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The presented dissertation grew out of the need to achieve a better understanding of the relationship between language processing and short-term memory (STM) in persons with aphasia (PWA). Deep dysphasia and phonological dysphasia form a classification of aphasia identified by a pattern of speech errors attributed to chronic verbal STM impairment. Exploring evidence demonstrating the pattern of speech errors mediated by STM impairment in PWA, research objectives were three-fold: • To add to the knowledge base on deep dysphasia and phonological dysphasia and extend the characteristic presentation of this; • To determine the characteristic profile of recovery in deep dysphasia, providing further evidence that deep dysphasia is an impairment that exists at the most severe point along a continuum of recovery which in milder form demonstrates phonological dysphasia. • To evaluate the efficacy of treatment approaches developed on the basis of Dell and O’Seaghdha’s (1992) two-step interactive activation model of word production and using repetition to improve verbal STM and word processing. First, a comprehensive systematic review of the deep dysphasia and phonological dysphasia literature base was conducted. This review addressed the paucity of case studies reporting the diagnoses of dysphasia. Studies investigated individuals with chronic STM impairment in the auditory modality as well as other characteristic markers of deep and phonological dysphasia, such as an imageability effect. In repetition tasks, an imageability effect indicates that concrete (high-image) words are repeated with greater ease when compared to accuracy in the repetition of abstract (low-image). The review supported the hypothesis that these profiles reflect a chronic impairment of auditory-verbal STM existing on a continuum of severity. Evidence from this review supports the hypothesis that deep dysphasia and phonological aphasia are two points on a continuum of an impairment mediated by verbal STM (Martin, Saffran & Dell, 2006, Willshire & Fisher, 2004). Second, with the insights from this review, a single-subject multiple-baseline, multiple-probe treatment study was undertaken. The participant LT presented with a pattern of repetition consistent with the continuum of deep-phonological dysphasia, including an imageability effect in repetition (Martin et al., 1996, Wilshire & Fisher, 2004). This treatment approach sought to directly remediate language and short-term memory abilities using a repetition task targeting imageability effect. In order to improve access to and repetition of low-image (LI) words, this approach aimed to enhance semantic context. Results of this study indicate improving LT’s access to abstract word pairs improved verbal STM as well as language processing. Third, the single-case study led to the development of a four-condition, multi-participant facilitation study that aimed to improve access to and repetition of LI words by embedding them in a context that enhanced their imageability. The goal of this manipulation was to increase the probability of accessing lexical and semantic representations of abstract words in repetition by enriching their semantic-syntactic context. Ten participants with chronic impairment in verbal STM demonstrated that this approach participants’ ability to repeat those words when presented in isolation. Evidence from PWA has confirmed that a damaged language processing system includes disruption to STM (Martin & Saffran, 1992; Martin et al., 1996; Martin & Saffran, 1997; Martin & Saffran, 1999; Martin & Ayala, 2004; Kalinyak-Fliszar, Kohen, & Martin, 2011; Allen et al., 2012). Recent investigations have provided evidence that STM tasks can be used as mechanisms to improve language processing (Kalinyak-Fliszar et al., 2011; Salis, 2012; Berthier et al., 2014). Despite the central role repetition plays in functional communication and the difficulties PWA encounter with repetition due to STM impairment, few treatment approaches have targeted STM and the language processes supporting repetition (Martin, Kohen, & Kalinyak-Fliszar, 2010; Kalinyak-Fliszar, 2011; Berthier et al., 2014). This dissertation research filled that void and demonstrated the promise of clinical approaches that directly target language processing and auditory verbal STM impairment in PWA. / Communication Sciences

In the early years of the AIDS epidemic, being infected with the virus that causes the disease was considered a virtual death sentence. But with the development of highly active antiretroviral therapy (HAART), many infected with HIV-1 are living much longer. In fact, it is estimated that by 2015, about half of all HIV-positive individuals will be older than 50. Yet those over 50 also progress to AIDS faster than adults in their 20s or 30s. And those in the younger age bracket, even those responding well to antiretroviral therapy, still exhibit illnesses and clinical conditions commonly associated with older people, such as HIV-associated neurocognitive disorders (HAND), certain cancers, liver and bones diseases. For the most part, the reasons for this have remained a mystery. However, one may ask, how in the absence of circulating detected virus, viral proteins could cause this kind of damage. The answer is that eradication of latent viruses still unsuccessful