Synaptic plasticity is proposed to be a molecular mechanism underlying multiple forms of learning. The modulation of synaptic plasticity, therefore, may have profound consequences over behavior and may mediate pathological conditions. This project examined synaptic modulation via the activation of á1-adrenergic receptors (á1-ARs) in the bed nucleus of the stria terminalis (BNST), a brain region involved in reward, addiction and anxiety behaviors. I found that in the BNST, á1-ARs induce a heterosynaptic long term depression (LTD) of excitatory transmission that is maintained postsynaptically and requires the activation of L-type voltage gated calcium channels and the functional desensitization of calcium permeable AMPA receptors. Furthermore, this plasticity is disrupted in both behavioral and genetic models of affective disorders suggesting it may play a role in the pathology of these disorders. These data add to the recent evidence suggesting that the á1-AR may be an excellent target for therapeutic intervention in several affective disorders.

Mammalian circadian rhythms are orchestrated by the suprachiasmatic nuclei (SCN) of the hypothalamus. The SCN are composed of circadian clock neurons but the mechanisms by which these neuronal oscillators encode circadian physiology are incompletely understood. The overall objective of this dissertation was to determine the genetic and molecular organization of the neural network within the SCN, and elucidate how the SCN interacts with the environment to produce measurable behavioral and physiological circadian rhythms. Using genetic knockouts, molecular reporters and behavioral analyses, this dissertation explores the relationship between circadian genes, the SCN neural network in which these genes are expressed, and finally the behavioral output that results from this network in mice and humans. Overall these results suggest that circadian genes and light act together to organize the mammalian SCN neural network, and thus affect behavioral and neurological phenotypes.

During development of the nervous system, about half of the neurons generated undergo apoptosis. How these neurons are cleared in the peripheral nervous system was largely unknown. Our lab discovered that clearance in the dorsal root ganglia of mouse embryos was achieved by amateur phagocytes, cells that have other important roles besides phagocytosis. Specifically, the resident glia, satellite glial cell precursors are responsible for engulfing neurons in developing dorsal root ganglia. In addition, we identified the novel receptor Jedi-1, and the purported engulfment receptor MEGF10, as two receptors expressed in the glia that are involved in engulfing apoptotic neurons. Although nothing was known about the signaling mechanisms of Jedi-1 or MEGF10, my dissertation work revealed that both receptors contain two intracellular Immune receptor Tyrosine-based Activation Motifs that are phosphorylated by Src Family Kinases and interact with the non-receptor tyrosine kinase Syk. This interaction was necessary for either receptor to promote phagocytosis. Here, I describe these interactions as well as the importance of apoptotic cell clearance, including possible links to autoimmune disorders.

The Met receptor tyrosine kinase is capable of mediating several neurodevelopmental events in vitro, but its functions in vivo are incompletely understood. Three alleles in the 5 regulatory region of the human ortholog, MET, increase the risk for autism spectrum disorder, suggesting that disruptions in Met expression may contribute to the aberrant circuit development characteristic of neurodevelopmental disorders. To gain insight into the functional roles for Met in vivo, we mapped Met transcript and protein expression throughout perinatal and postanatal development. This study revealed complex and dynamic spatiotemporal patterns of expression, suggesting a role for the receptor in neurite outgrowth and synaptogenesis, particularly in establishing connectivity in forebrain circuits relevant to the social and emotional dimensions of behavior. The complement of transcription factors controlling Met expression in the nervous system is poorly understood. Yet knowledge of these regulatory factors is critical to understanding how Met expression patterns are established in vivo, and gives insight into sources of vulnerability to disrupted expression and consequent aberrations in circuit patterning. Previous work has demonstrated that the ASD-associated MET allele alters Sp-family binding to the genes 5 regulatory region. In this study, we demonstrate that Sp4 in particular has a role in regulating Met expression in the mammalian forebrain. Further, we developed a mass-spectrometric-based screen to identify additional regulators of Met expression, and used this screen to identify 49 putative regulators from postnatal day 7 mouse cortical tissue. A novel bioinformatics approach provides new opportunities for determining the downstream targets of regulatory genes relevant to autism risk.

