Monoamine signaling in the central nervous system plays an essential role in circuits involving attention, mood, memory, and stress as well as providing pivotal support for autonomic function in the peripheral nervous system. The high affinity norepinephrine (NE) transporter (NET) is the primary mechanism by which noradrenergic synaptic transmission is terminated. Data indicates that NET function is regulated by insulin, a hormone critical for the regulation of metabolism. Given the high co-morbidity of metabolic disorders such as diabetes and obesity with mental disorders such as depression and schizophrenia we sought to determine how insulin signaling regulates NET function and noradrenergic homeostasis. We show that insulin, through the downstream kinase protein kinase B (Akt), significantly decreases NET surface expression in mouse hippocampal slices and superior cervical ganglion neuron (SCGN) boutons (sites of synaptic NE release). In vivo manipulation of insulin/Akt signaling, with streptozotocin (STZ), a drug that induces a Type 1-like diabetic state in mice, also results in aberrant NET function and NE homeostasis. These data suggest that peripheral disruptions in Akt signaling such as in diabetes and obesity have the potential to alter NET function and noradrenergic tone in the brain. To investigate more specifically the ability of Akt to impact NET function, monoamine homeostasis, and behavior, we disrupted its function independent of insulin signaling. We used conditional gene targeting in mice to eliminate the mammalian target of rapamycin (mTOR) complex 2 (mTORC2) regulatory protein rictor in neurons, leading to impairments in neuronal Akt Ser473 phosphorylation. Importantly, defective Akt phosphorylation at the Ser473 site has been linked to schizophrenia. Here, Rictor-null (KO) mice exhibit prepulse inhibition (PPI) deficits, a schizophrenia-associated behavior, as well as decreased prefrontal dopamine (DA) content, elevated cortical NE, and enhanced expression of NET. In addition, NET blockade in rictor KO mice reverses cortical deficits in DA content and PPI, suggesting that dysregulation of DA homeostasis is driven by alteration in NET expression. Thus, these data illuminate a molecular link, Akt regulation of NET, between the recognized association of Akt signaling deficits in schizophrenia with a specific mechanism for hallmarks of the disorder, cortical hypodopaminergia and hypofunction.

Many neuropsychiatric disorders involve disruptions in emotional regulation that are correlated with dysfunctions in the limbic circuitry of the brain. Improved treatment and outcome of disorders of emotional regulation will come from understanding both the changes in brain signaling that underlie the behavioral and physiological components of the disorders and the origin of those changes. The limbic system associated membrane protein (protein: LAMP, gene: Lsamp) is of interest to the study of the biological mechanisms of emotional regulation because it is a developmentally relevant cell adhesion molecule expressed primarily in limbic circuitry and human genetic studies have implicated Lsamp in disorders of mood. We developed mice in which the Lsamp gene is deleted, creating a unique model to examine the functional consequences of disrupting the circuits that underlie emotional regulation. Basic neuroanatomical organization and sensory and motor development are normal in Lsamp_/_ mice. However, both male and female Lsamp-/- mice demonstrate heightened behavioral reactivity to novelty. Lsamp-/- mice also display heightened reactivity of the hypothalamic-pituitary-adrenal axis in response to a novel environment and a corresponding hyper-activation of brain circuitry that regulates the stress response. Preliminary data suggests that Lsamp-/- mice may have alterations in GABAergic tone and signaling, which could be responsible for the changes in stress-responsiveness observed in Lsamp-/- mice. These studies establish a role for LAMP in modulating the development and function of the circuitry that mediates emotional regulation.

Gliomas are the predominant type of primary central nervous system tumors. They are highly invasive and notoriously refractory to current therapies. Patients diagnosed with a malignant glioma face a dismal prognosis that has remained relatively unchanged in the last three decades. A promising novel approach to glioma therapy is based upon modulating molecular mechanisms that regulate cell types critical for tumor growth. Recent identification of cancer stem cells that initiate and maintain tumor growth in gliomas has prompted investigation into the molecular signaling pathways that regulate this unique cell type. The Hedgehog (Hh) signaling pathway regulates stem cells and is activated in several cancer types. Prompted by the role of Hh signaling in regulating neural stem cell self-renewal, I investigated the potential role of the pathway in glioma growth. To address this question, I evaluated the status of Hh signaling in primary gliomas. Furthermore, I tested our hypothesis that Hh signaling regulates glioma growth in a relevant preclinical model.

