SKF83959 is a high affinity dopamine D1 receptor agonist that has been reported to preferentially activate D1 receptors coupled to G(alpha)q. This pathway results in phosphatidylinositol hydrolysis, intracellular calcium mobilization, and potential activation of Ca2+/calmodulin-dependent protein kinase II alpha (CaMKIIalpha), an important regulator of synaptic transmission. Although the exact mechanism remains unclear, one recent model suggested that SKF83959 activates a D1/D2 receptor heteromer complex coupled to G(alpha)q. Here, we have used genetic models to define the signaling specificity of SKF83959 using behavioral endpoints. Furthermore, we have extended the behavioral characterization of SKF83959 on motor output and additionally defined SKF83959-induced effects on anxiety and depressive-like behaviors. In wildtype mice, a peripheral injection of SKF83959 (1mg/kg) produced a modest but significant increase in horizontal locomotor activity and orofacial grooming. The SKF83959-induced responses were blocked by the D1-like receptor antagonist SCH23390 and absent in D1 receptor knockout mice, confirming the role of D1 receptors in mediating the effects. SKF83959-induced behaviors were largely intact in D5 receptor and G(alpha)q knockouts, suggesting that these proteins are not necessary for mediating SKF83959-induced actions. There was, however, a significant difference between the response of wildtype and D5 receptor knockouts suggesting that D5 receptors may play some role in the signaling. Additionally, these responses were intact in D2 receptor knockouts and autophosphorylation-deficient CaMKIIalpha-Thr286Ala knockin mice, also negating the necessity of these proteins in SKF83959-mediated actions. SKF83959 produced no significant effects in an elevated zero maze, a useful task for assessing anxiety in rodent models. There was, however, a reduction in immobility observed in SKF83959-treated mice in the forced swim test, suggesting reduced behavioral despair; an effect that was confirmed in a second measure of behavioral depression, the tail suspension test. Finally, we evaluated chronic SKF83959 exposure (0.5 mg/kg for 21 days) and observed differences between treatment groups in a novelty-induced food suppression test; a paradigm sensitive to chronic antidepressant treatment. Taken together, these studies define the specificity of receptor-G-protein signaling in the effects of SKF83959. Furthermore, our data indicate that SKF83959 may be useful in treating motor dysfunction and could potentially define a novel class of antidepressants targeting the dopamine system.

I have studied the neurophysiological bases of saccade execution and evaluation within the oculomotor system of Macaca mulatta and Macaca radiata. To better understand saccade execution, I recorded the electromyogram (EMG) from extraocular muscles and the occurrence of microsaccades while monkeys canceled responses in the saccade stop-signal task. I found reduction in extraocular EMG and in the occurrence of microsaccades when monkeys canceled eye movements. These results are unanticipated by a well-accepted, new theory of saccade execution. I conclude that the theory's proposed mechanism of fixation is incomplete. To study saccade evaluation, I recorded activity from single neurons, small neural ensembles, and large groups of neurons in and overlying an area of frontal medial cortex called the supplementary eye field (SEF) while monkeys viewed light flashes or made correct and errant responses during the saccade stop-signal task. I found 1) that SEF displays functional microcircuitry very similar to that described in early sensory cortex even though anatomy differs drastically between these areas, 2) that monkeys display electrophysiological correlates of error detection homologous to the error related negativity (ERN) and error related positivity (Pe) recorded in humans, and 3) that current flow in SEF contributes to, but is not the sole determinant of the ERN. This work highlights differences between the oculomotor and spinal motor systems, suggests the need for improvements on current models of fixation, and provides novel insight on the neural basis of performance monitoring.

This dissertation describes pharmacological studies of the functional consequences of the Cys23Ser single nucleotide polymorphism (SNP) in two different edited versions of the human 5-HT2C receptor as well as evaluation of this SNP in a population of well characterized depressed patients. I discovered that the Cys23Ser SNP has no functional consequences when expressed in a variety of cells in culture, although there were positive associations with endophenotypes of major depressive disorder (MDD). Upon examining other SNPs that could be in linkage disequilibrium with the Cys23Ser SNP, I found that a functional SNP in the 5-HT2C promoter (-697G/C) is closely linked to the Cys23Ser SNP. Therefore, these results suggest the non-functional Cys23Ser SNP associates with disease states because it is closely linked to the functional -697G/C promoter polymorphism in the 5-HT2C receptor.