and studies showed the persistence of HIV-1 in brain cells as well as the presence of viral proteins in CSF. This notion was supported by the compelling neuropathological data suggesting that the loss of Synaptic Plasticity occurs with the ongoing presence of virus and despite HAART. Clinically, these neuropathological data manifest by a gradual loss of working memory and learning disability, which promote alteration of synaptic plasticity that may manifest by symptoms similar to the ones observed in aged brain or what is called PREMATURE BRAIN AGING. Anatomically, working memory and learning ability functions are assured by neurons of the hippocampus, a brain area known-to-be affected by HIV-1 proteins. Mechanistically, several laboratories, including ours, demonstrated that viral proteins perform their functions through deregulation of several molecular pathways that can cause mitochondrial damage (such as depletion of mitochondrial calcium and release of ROS), inhibition of axonal transport leading to prevent neuronal communications or loss of long-term potentiation (LTP). Interestingly, CREB and BDNF proteins have been shown to play an important role in this phenomenon directly or through its downstream target genes. In here, we examined the impact of HIV-1 Tat on CREB-BDNF pathway and whether Tat is using this pathway to cause neuronal deregulation. / Cell Biology

Ethanol (EtOH) abuse is the third leading cause of preventable death in the United States. Mounting evidence indicates that EtOH-induced neuropathology may result from multicellular responses in which microglia cells play a prominent role. Purinergic receptor signaling plays a key role in regulating microglial function and, more importantly, mediates EtOH-induced effects. In our current study we sought to determine the effects of EtOH on microglial cell function, specifically the role of purinergic receptor X4 (P2X4) in EtOH-mediated microglial responses. Our results show a significant up-regulation of P2X4 gene expression as analyzed by real-time qPCR and protein expression as analyzed by flow cytometry in embryonic stem cell-derived microglial cells (ESdM) after 48 hours of EtOH treatment, as compared to untreated controls. Calcium mobilization in EtOH treated ESdM cells was found to be P2X4R- dependent using 5-BDBD, a selective P2X4R antagonist. Blocking P2X4R signaling with 5-BDBD decreased the level of calcium mobilization compared to EtOH treatment alone. EtOH decreased migration of microglia towards fractalkine (CX3CL1) by 75% following 48 hours of treatment compared to control. CX3CL1-dependent migration was confirmed to be P2X4 receptor-dependent using the antagonist 5-BDBD, which reversed the effects as compared to EtOH alone. Similarly, 48 hours of EtOH treatment significantly decreased phagocytosis of microglia by 15% compared to control. 5-BDBD pre-treatment prior to EtOH treatment significantly increased microglial phagocytosis. These findings demonstrate that P2X4 receptor may play a role in modulating important regulatory functions in microglia in the context of EtOH abuse. P2X4R plays an important regulatory function in microglia. P2X4 is involved in a myriad of molecular signaling such as proliferation, activation of transcription factors, specifically through the MAPK pathway, and ATP signaling. Here, we also investigated the intracellular signal transduction pathway that influences P2X4R expression in microglia in response to EtOH. We found EtOH (100 mM) decreased phosphorylated AKT and extracellular signal-regulated kinase (ERK) cascades in ESdM cells. EtOH effect on ERK phosphorylation was completely inhibited by U0126, an inhibitor of MEK 1 and 2. However, PD98095, a potent inhibitor of MEK1 but a weak inhibitor of MEK2 had modest effect on phosphorylated ERK1/2 suggesting a possible role of MEK2-dependent ERK signaling in modulating EtOH induced effects on microglia. Utilization of 5-BDBD, a selective P2X4R antagonist reversed the EtOH-induced effect on phosphorylated AKT and ERK. Next we wanted to examine the effects of EtOH on transcription factor activity to determine the signaling mediators, which may play a role in EtOH-induced increase in P2X4R in microglia. EtOH increased transcriptional activity of NFκB, NFAT, and CREB,, however 5-BDBD blocked the effect on CREB transcriptional activity alone, suggesting a specific role of CREB in EtOH-induced expression of P2X4R in microglia. In summary, EtOH affects the expression of P2X4R in microglial cells and contributes to aberrant microglial effector function including phagocytosis and migration as well as alterations in calcium mobilization. Furthermore, pharmacological blockade with a selective P2X4R antagonist reversed the action, suggesting that P2X4R may play a role in mediating EtOH-induced effects on microglia. EtOH decreased expression of ERK and AKT, which was blocked with the P2X4R antagonist, suggesting EtOH effect may contribute to irregular microglial signaling. Investigations regarding transcription factor NFκB, NFAT and CREB activity in response to EtOH, all showed an increase after EtOH treatment, however P2X4R antagonist only had an effect on CREB, blocking the effect of EtOH on its activity. Determining the mechanism underlying EtOH-induced increase in P2X4R expression still remains unclear. This research was conducted to investigate the importance of P2X4R signaling in EtOH-mediate microglial function. Although many more questions remain unanswered, these experiments have provided evidence to target purinergic receptor X4 as a potential mediator of EtOH-induced effects in microglia. / Pathology

