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Before hearing onset, synapses in the inhibitory pathway from the medial nucleus of the trapezoid body (MNTB) to the lateral superior olive (LSO) not only release GABA and glycine, but also glutamate. This transient glutamate release from nominally inhibitory synapses coincides with the period in which the MNTB-LSO pathway is tonotopically refined. I hypothesized that by releasing glutamate, developing MNTB-LSO synapses can elicit NMDA receptor (NMDAR) mediated calcium influx, which has been shown to underlie the refinement of excitatory topographic maps. To test my hypothesis, I designed and built a 2-photon microscopy system to perform two complementary series of calcium (Ca2+) imaging experiments. The objective of the first series of experiments was to investigate the potential of developing MNTB-LSO synapses to elicit NMDAR-mediated Ca2+ responses. Experiments were performed under conditions that maximized the ability for GABA/glycinergic conductances to aid in the relief of magnesium (Mg2+) block at NMDARs. Electrical stimulation of the MNTB-LSO pathway consistently elicited local Ca2+ transients in LSO dendrites that were significantly reduced by NMDAR antagonists. Despite their significant contribution to MNTB-elicited dendritic Ca2+ responses, NMDARs did not contribute to somatically recorded electrical responses. In the second series of experiments, I investigated the ability for MNTB-LSO synapses to activate NMDAR-mediated Ca2+ transients using only imaging techniques. Here we found that MNTB-LSO inputs by themselves could not elicit somatic NMDAR-mediated Ca2+ responses when GABA/glycinergic conductances were present with extracellular Mg+, despite having the potential to elicit these Ca2+ responses. These results suggest that GABA/glycinergic conductances shunt the somatically-recorded Ca2+ response. They raise the question of whether MNTB-LSO inputs can elicit local dendritic NMDAR-mediated Ca2+ responses independently from other LSO inputs. Together, these experiments demonstrate the ability of developing MNTB-LSO inputs to activate NMDAR Ca2+ signaling pathways during the time of topographic map refinement. Although this ability was only observed under conditions that altered or abolished GABA/glycinergic conductances or extracellular Mg2+, this ability could endow developing inhibitory synapses with a novel and potentially powerful refinement mechanism to be activated under physiological conditions.

The two major hallmarks of Alzheimers disease (AD) are amyloid plaques and neurofibrillary tangles (NFTs). Evidence suggests that the main component of amyloid plaques, â-amyloid peptide (Aâ) facilitates tau pathology via activation of specific kinases. Both glycogen synthase kinase-3â (GSK-3â) and cyclin-dependent kinase 5 (cdk5) have been demonstrated to be activated by Aâ and contribute to tau hyperphosphorylation. Recently, c-Abl has been implicated in Aâ-facilitated tau pathology by in vitro model systems. Alvarez et al. reported that c-Abl could be activated by Aâ in primary cultured neurons (Alvarez et al. 2004), and Derkinderen et al. found a novel phosphorylation site in paired helical filament tau (Tyr 394) that could be phosphorylated by c-Abl (Derkinderen et al. 2005). Moreover, Aâ has been shown to bind integrin receptors on the cell surface and transduce a signal from the extracellular space to the cell interior, regulating the cytoskeleton and/or gene transcription (Caltagarone, Jing et al. 2007). c-Abl can also be activated by integrin activation. Therefore, we hypothesize that c-Abl is associated with Aâ-facilitated tau phosphorylation via integrin binding and activation, contributing to the generation of AD pathology. We tested this hypothesis by examining the expression and distribution of c-Abl in the human hippocampus and by characterizing c-Abl interacting proteins in AD brain. We discovered that the activation state of c-Abl was altered during AD progression and c-Abl was associated with phospho-tau during AD. Preliminary co-immunoprecipitation data also suggested a possible association of c-Abl with another integrin signaling protein, paxillin. This study is the first to examine the expression and localization of c-Abl in healthy control and AD hippocampus, which contributes to our understanding of the functional role for c-Abl in AD pathogenesis. Interestingly, c-Abl was localized to granulovacuolar degeneration bodies (GVDs) during late-stage AD, a novel discovery that identifies a new protein component of GVDs in AD.