LOCALIZATION AND TRAFFICKING OF THE CAENORHABDITIS ELEGANS DOPAMINE TRANSPORTER (DAT-1). PAUL W. MCDONALD Dissertation under the direction of Professor Randy D. Blakely, Ph.D. Reuptake of dopamine (DA) through the dopamine transporter (DAT) is the primary mechanism by which DA signaling is terminated at the synapse. Drugs of abuse such as cocaine and amphetamine target DAT, altering DA signaling in the central nervous system. Mechanisms that localize DAT to synapses and control its activity remain ill defined. The soil nematode C. elegans, elaborates a cocaine-sensitive DAT (DAT-1), which is expressed in all the DA neurons of the worm and offers advantages over studies of DATs performed in mammalian systems. To advance studies of DAT-1 localization and trafficking mechanisms within an intact nervous system in vivo, I first generated DAT-1 specific antibodies to establish wild type DAT-1 localization. Once established, I expressed green fluorescent protein (GFP) fused to NH2-terminal of DAT-1 (GFP:DAT-1) in the DA neurons and examined localization in different compartments within a single neuron. Consistent with native antibody studies, GFP:DAT-1 is expressed throughout the DA neuron, with a synaptic accumulation of signal not seen with cytosolic GFP. Biophysical studies using fluorescent recovery after photobleaching (FRAP) reveal that this accumulation is predominantly immobile, whereas the cell body GFP:DAT-1 remains free to diffuse along the plasma membrane of the neuron. Trafficking studies examining GFP:DAT-1 localization using known synaptic determinant mutants and mutants generated in a forward genetic screen reveal that specific residues within DAT-1 are important for export to both dendritic and synaptic areas, whereas correct localization of DAT-1 is independent of the kinesin motor protein UNC-104. Examination of a truncated version of the GFP:DAT-1 fusion that lacks specific sequence implicated in both protein-protein interactions (GFP:DAT-1(delta IML)) reveal that expression of this mutant transporter in vivo results in protein instability and cell body accumulation, with little effect on synaptic targeting. Taken together, theses studies develop both a novel system for examination of fusion protein localization and function in vivo and they more specifically report, for the first time, the localization of a cocaine-sensitive DAT in intact DA neurons in vivo setting the stage for future targeted studies of DAT-1.

Our aged population is poised to expand dramatically within the next decade. In Alzheimers disease (AD) pathogenesis studies, the end point hallmarks or lesions are known and well studied; however, the exact processes leading to these lesions are not. Defining early pathological events at the molecular and protein level and targeting appropriate therapies to pre-clinical or early stage dementia is necessary to avert the coming public health crisis. This project showed that lipid peroxidation products can lead to microtubule dysfunction that is characteristic of AD and that this is associated with their accumulation on tau from among the cytoskeletal proteins investigated. In contrast, another type of protein oxidation was observed selectively on Beta-III tubulin using mass spectrometry. Together, these data indicate that multiple oxidative modifications to cytoskeletal proteins are likely occurring in AD and that these can contribute to cytoskeletal dysfunction, leading to a modified model of AD pathogenesis. Furthermore, the results suggest that approaches to limit protein oxidation may have the downstream effect of suppressing protein insolubility and its consequences. Perhaps, with further investigation, studies will be able to define drug-treatable targets to prevent and slow neurodegenerative disease progression.

This dissertation explores the anatomical and functional organization of the sensorimotor system of Bellangers tree shrews. A special emphasis is placed on establishing the organization of the motor cortex and thalamus in tree shrews using multiple criteria for comparison with primates and their more distant relatives. The results of microstimulation, architectonic, and corticospinal tracing studies are presented first, which provide evidence for a primary and secondary motor cortical area (M1 and M2 respectively), and a number of cortical areas in anterior parietal cortex. The results of tracer injections into these areas are presented in the subsequent two chapters. The corticocortical connections of motor cortex provide evidence for an additional medial motor area (MMA) in cortex, as well as a number of sensorimotor areas in posterior parietal and temporal cortex. The thalamocortical tracing results are combined with detailed histological analysis of thalamus, and provide evidence for a redefinition of a number of motor and somatosensory related thalamic nuclei. A comparison of the of the functional and anatomical organization of the motor cortex and thalamus in tree shrews reveals more shared features in common with primates, especially prosimians, than more distant relatives such as rats. The findings of this dissertation indicate that 1) previous assertions that the motor system of tree shrews contains only primitive features are incorrect, 2) the secondary motor area of tree shrew motor cortex (M2) may represent a transitional form of the premotor cortex of primates, and 3) comparative studies of the motor systems of both primates and other mammals will lead to a better reconstruction of the evolution of the motor system in primates.

THE STRUCTURE AND FUNCTION OF EIMERS ORGAN IN THE MOLE Paul D. Marasco Dissertation under the direction of Kenneth C. Catania This study examines the structure and function of Eimers organ in the mole. Eimers organ is a complex, well organized, and densely innervated mechanosensory structure found on the nose of the mole. Eimers organs appear to allow the mole make rapid sensory discriminations of objects. Labeling with AM1-43, substance P, neurofilament 200 and DiI revealed innervation patterns indicating that mechanosensory and nociceptive function is segregated to separate afferents within Eimers organ. Transmission electron microscopy revealed that the central column of Eimers organ may mechanically transduce stimuli and the intraepidermal free nerve endings do not appear to be active within the outer layer of the skin. The peripheral free nerve endings were structurally distinct from their central column counterparts and they were unmyelinated. The receptors in Eimers organ were similar in morphology to those found in a range of other mammalian skin. Electrophysiological examination revealed that the receptive fields for receptors in Eimers organ were small and response properties were consistent with an SA-1 receptor class, an FA-2 class. A second rapidly adapting class was hypothesized to represent activity of the mechanosensory free nerve endings. Most receptors were directionally sensitive and highly phase locked. Some receptors responded best when stimulated at a velocity matching the velocity of the nose in during foraging. The results support the hypothesis that Eimer's organ is a mechanosensory structure that functions to detect small surface features and textures by encoding and integrating deflection information for multiple Eimer's organs during brief touches.

