The flow of information through neuronal circuits relies on the ability of neurons to form synaptic connections with specific temporal and spatial properties. These properties are not static but have the ability to change, allowing synaptic connections to be strengthened or weakened. It is this plastic nature of synapses that is central to higher order processes such as learning and memory. However, major gaps remain in our understanding of this process. Throughout my dissertation work I have examined mechanisms of a form of structural synaptic plasticity by analyzing the roles of a variety of proteins that we have found serve to regulate the formation and maintenance of glutamatergic synapses at the Drosophila NMJ. These proteins include APPL, the Drosophila homolog of Alzheimer's disease-associated β-Amyloid Precursor Protein (APP), the tumor suppressor protein Scribble (Scrib), the secreted signaling molecule Wingless (Wg), and the cell adhesion molecule Fasciclin II (FasII). In this work, in collaboration with members of the labs of Dr. Vivian Budnik, Dr. Kalpana White, and Dr. Susan Cumberledge, I have demonstrated that Wingless (Wg) provides a secreted signal that is required to initiate the formation of pre- and postsynaptic structures. Further, I have also demonstrated that once synapse formation is initiated, presumably by Wg signaling, APPL regulates synaptic bouton proliferation. This process also involves signaling by FasII, a protein required for synapse maintenance, and growth. Moreover, I have also demonstrated that Scrib is a scaffolding protein that plays a key role at these synapses in influencing neurotransmitter release.

Assessments of stress typically involve either point samples (blood or saliva) reflecting a moment in time or state samples (urine of feces) reflecting several hours or a day. Currently, there is no way to assess chronic levels of stress without using repeated sampling procedures which are both time consuming, expensive, and possibly stressful. The purpose of this dissertation was three fold: (1) to develop a more chronic estimate of stress by measuring cortisol concentrations in hair, (2) to determine using this measure, how rhesus macaques responded to the prolonged stress of relocation, and (3) to determine the role of a proposed modulatory protein corticosteroid binding globulin (CBG) in measuring stress. Results demonstrate that cortisol can be quantified in hair and that stress reactivity can also be measured in hair. Rhesus monkeys responded to a major life stress (relocation) with a significant increase in cortisol in both hair and serum and with substantial behavioral changes. CBG concentrations failed to show alteration initially following relocation, however a significant increase was observed one year later. The findings demonstrate the importance of the cortisol/CBG relationship during a prolonged stress paradigm. Our data also provide further evidence that the free cortisol index (cortisol:CBG ratio) may be a better indicator of stress reactivity when compared to the commonly used serum cortisol concentrations.

Variability in the discharge of human motor neurons was investigated in the context of aging and skill acquisition. In the aging experiment, young and older adults performed constant-force and complex sinusoidal isometric force-matching tasks while motor unit action potentials were recorded from the first dorsal interosseous (FDI) muscle. In three manuscript chapters, the characteristics of motor unit inter-spike interval (ISI) distributions were compared, patterns in the time series of ISIs were investigated with two experimental measures of complexity, and the extent of motor unit synchronization and common drive was compared between young and older adults. The sinusoidal force-matching conditions required substantial motor unit rate coding compared to the constant-force task. In both tasks, older adults utilized a greater proportion of motor units that were discharging at lower rates and with greater discharge variability. Positive skewness of ISI distributions from older adults indicated that motor unit discharge rates varied more freely towards lower rates and de-recruitment. That skewness was greater in older adults indicates that disruption of consistent discharge through de-recruitment and subsequent recruitment may be one reason why force fluctuations are greater and force-matching performance was poor in older adults. The complexity measures indicated that the fluctuations in motor unit discharge during constant-force contractions were random. Detrended fluctuation analysis, but not approximate entropy, was able to distinguish between the constant-force and sinusoidal force-matching tasks. It was demonstrated with simulated data that these techniques are sensitive to the manner in which motor unit data are pre-conditioned. Indeed, different methods of creating firing rate time series resulted in opposite conclusions regarding complexity. These complexity measures should be further evaluated with discharge data from patient populations in which patterns might be expected due to altered neural function. Motor unit synchronization and common drive were similar in young and older adults and greater synchronization was associated with greater discharge variability. (Abstract shortened by UMI.)

