the rapid repolarization of photoreceptors at the end of the light response. From an RNAi-based screen, I identified a vesicular glutamate transporter (vGluT) in amacrine cells as an indispensable factor for the rapid repolarization of the photoreceptor, suggesting a critical role of glutamatergic transmission from amacrine cells in this retrograde regulation. Further, I found that loss of a glutamate-gated chloride channel GluCl phenocopies vGluT downregulation. Cell specific knockdown indicates that GluCl functions in both neurons and glia. In the lamina, a FLAG-tagged GluCl colocalized with the SLRP protein in the gnarl-like structures, and this localization pattern of GluCl depends on SLRP, suggesting that lamina epithelial glia receive glutamatergic input from amacrine cells through GluCl at the site of gnarl. Since the amacrine cell itself is innervated by photoreceptors, these observations suggest that a photoreceptor — amacrine cell — epithelial glia — photoreceptor feedback loop facilitates rapid repolarization of photoreceptors at the end of the light response. In summary, my thesis research has revealed a feedback regulation mechanism that helps to achieve rapid kinetics of photoreceptor response. This visual regulation contributes to the temporal resolution of the visual system, and may be important for vision during movement and for motion detection. In addition, this work may also advance our understanding of glial function, and change our concept about the effect of glutamatergic transmission.

<p>Infants must learn to discriminate between individuals in order to determine who is familiar (and likely to provide their basic needs) from those who are unfamiliar (and possibly a threat to their survival). One cue that humans use to discriminate others is the unique sound of each individual’s voice. Until the present thesis, little research existed on the topic of voice discrimination development during infancy. I conducted the first set of studies to investigate whether voice discrimination develops through a process of perceptual narrowing. Perceptual narrowing is defined as an experience-dependent increase in sensitivity to distinctions important in the native environment and a decrease in sensitivity to distinctions not important (often foreign to) the native environment across the first year. It has been described in previous research for the processing of a number of socially-relevant stimuli in the auditory (e.g., musical rhythms and pitches, linguistic phonemes) and visual (e.g., faces) domains. In Chapter 2, I provide the first evidence that narrowing occurs for voice discrimination, with infants specializing for the discrimination of native (human, English-speaking) over foreign (rhesus monkey) vocalizations between 6 and 12 months. In Chapter 3, I establish that the specialization demonstrated in Chapter 2 resulted primarily from familiarity with the vocalization from the human species, rather than the particular language spoken. In Chapter 4, I show that sensitivity to distinctions between monkey voices can be reinstated at 12 months of age, after narrowing has taken place, with two weeks of exposure to monkey voices. Together, these findings indicate that infants become attuned to individual distinctions between human voices by the end of their first year, but that plasticity remains such that sensitivity to distinctions between voices from rarely-heard species can be reinstated with exposure, at least until the end of the first year.</p> / Doctor of Philosophy (PhD)

We introduce a novel integration of real-time, predictive eye-gaze tracking models into a multimodal dialogue system tailored for remote health assessments. This system is designed to be highly accessible requiring only a conventional webcam for video input along with minimal cursor interaction and utilizes engaging gaze-based tasks that can be performed directly in a web browser. We have crafted dynamic subsystems that capture high-quality data efficiently and maintain quality through instances of user attrition and incomplete calls. Additionally, these subsystems are designed with the foresight to allow for future re-analysis using improved predictive models, as well as enable the creation and training of new eye-gaze tracking datasets. As we explored gaze patterns for various user-performed tasks, we developed generalizable eye-gaze metrics that capture and reflect the distinct gaze trends among different cohorts. And through testing various feature extraction and classification methods, we have found promising results that have enabled us to effectively classify individuals with Mild Neurocognitive Disorder (MiNCD) / Mild Cognitive Impairment (MCI) in a crowdsourced pilot study (N = 35) with an average accuracy of 0.94 (f1 = 0.83). Although just the beginning, this work represents the first step towards establishing predictive eye-gaze tracking as an accessible and important modality for healthcare applications moving forward, with the potential to significantly impact remote screening and monitoring of neurocognitive health.

This thesis examines the methods used by the Ministerium für Staatssicherheit (MfS), more commonly known as the Stasi, or East German secret police, for extraction of information from citizens of the German Democratic Republic for the purpose of espionage and covert operations inside East Germany, as it pertains to the deliberate brainwashing of East German citizens. As one of the most efficient intelligence agencies to ever exist, the Stasi’s main purpose was to monitor the population, gather intelligence, and collect or turn informants. They used brainwashing techniques to control the people of the GDR, keeping the populace paralyzed with fear and paranoia. By surrounding themselves with a network of informants they prevented actions against the dictatorial communist regime. Using the video testimonies of former prisoners, and former confidential informants who worked closely with and collaborated with Stasi agents, in combination with periodicals and previous historical studies, this work argues that the East German Police State’s brainwashing techniques had long and lasting consequences both for German citizens, and for the psychiatric health of former GDR citizens. The scope and breadth of the techniques and data compiled for use by the Stasi were exhaustive, and the repercussions of their use are still being felt and discovered twenty five years after the fall of the Berlin Wall. This study aims to show the lasting effects brainwashing had on former informants and the Stasi’s victims.