Identification of homologous primate visual areas and common organizational strategies among visual areas are essential to determining if a basic plan for primate visual cortex and visual areas exists. In this dissertation, we argue that V3 should be considered a homologous area among primates and that the study of V4 modularity provides important insight into how visual areas organize and process information. Because galagos are part of the prosimian radiation of surviving primates, cortical areas that bear strong resemblances in prosimians and other primates provide a strong argument for their conserved existence. Using intrinsic signal optical imaging (IOI) and 100 electrode array, our data support the presence of V3 in galago visual cortex, thus supporting its inclusion as a homologous primate visual area. It is also critical to study how visual areas organize and process information in order to identify possible themes across visual areas. Using IOI in awake macaques, we uniquely examined organizational features and properties of highly foveal representations in macaque V4. We found that bands of color and orientation modules in foveal V4 repeat visuotopic positions with similar cortical magnification factors, analogous to the modality specific representations of the visual field in V2 stripes. These visuotopic relationships could be a common strategy used by all visual areas to efficiently process information. We also investigated the size and spatial frequency preferences for color and orientation selective domains in foveal V4. For size, we found that surround suppression in orientation domains tended to be greater than color domains inV4. We also discovered a bimodal Gaussian distribution in our optical imaging signal in response to changing spatial frequency for both color and orientation selective domains of V4. While the source for this bimodal finding is currently unclear, it is clear that our modular level study of the foveal representation in V4 has revealed organizational properties and response properties that will impact our understanding of how a visual area organizes and processes information.

Our world constantly bombards us with more information than we can process. To ensure that we can act in accordance with our goals and relevant events, we use attention to select and enhance aspects of our environment. Attention is not unitary, able to be captured by salient events (stimulus-driven) or deployed under voluntary control (goal-directed). These two forms of attention rely on largely distinct ventral and dorsal fronto-parietal networks. While these networks have been extensively studied, their control and coordination is poorly understood. Using functional neuroimaging and a variety of behavioral tasks, this dissertation shows that the inferior frontal junction (IFJ) of the prefrontal cortex may be a key region in the control and coordination of attention. I employ a novel Surprise-induced Blindness paradigm to show that the IFJ and temporo-parietal junction (TPJ), core members of the ventral attention network, support stimulus-driven attention. I then demonstrate that the IFJ co-activates with the dorsal attention network--including the frontal eye field (FEF) and intra-parietal sulcus (IPS)--for goal-directed attention. This result suggests that the IFJ may coordinate stimulus-driven and goal-directed attention. The remainder of the dissertation functionally distinguishes the IFJ from other regions within the ventral and dorsal attention networks. I first dissociate the function of the IFJ from the ventral attention networks TPJ by demonstrating that only the latter is activated during a task in which participants reason about others' mental states (Theory of Mind) and while resting between the attention-demanding periods of a search task (Default Mode of Processing). Using an endogenous Posner cueing task, I next show that the IFJ is involved in cue interpretation for setting goal-directed attentional weights, but not in the maintenance of these weights. The FEF and IPS, by contrast, are involved in both processes. I conclude that the IFJs primary function may be to connect incoming sensory information with appropriate behavioral or dispositional responses, coordinating the activity of widespread brain regions to do so. Consequently, the IFJ influences central aspects of human information processing and even the contents of consciousness.