Cortical malformations arise from defects in any stage of brain development and often result in life-long disability ranging from epilepsy to developmental delay and even perinatal lethality. The neuroepithelium of the emergent cortex lays the foundation on which the future cortex will develop, and as such, neuroepithelial tissue and the neural progenitor cells (NPCs) which comprise it are critical to the proper growth and development of the cortex. Here I demonstrate the significance of neuroepithelial cell polarity determinants in cortical development and how they affect both junctional integrity and the regulation of NPC proliferation leading to a variety of cortical malformations. Until now, the role of basal polarity complex protein Lgl1 in cortical development remained elusive due to perinatal lethality in animal models. To bypass this, we developed a novel conditional knockout mouse model of Lgl1 in the neuroepithelium and show that Lgl1 is essential to the maintenance of neuroepithelial integrity and regulation of NPC proliferation. Loss of Lgl1 results in a displaced ventricular zone with widespread ectopic proliferation resulting in severe periventricular nodular heterotopia (PNH). Furthermore, Lgl1 loss reduces the cell cycle length resulting in hyperproliferation leading to neuronal overproduction. Together, this work identifies a novel genetic cause of PNH. Next, I aimed to characterize the interaction of Lgl1 with other polarity proteins and downstream signaling pathways in cortical development. Apical and basal polarity proteins have demonstrated mutual antagonism in the establishment/maintenance of epithelial polarity; however, little is known about the role of this antagonism on cortical size and structure or the signaling pathways through which it acts. To address these questions we generated multiple genetic mouse models to investigate the opposing roles of basal protein, Lgl1, and either apical proteins Pals1 or Crb2. Concurrent loss of Pals1 and Lgl1 was able to prevent heterotopic nodules and increase proliferation compared to loss of Pals1 alone. However, cortical size was severely diminished due to overriding effects of Pals1 on cell survival that was unmitigated by Lgl1 loss. Remarkably, loss of both Crb2 and Lgl1 restored the cortex and hippocampus to near normal morphology with a profound rescue of cortical size, suggesting their essential antagonism in both cortical and hippocampal development. Importantly, genetic manipulation through reduction of YAP/TAZ expression in the Lgl1 CKO eliminates periventricular nodules and restores cortical thickness to that of WT cortices. This important finding implicates Lgl1 in the regulation of YAP/TAZ in cortical development. Finally, we investigated a possible downstream target of Pals1 in cell survival, BubR1. My work demonstrates that loss of Pals1 reduces BubR1 expression, which is an essential regulator of the mitotic checkpoint and causative gene of the human disorder Mosaic Variegated Aneuploidy. I show that loss of BubR1 results in significant apoptosis across all cell types in the cortex leading to microcephaly. These data provide the first link between cell polarity determinants and mitotic regulation in the cortex and suggests that BubR1 reduction likely contributes to the decreased cell survival following Pals1 loss. Overall these findings implicate impaired polarity complex function in a wide variety of NPC defects resulting in multiple cortical malformations. My work shows that polarity proteins regulate every stage of the NPCs life cycle from cell division and proliferation to cell survival through regulation of mitosis and YAP/TAZ signaling. / Biomedical Sciences

Alcohol is a psychoactive substance with dependence-producing properties that has been used by many cultures in human society for millennia. Among people 20-39 years old, 13.5% of total deaths are attributed to alcohol, and harmful use is a causal factor in more than 200 diseases and injury conditions. Current pharmacological treatments of alcohol use disorder mainly target the positive reinforcing properties of alcohol or increase sensitivity to the adverse effects of alcohol. However, these medications have limited efficacy in reducing alcohol use and maintaining sobriety. The majority of these treatments stem from data conducted solely in males and fail to target the psychological distress underlying alcohol motivation in those assigned female at birth and individuals with adverse childhood experiences who are more apt to drink to relieve negative affective states. The dorsal raphe nucleus (DRN)-serotonin (5-HT) system has an important role in mood regulation, is sexually dimorphic, vulnerable to stress-induced perturbations during development, and shows an inverse relationship with ethanol (EtOH) consumption. These characteristics make the 5-HT system an ideal target to investigate the mechanisms underlying aberrant alcohol consumption and drinking despite negative consequences, especially in at-risk populations. Our guiding hypothesis was that DRN 5-HT neurotransmission would underlie the