Many have suggested that executive control over the perception, selection, and production systems is a central component of human cognition required for monitoring performance and compensatory adjustments in response time following errors. Evidence from human brain imaging, human event related potentials, and single units in non-human primates have implicated the medial frontal lobe as part of this monitoring system. We have used a stop signal or countermanding task to probe the ability to control action by requiring subjects to withhold a planned movement in response to an infrequent stop signal which they do with variable success depending on the delay of the stop signal. Emeric et al (2006) provided evidence that the compensatory adjustments of response time of both humans and macaque monkeys in a saccade countermanding task is influenced by stimulus and performance history. We have established a bridge between event-related potential and functional brain-imaging studies in humans and neurophysiology studies in non-human primates with event related intracranial local field potentials (LFPs) recorded in the anterior cingulate cortex and supplementary eye fields of macaque monkeys performing this task. The results provide clear evidence that error-, feedback-, and conflict-related, signals are carried by the LFP in the medial frontal lobe of macaques (Emeric et al 2008). Finally, we have examined the characteristics of extracranial field potentials from monkeys performing the saccade stop signal task and have identified putative performance monitoring potentials consistent with the human event related potential literature. Taken all together, this body of work has contributed to the groundwork for examining neural signals that are homologous to human event related potentials in the nonhuman primate.

<p>Expansion in the glutamine encoding CAG triplet-repeat in the Huntingtin (HTT) gene causes Huntingtons disease (HD). The susceptibility, age of onset, and severity of HD is modified by ill-defined genetic and/or environmental factors. We have previously reported that expression of mutant HTT is associated with impaired Mn accumulation in a striatal cell line and mouse models of HD. Recognizing the regulatory relationship between HTT protein and metal transporters, I hypothesized that mutant HTT alters regulation of cellular Mn by aberrant interactions with the normal cellular Mn transport system. <p>To identify the mechanism, I have developed and validated a high-throughput fluorescence-quenching based assay (cellular fura-2 manganese extraction assay - CFMEA) to measure Mn transport kinetics and storage properties in cultured striatal cells and rodent tissues. I tested the hypothesis that expression of mutant HTT impairs Mn accumulation by (i) modulating Mn uptake, (ii) intracellular trafficking dynamics and accumulation, and (iii) efflux transport mechanisms. I demonstrate that expression of mutant HTT alters instantaneous Mn uptake kinetics. I observed a significant decrease in net Mn storage capacity in mutant compared to wild-type striatal cells, despite the similar net Mn efflux rates between both genotypes. <p>To dissect the metal transporter system(s) underlying altered mutant Mn homeostasis, I examined several known Mn transporters and failed to observe their role in mutant Mn uptake deficits. However, I demonstrate that the putative Mn transporter, Huntingtin interacting protein (HIP14), protein levels is significantly decreased in mutant compared to wild-type striatal cells. Furthermore, I observed a wild-type specific down regulation of HIP14 protein levels in response to excess Mn, which is consistent with a role of HIP14 in regulating Mn transport in the striatal cells. In addition, I demonstrate that Mn uptake in the striatal cells is substantially inhibited by the two highest affinity cations (Mg2+ and Sr2+) for the HIP14 transporter and its specific inhibitor (Ca2+). Finally, I show a functional relationship between the previously reported differential Mn induced cytotoxicity between wild-type and mutant HTT striatal cells and the cellular Mn pools that contribute to oxidative injury. <p>In conclusion, I have developed a novel high throughput assay for assessing Mn transport dynamics and my findings suggest that the Mn accumulation deficit in mutant cells is due to impairment in Mn uptake and storage capabilities and not alteration in Mn efflux. In addition, mutant HTT impairment of HIP14 transporter function points to its responsibility for disruptions of Mn homeostasis and this may contribute to environmental modulation in HD.

Embryonic dopamine (DA) receptor (DR) expression in the medial frontal cortex (mFC) coincides with the formation of neural circuits during the period of axon pathfinding but the signaling properties of DRs and their effect on axon guidance events has not been examined. We used in situ hybridization and quantitative PCR to map DR mRNA transcripts in the medial frontal cortex (mFC) of the rat from E15 to E21. The developmental trajectory of DR mRNAs revealed distinct patterns of DA receptors 1 and 2 (DRD1, DRD2). To identify the functional properties of DRs during embryonic development, the phosphorylation states of CREB, ERK1/2, and GSK3β were examined after DR stimulation in primary neuronal cultures obtained from E15 and E18 embryos and cultured for three days to ensure a stable baseline level. DR-mediated signaling cascades were functional in E15 cultures. Because DA fibers do not reach the mFC by E15, and DA was not present in cultures, these data indicate that DRs can become functional in the absence of DA innervation and during axon pathfinding. We assessed the consequences of DR stimulation during Netrin-1-mediated axon guidance. Netrin-1 is a secreted axon guidance molecule expressed at the ventricular zone that can both attract or repel axons. We examined primary neuronal cultures of the mFC and found DRs co-localized with netrin-1 receptors DCC and UNC5C. In neuronal outgrowth assays as well as tissue explants, DR agonists interfered with the attraction of neurites to netrin-1. Stimulation of DRs in mFC cultures altered mRNA expression of UNC5C, suggesting that DR activation may modify the growth cone response to netrin-1 by regulating receptor abundance. By disrupting the trajectory of axon outgrowth, abnormal DR activity during embryogenesis may impact neuronal circuit formation in the forebrain and mFC function later in life.