ROLE OF C-TERMINUS OF HSC70 INTERACTING PROTEIN IN DETERMINING NEURONAL FATE IN ACUTE INJURY JEANNETTE N. STANKOWSKI Dissertation under the direction of Professor BethAnn McLaughlin The decision to remove or refold oxidized, denatured or misfolded proteins by heat shock protein 70 (HSP70) and its binding partners is critical to determine cell fate. Acute overexpression of the ubiquitin ligase C-terminus of HSC70 interacting protein (CHIP) can compensate for failure of other ubiquitin ligases and enhance protein turnover and survival under chronic neurological stress. CHIPs ability to alter cell fate following acute neurological injury has however, not been assessed. Using post-mortem human tissue samples, we provide first evidence that cortical CHIP expression is increased following ischemic stroke. Oxygen glucose deprivation in vitro led to rapid protein oxidation, antioxidant depletion, proteasome dysfunction and a significant increase in CHIP. To determine if CHIP upregulation enhances neural survival, we overexpressed CHIP in vitro and evaluated cell fate 24hr following oxidative stress. Surprisingly, we observed that CHIP overexpressing cell lines fared worse against acute oxidative injury, accumulated more ubiquitinated and oxidized proteins and experienced decreased baseline proteasome activity suggesting that long-term upregulation of CHIP can be maladaptive. Conversely, decreasing CHIP expression in primary neuronal cultures using siRNA improved survival following oxidative stress, suggesting that the observed increase in CHIP following stroke-like injuries may negatively impact the neuroprotective potential of HSP70. To determine if cellular outcome could be further increased in an acute injury setting, we moved to a CHIP knockout model system and found increased levels of total oxidized proteins in brain tissue and accelerated calcium-induced mitochondrial permeability transition activities in these mice. Using the biotin-avidin-capture methodology to identify specific protein targets of oxidative stress, we found that known modulators of mitochondrial homeostasis or dynamics were not oxidatively modified. These results support previous findings of decreased lifespan and impaired survival upon injury observed in CHIP deficient animals. Together, these data suggest that CHIP expression must be tightly regulated in an acute injury setting as CHIP plays an essential role in regulating neuronal redox tone, and that strategies aimed at increasing CHIP expression levels may have previously unappreciated deleterious effects.

Rolling Blackout (RBO), a Drosophila EFR3 homolog, is an integral membrane protein predicted to be a lipase. A conditional temperature-sensitive (TS) mutant (rbots) displays paralysis following a 25-37oC temperature shift, an impairment previously attributed to blocked synaptic vesicle exocytosis. rbots displays a synergistic interaction with a dominant positive Syntaxin-1A TS allele, syx3-69. At neuromuscular synapses, rbots shows a defect in styryl FM dye endocytosis, and rbots;syx3-69 double mutants display a synergistic, more severe endocytosis impairment. rbots synapses in primary brain culture show defective FM endocytosis. Nephrocyte Garland cells show the same endocytosis defect in tracer uptake assays. Ultrastructurally, rbots displays a defect in tracer uptake into endosomes in both neuronal and non-neuronal cells. At the rbots synapse, there is total blockade of endosome formation possibly via activity-dependent bulk endocytosis. A point mutation in the putative lipase active site fails to rescue embryonic lethality of the null mutants and garland cell endocytosis defects in the TS background, suggesting that the mutation disrupts some vital aspect of protein function and that lipase activity may be required for membrane trafficking and viability. However, the putative lipase-dead mutant is able to fully rescue paralysis and NMJ endocytic defects in rbots possibly indicative of interallelic complementation. Further, we identified a synthetic lethal interaction between rbots and the dynamin GTPase mutant shits1. In all assays, both in non-neuronal cells and neuronal synapses, shits1; rbots phenocopies shits1 endocytic defects, indicating that dynamin and RBO act in the same pathway. Together, these results demonstrate that RBO is required for dynamin-dependent bulk endocytosis/macropinocytosis in both neuronal and non-neuronal cells, and at the synapse this mechanism is responsive to the rate of Syntaxin-1A-dependent exocytosis.

This project is concerned with identifying and characterizing structural determinants of GABAA receptor biogenesis. I used flow cytometry to measure surface levels of GABAA receptor subunits on HEK293T cells coexpressing wild-type alpha1 subunits and mutant beta2 subunits containing segmental deletions or point mutations to identify beta2 subunit structural determinants that are important for biogenesis of alpha1beta2 GABAA receptors. I located novel sites in the beta2 subunit N-terminal domain and major M3-M4 cytoplasmic loop that are necessary to attain maximal surface GABAA receptor levels. I used a combination of multiple sequence alignment, glycosidase digestion, brefeldin A treatment and analytic centrifugation and demonstrated that a structural determinant (D450) at the boundary of the beta2 subunit major cytoplasmic loop and M4 transmembrane domain is conserved among all subunits of the Cys-loop superfamily and is required for receptor assembly. Furthermore, using homology modeling and glycosidase digestion, I found that beta2 subunit N-terminal residue, N104, is a glycosylation site that is located on the minus side of the subunit-subunit interaction region and that its N-glycan processing in the Golgi apparatus is affected by the incorporation of gamma2 subunits into alpha1beta2gamma2 pentamers. Together, my studies demonstrated that both beta2 subunit N-termini and M3-M4 cytoplasmic loops contain structural determinants for GABAA receptor biogenesis.