The liberation of zinc from intracellular stores during pathophysiological conditions, especially those in which reactive oxygen and nitrogen species have been implicated, is central to the progression of neuronal injury. In addition, cellular efflux of a second ionic species, potassium, is similarly responsible for facilitating apoptotic cell death downstream of the initial oxidant-induced zinc liberation. Potassium efflux is mediated by Kv2.1-encoded potassium channels in apoptotic neurons. Here, I have characterized several critical molecular components that link zinc liberation to the loss of cytoplasmic potassium following oxidative injury. First, electrophysiological and viability studies using Kv2.1 channel mutants identified a p38 phosphorylation site at serine 800 (S800) that is required for Kv2.1 membrane insertion, potassium current enhancement, and cell death. In addition, a phospho-specific antibody for S800 detected a p38-dependent increase in Kv2.1 phosphorylation in apoptotic neurons, and reveals phosphorylation of S800 in immunopurified channels incubated with active p38. Next, I present data indicating that an N-terminal tyrosine of Kv2.1 (Y124), which is targeted by src, is also critical for the apoptotic current surge. These latter studies suggest that Y124 works in concert with the C-terminal serine (S800) target of p38 MAPK to regulate Kv2.1-mediated current enhancement. While zinc was previously shown to activate p38 and src, I demonstrate here that this metal inhibits cytoplasmic protein tyrosine phosphatase å (Cyt-PTPå), which targets Y124 and antagonizes the actions of src. Therefore, I have identified two requisite phosphorylation sites on Kv2.1, at Y124 and S800, that cooperatively mediate apoptotic potassium current enhancement. Importantly, disruption of either phosphorylation event is neuroprotective. The work presented here provides a more complete understanding of neuronal apoptotic processes by revealing the intracellular signaling events linking intracellular zinc liberation and cytoplasmic potassium efflux.

Vesicular neurotransmitter transporters (VNTs) are a small family of proteins responsible for packaging neurotransmitter into secretory vesicles. Their presence and function are required for regulated secretion from neuronal and neuroendocrine cells. During both the biogenesis and the activity-dependent recycling of secretory vesicles, VNTs undergo trafficking that can determine the quality, quantity, and location of packaged neurotransmitter. Thus understanding the signals and mechanisms of VNT trafficking is essential to understanding the regulation of neurotransmission. Here, the synaptic vesicle specific trafficking of Vesicular Acetylcholine Transporter (VAChT) is investigated. A dileucine containing targeting motif, with dual properties for internalization and synaptic vesicle targeting, is identified in the C-terminus of VAChT. Chimeras between this motif and an unrelated plasma membrane protein localize to synaptic-vesicle-like vesicles in a neuroendocrine cell line. The specificity and generalization of this motif is assessed. Next, sorting nexin 5 (SNX5), implicated in the regulation of membrane traffic, is identified as a novel regulator of VAChT targeting to synaptic vesicles. Disruption of SNX5 function leads to a decrease in VAChT-directed synaptic vesicle targeting and a concomitant increase in targeting to large dense core vesicles. This shift between secretory granules suggests an important mechanism of VNT regulation with the potential to shape properties of neurotransmission. In order to understand the physiologic importance of VNT regulation, vesicular transport and its influence on activity-dependent release must be assessed in living neurons. However, this has not been possible. Therefore, a live cell assay was established to measure vesicular transport and its contributions to release in brain slice. Using a pH sensitive, fluorescent serotonin analog visualized by two-photon microscopy, activity dependent somatic release and vesicular monoamine transporter (VMAT) activity were measured in the dorsal raphe nucleus. Interestingly, while a portion of monoamine packaged at rest was held in reserve, monoamine packaged during stimulation was released efficiently. The work presented in this thesis provides a greater understanding of VNT trafficking and activity-dependent function. Furthermore, it provides the foundation for the comprehensive study of the active role of VNTs in shaping the properties of neurotransmission.