The frog neuromuscular junction is a unique model that allowed us to selectively remove cellular components from the neuromuscular junction and create preparations with varying degrees of nerve terminal stability. We found further evidence that frog terminal Schwann cells communicate with their cellular partners, as terminal Schwann cells responded with changes in number or morphology as a result of changes in synaptic integrity. Terminal Schwann cells divided at synaptic sites in response to a regenerating nerve terminal. Terminal Schwann cells also had morphological changes in response to changes in status of their cellular partners; they extended processes in response to removal of the nerve terminal. Orientation and length of these processes was profoundly affected by the presence or absence of muscle fiber and nerve terminal. Similar to observations at the mammalian neuromuscular junction, terminal Schwann cells appear to play a role in reinnervation, as frequently regenerating nerve terminals were within the confines of terminal Schwann cells and their processes. I also investigated the organization of actin within preparations with varying amounts of nerve terminal stability, including developing nerve terminals and regenerating adult nerve terminals that were forming either stable or unstable connections. Previously, F-actin stained target-deprived nerve terminals in a ladder-like pattern and was concentrated in the nonrelease domains (Dunaevsky and Connor 2000). I found that β-actin was similarly distributed and localized to the nonrelease domains of nerve terminals at intact neuromuscular junctions. Further, association of actin with these particular domains appeared to be important for nerve terminal stability. As nerve terminals acquired increasing stability during development, they acquired this domain specific distribution of F-actin. Additionally, although synaptic sites with stable regenerating nerve terminal acquired this ladder-like pattern of F-actin, it was very rare for unstable regenerating nerve terminals to do so. I also tested the dynamic nature of F-actin with pharmacological perturbation. F-actin at nonrelease domains was found to be very stable. This stability of the F-actin based cytoskeleton further suggests that F-actin at the nonrelease domains of nerve terminals may play a role in the stability of motor nerve terminals.

Synapses are the functional units of neuronal circuits and the sites of integration for multiple signaling pathways. Understanding the molecular basis of synaptic function is critical to understanding the bigger picture of how we think, learn, remember and how neurological diseases and disorders disrupt these faculties. Here we characterize the role of bendless (ben) in central synapse formation by utilizing the giant fiber system (GFS), a well established neuronal network in Drosophila melanogaster. Ben is an E2 conjugase and a key member of the enzyme cascade involved in ubiquitin dependent regulation. Ben was originally identified more than two decades ago and was believed to be involved in axon guidance as mutants lacked a synaptic connection between the giant fiber (GF) and its target, the jump motor neuron (TTMn). We have been able to redefine Ben function by demonstrating that an incipient GF-TTMn synaptic connection is present in ben mutants. We have also analyzed the synapse with the help of synaptic markers as well as studied its features at an ultrastructural level. By conducting cell autonomous rescue experiments we have spatially determined that ben has a presynaptic function in the GFS. We then used the TARGET system to temporally characterize the gene and have isolated a critical period for Ben function during development. We further assayed protein localization by generating GFP-tagged Ben constructs and have found the protein to be nuclear as well as cytoplasmic. Subsequent studies have identified two multifunctional proteins—Semaphorin1a and Distracted—to be putative targets of Ben action. We have also carried out a preliminary characterization of the synaptic roles other components of the ubiquitin system have in the GFS, such as the ubiquitin ligase highwire and the deubiquitinating proteases, fat facets and UBP2. In summary, we have found the ubiquitin conjugase Ben to have a novel and distinct role as a developmental switch in the establishment of a central synapse. The identification of likely downstream targets of Ben and comparison with related ubiquitin associated proteins suggest that a delicate regulatory balance has to be maintained in order for a synapse that is functionally and morphologically normal to be sculpted.