In order to form synaptic connections, an individual neuron needs to coordinate the activities of a large number of proteins. The neuron can modulate the activity of these proteins in a variety of ways. For instance, the neuron can directly target proteins for degradation at the synapse. The neuron can also modulate the activity state of proteins at the synapse through chemical modifications (phosphorylation, glycosylation, etc.). A third way a neuron can modulate synapse structure and connectivity is by regulating transcription. My work has focused on the protein RPM-1, an E3 ubiquitin ligase that modulates protein activity at the synapse in a number of ways (through regulating protein degradation, and indirectly regulating phosphorylation state and transcription). RPM-1 is localized at the synapse directly adjacent to the active zone. Mutations in rpm-1 and its orthologues Highwire/Phr1 disrupt synaptic structure and signaling. Genetic epistasis experiments suggest that RPM-1 controls synaptic development by downregulating MAP Kinase signaling. We have shown that RPM-1 regulates transcription through MAP Kinase. Our data, together with data from Drosophila, suggest that RPM-1/Highwire regulates synaptic morphology by regulating transcription. We have exploited a powerful new technology, mRNA-tagging, to identify the transcriptional targets of RPM-1. A profile of the entire C. elegans nervous system revealed 558 enriched transcripts in an rpm-1 mutant dataset. In vivo GFP imaging confirmed the enrichment of one of these targets, tbb-6. Further work is needed to determine the role these targets play in synaptic development. C. elegans, with its fully defined neural architecture and powerful genetic and genomic strategies, is an excellent model system for functional and developmental studies of the nervous system. We have exploited the mRNA-tagging technique to generate a gene expression profile of the worm nervous system. We have identified a total of 2149 enriched genes using two different amplification methods. We have confirmed significant enrichment for known neural genes (~90%). These data have also revealed ~30 previously uncharacterized genes that are enriched in the C. elegans nervous system and are conserved in humans. This suggests that these uncharacterized proteins may also function in the human nervous system. In addition to profiling all neurons in the worm, we have utilized the mRNA-tagging technique to generate a gene expression profile of a specific neural class (A-class motor neurons). The use of higher resolution profiles may help direct gene discovery in mammalian systems and may also suggest functions for uncharacterized proteins.

During postnatal development of the central nervous system, the response of GABAA receptor to its agonist undergoes a switch from excitatory to inhibitory, due to a developmental decrease in the intracellular Cl- concentration. In this thesis, I show that the cation chloride cotransporters KCC2 and NKCC1 regulate intracellular chloride and brain excitability. First, I provide evidence that KCC2 is responsible for the developmental decrease of intracellular [Cl-]. Second, I demonstrate that in the young adult, KCC2 is responsible for the rapid removal of chloride that is accumulated in the cell either by prolonged GABAA activation, prolonged membrane depolarization, or co-activation of GABA and glutamate receptors. Third, I show that excitability and seizure susceptibility are both increased in KCC2+/- brain slices. Finally, I demonstrate that NKCC1 decreases neuronal excitability and prevents 4-aminopyridine-induced epileptic seizure in young mice. During the course of this research, I also developed a new method to measure the membrane potential of neurons in brain slices.

DOPAMINE AND THE POSITIVELY REINFORCING PROPERTIES OF AGGRESSION MARIA H. COUPPIS Dissertation under the direction of Professor Craig H. Kennedy Aggression is a necessary behavioral response aimed at securing survival. However, when aggressive topographies exceed species typical norms, they become pathological and problematic to society. It has been hypothesized that aggression may be positively reinforcing and that these positively reinforcing characteristics are modulated by mesocorticolimbic dopamine systems. In order to gain a more comprehensive understanding of the rewarding properties of aggression and their biological substrates, a series of experiments were conducted to address the questions: 1) Is aggression positively reinforcing? If so, what part of the aggressive encounter serves as the positively reinforcing event? 2) Do DA1/5 and/or DA2/3 receptors in the NAC mediate access to aggression as positive reinforcement in mice? and 3) Are there any endogenous differences between aggressive and non-aggressive individuals? It was concluded from these experiments that physical aggression can be positively reinforcing, that these positively reinforcing properties are modulated by mesocorticolimbic dopamine and that there are endogenous differences in mesocorticolimbic dopamine systems between aggressive and non-aggressive individuals.