Stroke is the third leading cause of death in the United States and the second leading cause of death in the world. Despite the epidemiological significance of this disease, there are few treatment options. The purpose of this dissertation is to expand the understanding of underlying mechanisms mediating neuronal death caused by stroke, or cerebral ischemia. Two major metabolic disturbances occur due to ischemiapersistent protein synthesis inhibition and secondary energy depletion. All ischemia-affected neurons experience protein synthesis inhibition. However, neurons that recover protein synthesis live, while neurons that fail to recover die. This makes protein synthesis a robust predictor of neuronal death. However, the underlying mechanisms of persistent protein synthesis inhibition remain unknown. The hypothesis of this dissertation is that persistent protein synthesis inhibition is caused by activation of the calcium-sensitive protease calpain, which degrades eukaryotic translation initiation factor (eIF) 4G. Inhibition of calpain or overexpression of eIF4G results in increased protein synthesis and increased neuronal viability following the in vitro model of ischemia oxygen glucose deprivation in rat primary cortical neurons. Importantly, the neuroprotective effect of preservation of eIF4G is only partly due to its restoration of protein synthesis. Potential protein synthesis-independent mechanisms eIF4G-mediated protection are discussed. Neurons subjected to ischemia suffer an initial loss of energy in the form of ATP, which returns to baseline within fifteen minutes of restoration of blood flow. However, ischemia-sensitive neurons undergo secondary energy depletion prior to delayed neuronal death. The cause of secondary energy failure is hypothesized to be due to DNA recognition enzyme poly(ADP)-ribose polymerase (PARP)-1 depletion of the energy substrate NAD+. Evidence is presented linking PARP-1 activation to mitochondrial calcium dysregulation with subsequent calpain activation and apoptosis-inducing factor release. The results of these two findings are discussed in depth and future experiments are outlined. The potential of role of eIF4G in mitochondrial biogenesis, inhibition of autophagy and prevention of secondary energy loss is postulated. The research presented in this dissertation provides a novel perspective regarding the mechanisms underlying delayed neuronal death and may eventually lead to the development of clinically applicable neuroprotective strategies.

Hox transcription factors have been implicated in many aspects of embryonic rostrocaudal (or anteroposterior) patterning. I have examined the roles of two members of this family, Hoxd10 and Hoxd11, in the development of the lumbosacral (LS) region of the embryonic chick spinal cord. Hoxd10 is expressed uniformly throughout the LS spinal cord at early stages of motoneuron development, but later restricted to subsets of motoneurons in rostral segments. In contrast, Hoxd11 is expressed exclusively in caudal LS segments. Data presented here from overexpression experiments provide evidence that Hoxd10 promotes the development of motoneurons of the lateral subdivision of the lateral motor column (LMCl). Motoneurons transfected with Hoxd10 were likely to acquire a molecular profile, position, and peripheral axonal trajectory consistent with an LMCl identity. In contrast, Hoxd11 suppresses LMCl formation, and imparts a caudal and medial identity upon motoneurons, most likely via repressive interactions with Hoxd10 and the retinoic acid synthetic enzyme RALDH2. To further elucidate the mechanisms governing these opposing actions, I have also created a hybrid protein in which the DNA-binding homeodomain of Hoxd10 is replaced with that of Hoxd11 (Hoxd10d11HD). Hoxd10d11HD, when expressed in LS segments, behaves in a manner similar to Hoxd11, and in direct opposition to Hoxd10, by suppressing the development of the LMCl. It is therefore likely that some of the functionally specific actions of Hoxd11 are governed by the properties of its homeodomain.