In addition to physiological functions, the neuropeptide hormone, arginine vasopressin (AVP) facilitates the mammalian central nervous system (CNS) regulation of cognitive function and social behaviors. V 1A receptors mediate these effects. Although distribution patterns of AVP-producing neurons and fibers are similar among mammals, CNS V1A receptor patterns are species-specific. Here, rhesus (Macaca mulatta) central V1A receptors were mapped using receptor autoradiography techniques. By incubating 20 μm tissue sections with 125I-lin-AVP (125I-Phenylacetyl-D-Tyr(Me)-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH 2 linear vasopressin V1A receptor antagonist) AVP binding sites were detected. A V1A agonist ([d(Ch2)5[Tyr(Me)]AVP) displaced the 125I-lin-AVP tracer in all sites, providing support for verification of the sites as V1A receptors. V1A receptors were localized within pyriform, entorhinal, cingulate and insular cortices, the presubiculum, bed nucleus of the stria terminalis (BNST), lateral septum (LS), mammillary bodies and within several structures of the amygdala, hypothalamus, hippocampus, thalamus and brain stem. The rhesus V1A receptor map is relevant to recent research in human learning, memory and emotion. Particularly pertinent are those receptors found in the amygdala, mammillary bodies, medial temporal lobe structures, and cingulate cortex. Comparison of this rhesus V1A receptor map to those previously reported for nonprimates revealed that all had V1A receptors in the BNST, central amygdala, diagonal band of Broca, LS, and solitary tract. Marmosets shared similar binding patterns in the BNST, but densities differed in SCN, LS, and various amygdala and hypothalamus regions. In presubiculum, supraoptic nucleus, cortical regions, and mammillary bodies, areas dense with V1A receptors in rhesus, marmosets revealed none. Such differences may reflect species-specific memory capabilities.

Introduction: Restless legs syndrome (RLS) is a common neurological disorder affecting 10% of the population. Most antidepressants exacerbate symptoms; however, correlational studies have noted symptom improvement with bupropion. The purpose of the current study was to examine whether, in a controlled study, bupropion would improve the symptoms of RLS, or at least not exacerbate them. Methods: This was a double-blinded, randomized controlled trial. Twenty-nine participants with moderate to severe RLS received 150 mg sustained-release bupropion once daily, and 31 control participants received a placebo. Participants were followed for 6 weeks and completed standardized tools, including the International Restless Legs Syndrome Study Group (IRLSSG) severity scale. Results: The primary outcome was change from baseline in IRLSSG severity score; lower scores were associated with improved symptoms. At 3 weeks, IRLSSG scores were 10.8 points lower in the bupropion group and 6.0 points lower in the placebo group (P = .016). At 6 weeks, IRLSSG scores were 10.4 points lower in the bupropion group and 7.6 points lower in the placebo group (P = .108). Bupropion was more effective than placebo in the treatment of RLS at 3 weeks; however, this difference was not statistically significant at 6 weeks. Conclusions: The data from our study suggest that bupropion does not exacerbate the symptoms of RLS and may be a reasonable choice if an antidepressant is needed in individuals with RLS. Larger studies that include titration of bupropion should be considered to determine if bupropion is appropriate for primary treatment of RLS, particularly considering the lower cost and favorable side effect profile compared with currently recommended first-line dopamine agonists.