The presynaptic choline transporter (CHT, SLC5A7) supplies choline for acetylcholine (ACh) synthesis in cholinergic neurons. CHT is particularly important as the demands of cholinergic signaling increase and ACh stores are depleted is vital for sustaining normal autonomic and cognitive function. Despite its role at the plasma membrane in importing choline, CHT is predominately localized at neuromuscular and brain synapses on intracellular synaptic vesicles. Whether augmentation of CHT through genetic or pharmacological approaches will enhance cholinergic tone is presently unknown. This dissertation examines models providing for genetically-instated reduced and elevated CHT expression generated to evaluate more clearly how such changes impact cholinergic signaling. Brain slice studies in CHT heterozygous mice are used to evaluate the contribution of loss of transporters on CNS ACh release and to evaluate whether alterations observed derive from compromised ACh synthesis or altered vesicular CHT support for release. A second model produces specific overexpression of CHT in motor neurons using the motor neuron specific promoter Hb9. I provide evidence that motor neuron overexpression of CHT can modestly extend life of CHT knockout mice and when combined with expression of native genomic expression of CHT results in enhanced endurance in a forced exercise paradigm. Surprisingly, analysis of stimulated muscle action potentials in these mice reveals a change of muscle responses from the unimodal wild-type distribution to a bimodal distribution where each population of the transgenic distribution exhibits different recovery kinetics following high frequency stimulation. A third model uses multiple copies of the genomic region encoded by a Bacterial Artificial Chromosome (BAC) for CHT to produce constitutive CHT overexpression. Although finding that these mice express substantially more CHT, such expression does not result in a commensurate increase in uptake. An initial behavioral battery has suggested that these mice have more anxiety and reduced working memory. Together, the results of this dissertation support both a classical role of CHT in sustaining ACh synthesis but also a novel role in contributing to the composition or mobilization of cholinergic vesicle pools.

The primary somatosensory cortex (SI) plays a principal role in the processing of both somatosensory and pain information. The organization and function of this cortical region have been studied for over a century using invasive anatomical and physiological methods that give us insight into the architecture of the human cortex and are practical for studies in most mammalian species, but are unfeasible approaches to studying this region in most people. With the advent of ultra-high (7-Tesla) non-invasive magnetic resonance imaging (MRI) we are now able to study the cortex with the resolution and precision once available by invasive techniques. In this work we use 7-T MRI to explore how human SI responds to and processes somatosensory and noxious stimuli. We aimed to evaluate the ability of BOLD signals at high MRI field (7 T) to map fine-scale activations in subdivisions of SI in individual subjects. We acquired BOLD fMRI data from cortical areas around the central suclus in healthy human subjects while stimulating individual finger pads with innocuous tactile or noxious thermal stimuli. In both areas 3b and 1 of contralateral SI, the responses to tactile stimulation of individual digits organized somatotopically. The spatial and temporal profiles of the BOLD signal showed greater overlap of digit responses in area 1 than area 3b, suggesting that these areas play different roles in touch perception. Within individual subjects, the cortical responses to tactile stimulations and the magnitude of the BOLD signals were reproducible across imaging runs and were comparable across subjects. We also identified cortical activation in bilateral areas of SI (3a, 3b, 1, and 2) that correlated to intensity estimation of pain. Functional connectivity between these areas was significant in the resting state, suggesting an intrinsic pain network. Moreover, it appears that resting state correlations may vary with time before and after a painful episode and thus show dynamic properties that merit further study. Our findings demonstrate that BOLD fMRI at 7T is capable of revealing the functional organization of areas 3b and 1 of SI with good within-subject reliability and reproducibility, and activation and correlation maps can be acquired within a reasonably short time window, which are essential characteristics for several neurological and psychiatric applications within patient populations.