Tlx (Nr2E1) is an orphan nuclear receptor transcription factor expressed in neural progenitor cells (PCs) in the forebrain throughout development and in regions of adult neurogenesis. Tlx regulates proliferation in both embryonic and adult PCs. Loss of tlx leads to abnormalities in limbic structures resulting in alterations in emotional and cognitive behaviors. However, the precise role of tlx in the developing forebrain and how tlx contributes to the normal development of adult anatomy and behavior are not fully understood. Tlx is expressed in PCs throughout the dorsal and ventral telencephalon and the diencephalon that give rise to structures including the cerebral cortex, hippocampus, amygdala, septum, striatum, and hypothalamus. Detailed examination of tlx expression revealed that within the dorsal PC population tlx is expressed specifically in radial glial progenitors but is absent from intermediate progenitor cells (IPCs). However, in the absence of tlx IPCs are reduced throughout development, suggesting that tlx promotes the production of IPCs. To examine the role of tlx specifically in dorsal PCs we generated mice with a conditional mutation of tlx in cortical, Emx1-expressing PCs (tlxcKO). In these animals functional recombination of the floxed tlx allele occurs prior to embryonic day 12.5. TlxcKO animals show similar changes in PCs as nulls, indicating a requirement for tlx within dorsal PCs. The cerebral cortex of tlxcKO animals is reduced in surface area and thickness from birth, persisting into adulthood. As in tlx null mutants, superficial layers are specifically affected and caudal functional cortical areas, including visual cortex, are disproportionately reduced. Other dorsally-derived structures, including the hippocampus, specific nuclei of the amygdala, and the septum are reduced, whereas ventrally-derived structures are relatively unaffected. These animals exhibit a subset of the behavioral abnormalities observed in nulls, with the primary phenotype being a reduction in anxiety. Together, these findings suggest an important role for tlx in the regulation of dorsal PCs. I propose that tlx promotes divisions that produce IPCs, and that disruption of this population leads to specific alterations in adult brain structure and behavior. This model allows us to begin to make connections between early development and behavior.

Change is inevitable, changes in neuronal function occur in physiologic and pathologic processes. The ability to reliably analyze and quantify those changes in neuronal morphology and function has been an important part of technical developments in Neuroscience. A key innovation in the Neuroscience was the development of primary neuronal cultures. Primary neuronal cultures allow neurons to be dissociated and studied as individual components. The study of specific pathologic processes associated with neurodegeneration have benefited greatly from the development and characterization of dissociated primary neuronal cultures. Human Immunodeficiency Virus can lead to a neurodegenerative process. Establishing a consistent model for studying the effects of HIV infection in the brain has provided a unique challenge. The use of analysis of quantification of neuronal changes in dissociated primary neurons modeling HIV dementia has proven useful. As the study of this disorder continues the characterization of the model system will become increasing important. This review will focus on analysis of specific techniques used to quantify specific changes in neurons in this model system. As this field moves forward it will be important to specifically focus on techniques involved in cell viability, morphologic changes, and synaptic formation

Sub-lethal preconditioning stimuli can confer neuronal tolerance by triggering the activation of endogenous survival pathways that limit or resist subsequent injury. Recent evidence has demonstrated that neuroprotection is paradoxically dependent on the sub-lethal activation of cell death mediators. As intracellular Zn2+ accumulation has been closely associated with neuronal cell death pathways, I tested the hypothesis that neuronal tolerance is also dependent on sub-lethal Zn2+ signals. I found that preconditioning triggered an immediate transient rise in neuronal free Zn2+, while lethal excitotoxicity led to a delayed accumulation of the metal. The sub-lethal rise in Zn2+ was necessary and sufficient in attenuating subsequent Zn2+-dependent toxicity in preconditioned neurons. Chelating Zn2+ during the preconditioning stimulus restored the lethal excitotoxic accumulation in neuronal Zn2+ and abolished neuronal tolerance. These data suggested that preconditioning-induced Zn2+ could trigger mechanisms for preventing subsequent Zn2+-dependent cell death. Indeed, preconditioning triggered protein kinase C (PKC)-dependent Zn2+-regulated gene expression in neurons. Examination of the mechanism involved in modulating Zn2+-regulated gene expression revealed a surprisingly early role for PKC in directly modifying the intracellular source of Zn2+. A conserved PKC phosphorylation site was identified at serine 32 of the metal binding protein metallothionein, which was important in modulating Zn2+ regulated gene expression and ultimately conferring neuronal tolerance. In addition to modulating gene expression, Zn2+ signals may also be important in mediating the acute cellular response to stress. Here, I found a critical role for the transient Zn2+ rise in modulating changes in voltage-gated potassium channel activity and localization following ischemia. Together, these data strongly suggest that a transient rise in neuronal free Zn2+ is an important early signal in conferring neuronal tolerance and in mediating acute cellular adaptive responses to stress. Thus, Zn2+ is a previously unrecognized, highly regulated signaling component in the initiation of survival pathways in neurons.