Includes bibliographical references / Aims: To assess the clinical and cerebrospinal fluid characteristics, and the role of tuberculous meningitis (TBM) as a confounder, in a cohort of HIV positive individuals with positive varicella zoster virus (VZV) positive cerebrospinal fluid PCR. Methods: Patients in the NHLS database at Groote Schuur Hospital with positive CSF VZV PCR who were also HIV co-infected and whose folders were available for clinical review were reviewed. Clinical and biochemical data were collected. Patients were divided into two groups based an accepted case definition for TBM. Differences between groups were assessed using Mann-Whitney U or Chi squared tests as appropriate. Results: There were 437 for VZV PCR over three years. Of these 98 were positive and, after exclusions, 31 HIV positive patients were included for further analysis. Median age was 31 and median CD4 count was 146 cells/mm³. 11 (35%) had meningitis and 8 (25%) had encephalitis. 13 (42%) met the case definition for TBM. Patients with CNS varicella were frequently confused whereas those with TBM presented sub-acutely. There were no differences in CSF characteristics. Additional organisms were detected 6 (19%) patients. 4 (13%) patients died in hospital. CSF TB culture was requested in 24 (77%) patients and extra CNS samples were sent in only 4 patients. Conclusion: The clinical and CSF presentation of CNS Varicella and TBM overlap and in this cohort patients were under investigated for TB. In settings of high TB prevalence the possibility of false positive PCR or incidental varicella reactivation should be considered.

No description available.

Neuromuscular junction (NMJ) dysfunction has been identified as one of the earliest events in Amyotrophic Lateral Sclerosis (ALS) pathology. However, which tissue type induces NMJ disruption; be it the motoneurons (hMN), Schwann cells or skeletal muscle (hSKM) remains unresolved. While mechanisms by which ALS hMN contribute to NMJ dysfunction are well-described in literature, limited information exist on how the other tissue types in the tripartite synapse (hSKM and Schwann cells) induce and/or contribute to ALS NMJ disruption. A fair understanding of the role of each tissue type in NMJ dysfunction would help shape the trajectory of future ALS research and drug discovery. It is generally accepted that the observed ALS hSKM weakness and atrophy are a result of hMN axonal retraction. While recent findings postulate the active pathologic involvement of the hSKM in ALS onset via NMJ disruption, some scientists have questioned the validity of the transgenic model used in most studies and the translatability of their results. Contrarily, in vitro modeling of this phenomenon using patient hSKM samples has proved challenging due to the inability to reliably expand and maintain them before becoming senescent. Thus, this study sought to investigate the pathology of hSKM derived from patient ALS (ALS hSKM), its possible contribution to NMJ dysfunction, and whether hSKM-specific treatment confer any therapeutic benefit to the NMJ. To avoid the main challenges associated with human-based ALS hSKM studies i.e., tissue scarcity and established culturing challenges, patient iPSCs were utilized as the tissue source. This assured a single source for obtaining both cell types necessary for modeling the ALS NMJ. IPSC-derived wild type (WT) and ALS hSKM were differentiated using a serum-free, small molecule-directed protocol which centered on the concurrent modulation of the Wnt and bone morphogenetic protein pathways. To determine whether the ALS hSKM has intrinsic deficits independent of hMN, comparative assessment of WT and ALS hSKM were carried out. The myogenicity of resultant WT and ALS progenitors were confirmed via phase-contrast microscopy, immunocytochemistry, and flow cytometry, after which they were terminally differentiated in myotubes. WT and ALS hSKM were compared morphologically and functionally. The inner mitochondrial membrane potential (ΔΨM) and metabolic plasticity of both WT and ALS hSKM were also evaluated. After the hSKM-only characterization, both ALS and WT hSKM were co-cultured with either ALS or WT hMNs. This was to study how the previously outlined hSKM deficits affect NMJ formation, integrity and function in an effort to delineate the sole contribution to NMJ disruption. With knowledge of the pathologic contribution to NMJ dysfunction, hSKM-specific Creatine treatments were performed to investigate its therapeutic benefit to the ALS NMJ. This project advances the field's knowledge on the hSKM's role to ALS NMJ disruption and creates awareness about considering the hSKM in future research and drug discovery exploits. Additionally, this project resulted in the development of microphysiological platforms that recapitulate known phenotypic parameters of ALS while allowing the independent treatment and/or interrogation of each tissue type in the co-cultures.