sex-dependent effects of early life stress on EtOH intake and consumption despite negative consequences. To test this hypothesis, we first investigated the effects of sex and early life stress on EtOH-motivated behaviors in rats during adulthood. Next, we examined the role of the DRN 5-HT system in sex- and stress-dependent EtOH-motivated behaviors. Adolescent social isolation stress (SIS) was used as a translational model of early life stress. Male and female rats were group housed or isolated postweaning before undergoing voluntary EtOH consumption (i.e., homecage drinking, self-administration (SA), footshock-punished SA) paradigms in adulthood to test the effects of sex and stress on EtOH consumption and consumption despite negative consequences. Consistent with our hypothesis, behavioral results indicated that SIS increased homecage EtOH preference and consumption, as well as responding during SA, particularly in females. Females also showed more punishment-resistant responding for EtOH than males. To complement these behavioral findings, we employed a combination of electrophysiological, functional neuroanatomy, and chemogenetic strategies to explore the role of the DRN 5-HT system in sex- and stress-dependent EtOH-motivated behaviors. Both SIS and EtOH exposure induced hypofunction of 5-HT neurons, particularly in females, paralleling SIS-induced increases in EtOH-motivated behaviors. Chemogenetic strategies expanded upon the sex- and stress-dependent nature of this relationship. Chemogenetic activation of DRN 5-HT neurons in Tph2-iCre rats reduced responding for both natural and EtOH reward and elevated punished responding for EtOH, indicating a causal connection between DRN 5-HT signaling and acute responding to rewards and punishment. Collectively, our findings reveal an inverse relationship between EtOH intake and 5-HT neurotransmission, further implicating EtOH consumption as an important negative reinforcer to “normalize” SIS-induced hypofunction of the DRN 5-HT system, particularly in stress-sensitive females. Furthermore, our conclusions highlight the DRN 5-HT system as a potential pharmacotherapeutic target to treat aberrant alcohol consumption as a means to “self-medicate” hypofunction of the 5-HT system in at-risk populations. / Biomedical Neuroscience

Locomotion in vertebrates depends on proper formation and maintenance of neuronal networks in the hind-brain and spinal cord. Malformation or loss of factors required for proper maintenance of these networks can lead to severe neurodegenerative diseases limiting or preventing locomotion. A powerful tool to investigate the genetic and cellular requirements for development and/or maintenance of these networks is a collection of zebrafish mutants with defects in motility. The zebrafish mutant quetschkommode ( que) harbors a previously unknown gene defect leading to abnormal locomotor behavior. Here I show that the que mutants display a seizure-like behavior starting around four days post fertilization (dpf) that is characterized by a lack of an initial high amplitude body bend (C-bend) and simultaneous contra-lateral contractions leading to a seizure-like phenotype and paralysis. Peripheral nerve recordings show a significant increase in the number of initiated swimming bouts and overlap between left and right motor neuron activity. These data suggest that the que mutation leads to defects in nervous system function, at the level of motor neurons or central control of motor neurons. I have genetically mapped the que locus to a 0.36cM interval on chromosome 22 using meiotic mapping. I identified a splice mutation in the gene `dihydrolipoamide branched-chain transacylase E2' (dbt) as defective in que mutants. An orthologous mutation in humans lead to Maple Syrup Urine Disease (MSUD), a devastating metabolic disorder leading to seizures, mental retardation, coma and neonatal death if untreated. In zebrafish, dbt is expressed throughout early development and dbt transcripts become enriched in the hind-brain as well as in the gut and liver by 96 hpf. In MSUD patients levels of branched chain amino acids (BCAA) and their keto acids are significantly increased due to the essential role of the dbt enzyme for the BCAA metabolic pathway. The que mutation causes a significant increase of branched chain amino acids in the zebrafish mutant and a strong decrease of neurotransmitters such as glutamate and GABA as well as precursors like glutamine. I hypothesize that reduced neurotransmitter levels in que lead to the observed motility phenotype. Consistent with this hypothesis, I show a tissue specific reduction of glutamate in the hind-brain and spinal cord of que mutants. To evaluate the que mutant's potential as a vertebrate model for MSUD I performed a pilot drug screen using a selection of metabolites of the pathway as well as diet additives currently evaluated in clinical trials. Conversely, application of phenylbutyrate, one of the diet additives, had a beneficial influence on swimming abilities of que mutant embryos, while the keto acid α-ketoisocaproate (KIC), one of the elevated keto acids in human patients, decreased the percentage of larvae capable of swimming. These results help establish the zebrafish que mutant as a new model for MSUD disease that can be used to further the understanding of this disorder and to help identify therapeutic agents.