Mild, moderate, and vigorous physical activity are associated with decreased risks of Alzheimers disease, Parkinsons disease, and a decreased infarct from stroke. Physical activity increases neurogenesis in the hippocampus and upregulates neurotrophic factors throughout the brain, but it largely has been understudied in motor brain regions related to Parkinsons disease. We hypothesized that aerobic exercise would be protective against a MPTP lesion in Rhesus monkeys. We used three cohorts of monkeys to study gene expression associated with physical activity and exercise in motor regions of the brain: (1) spontaneously physically active rhesus monkeys, (2) exercised rhesus monkeys, and (3) spontaneously physically active and exercised rhesus monkeys before and after receiving a MPTP lesion. We found that physical activity as measured by accelerometers predicts the size the MPTP lesion, as monkeys with high levels of physical activity were protected from the lesion. Furthermore, the activity-associated neuroprotective gene expression signature involved transcripts associated with chromatin remodeling, neuronal survival, and specifically syntaxin 3. These studies suggest that being physically active is protective against Parkinsons disease.

Autism spectrum disorders (ASDs) are heterogeneous neurodevelopmental disorders. Although research on ASDs has implicated multiple genes in pathogenesis, etiology for the majority of cases remains unknown. In the context of profound etiological and phenotypic complexity, one strategy to seek insight into risk and pathogenesis is to stratify a heterogeneous population based on a prominent feature, resulting in increased homogeneity within population strata. One such feature is gastrointestinal dysfunction (GID), which is present in a subpopulation of individuals with ASDs. A previous report demonstrated signal enrichment for an ASD risk variant in the MET gene specifically in individuals with co-occurring ASD and GID (ASD-GID), suggesting shared genetic risk for both the nervous and gastrointestinal systems due to pleiotropic expression of MET. The objectives of the current studies were to examine relationships between proposed causes of GID and GID outcomes in ASD; to investigate clinical and biological features specific to ASD-GID; and to study the biological roles played by Met in the gastrointestinal system of conditional knockout mice. Children with ASDs, with and without GID, as well as children with GID only, donated a blood sample and were assessed for language and social function, dietary habits and GID status. Met conditional null mice, lacking Met signaling capacity in the GI epithelium, were assessed for repair ability in response to acute epithelial injury. In children with ASDs, no association was found between dietary habits or medication status, and GID outcome. A significant fraction of children with ASD-GID had no expressive language, and had greater social impairment, compared to children with ASD only or GID only. Prevalence of the MET risk variant was increased in the ASD groups. The possibility that children with ASD-GID are more severely impacted is supported by data demonstrating significant elevation of a marker of oxidative stress specifically in this group. In the animal model studies, Met conditional null mice demonstrated impaired healing capacity in response to GI injury. These findings have important implications both for improving clinical care of children with ASDs and co-occurring GID, and for further understanding risk and pathogenesis in a subpopulation of individuals with ASDs.

I investigated how tactile stimuli applied to different parts of one hand and to both hands affect neuronal response properties in primary somatosensory cortex (area 3b) of owl monkeys. The premise of this research is that object discrimination requires integration of information from across the hand, and when used together, from both hands; and this integration is likely to involve area 3b. With colleagues, I recorded neuronal activity from 100-electrode arrays implanted in the hand representation of area 3b of five owl monkeys. From these large samples of simultaneous neuronal activity, I analyzed the effects of spatiotemporal tactile stimulation on three basic measures: firing rate, response latency, and spike timing correlations between neuron pairs. From these studies, I concluded that widespread spatial and temporal integration occurs within the area 3b hand representation and across the two hemispheres; and the results helped to quantify the extent and strength of these spatiotemporal interactions.