Harmonic functions are presented as a mechanism for robot control and planning. These functions produce smooth trajectories which are amenable to fast control. Obstacle avoidance based on harmonic functions is complete: If a path to the goal exists, the technique described here will find that path. Otherwise, it will detect that no such path exists. Harmonic functions have a number of other properties which are useful for robotics applications: they can be used to construct admittance controllers, they can be implemented using resistive networks, and they can be used for planning the trajectories of nonholonomic systems. Finally, recent results in neuroscience regarding the basal ganglia allow the formulation of a theory of basal ganglia function based in part on harmonic functions computed over various state spaces.

<p>Several classes of psychotropic medications are known to produce neurological side effects. It has long been recognized that antipsychotic drugs classically block the D₂ subtype of DA receptors inducing a range of acute and subacute extrapyramidal syndromes (EPS), including parkinsonism and akathisia, as well as chronic syndromes such as tardive dyskinesia. More recently, SSRI-type drugs, which, as the name suggests, inhibit the serotonin transporter (SERT), and have been found to induce a similar profile of EPS. It is unclear how medications with such different pharmacological actions can produce similar neurological side effects. The goal of this thesis was to study the neurochemical alterations induced by antipsychotic and SSRI medications, with a specific focus on the nigrostriatal pathway, the causative location of parkinsonism.</p> <p>Environmental enrichment and exercise (EE) has been shown to have protective effects in various neurological settings. In the first experiment, we studied the changes induced by SERT inhibition compared to those induced by a non-pharmacological form of therapy, namely, environmental enrichment with exercise. The SSRI, fluoxetine (FLX) significantly reduced the levels of tyrosine hydroxylase (TH) and phosphorylated glycogen synthase kinase-3β (pGSK-3β-inactive), while increasing phosphorylated TH (pTH) in the striatum (STR). EE also reduced TH and increased pTH, but contrary to FLX, it significantly increased striatal pGSK-3β protein expression.</p> <p>Microglia, the brain’s primary immune cells, have been implicated in several neuroinflammatory conditions, including Parkinson’s disease. The purpose of the second experiment was to explore the modulatory effects of microglia on neuroleptic-induced changes in the nigrostriatal system. The typical antipsychotic, haloperidol (HAL), did not affect the overall levels of TH, though it did induce a robust increase in pTH. The microglial NADPH oxidase inhibitor, apocynin (APO), significantly attenuated this increase in pTH. HAL also induced a significant increase in striatal pGSK-3β, while apocynin, rather surprisingly, induced a stark decrease in pGSK-3β protein expression.</p> <p>The results of this thesis indicate that both pTH and pGSK-3β are intriguing markers to study in the context of dopamine neurotransmission. In addition, EE proved to be a valuable modality in which to compare the downstream effects of pharmacological treatment. It is also clear that microglia fulfill an undefined, but fascinating role as modulators of neural transmission.</p> / Master of Science in Medical Sciences (MSMS)

<p>Trafficking of the glutamatergic AMPA receptors (AMPARs) has been implicated in synaptic plasticity regulation, including long-term potentiation, long-term depression, and synaptic scaling. Two proteins, GRIP for stabilization at the synapse and PICK for internalization, are involved in trafficking GluR2-containing AMPARs in and out of the synapse. In this thesis, I addressed the changes in the mechanisms of AMPAR trafficking by characterizing the developmental trajectories of GluR2, the phosphorylated form pGluR2, GRIP, and PICK and comparing expression in visual vs. frontal cortex. I found significant differences between cortical areas in the developmental trajectories of GluR2 and pGluR2. In visual cortex, expression levels exhibited smooth developmental increases. In frontal cortex, GluR2 and pGluR2 rose to an exuberant expression between P18 and P35. Developmental trajectories for GRIP and PICK showed smooth increases that were consistent across cortical areas. Furthermore, looking at the correlation between the surface components (GluR2 and GRIP) and internalized components (pGluR2 and PICK), I found that the development of AMPAR trafficking components is tightly regulated across the cortex.</p> <p>In this thesis, I also looked at AMPAR expression in adult cortex. Fluoxetine has previously been reported to induce a juvenile like state of plasticity in visual cortex and this plasticity was assessed through monocular deprivation. My results indicated that fluoxetine administration was not associated with significant changes in AMPAR expression levels. However, monocular deprivation induced significant upregulation in expression levels of all four proteins. These results imply the presence of AMPAR-mediated plasticity in the adult brain.</p> / Master of Science (MSc)