Previously, we found that 5-25 μg/ml safrole oxide could inhibit apoptosis and dramatically make a morphological change in human umbilical vein vascular endothelial cells (HUVECs). But the possible mechanism by which safrole oxide function is unknown. To answer this question, in this study, we first investigated the effects of it on the activity of nitric oxide synthetase (NOS), the expressions of Fas and integrin β4, which play important roles in HUVEC growth and apoptosis, respectively. The results showed that, at the low concentration (10 μg/ml), safrole oxide had no effects on NOS activity and the expressions of Fas and integrin β4. Then, we investigated whether HUVECs underwent differentiation. We examined the expressions of neuron-specific enolase (NSE) and neurofilament-L (NF-L). Furthermore, we analyzed the changes of intracellular reactive oxygen species (ROS). After 10 h of treatment with 10 μg/ml safrole oxide, some HUVECs became neuron-like cells in morphology, and intensively displayed positive NSE and NF-L. Simultaneously, ROS levels dramatically decreased during HUVECs differentiation towards neuron-like cells. At the low concentration, safrole oxide induced HUVECs differentiation into neuron-like cells. Furthermore, our data suggested that safrole oxide might perform this function by depressing intracellular ROS levels instead of by affecting cell growth or apoptosis signal pathways.

Indiana University-Purdue University Indianapolis (IUPUI) / The serotonergic system in the brain is necessary for neurophysiological processes related to mood, sleep, and cognitive regulation. This system is primarily regulated through the transport of extracellular serotonin (5-HT) into neuron terminals by the serotonin transporter (SERT). The activity of SERT is thought to be modulated in part by cholesterol and lipid rich microdomains within the plasma membrane where SERT localizes. However, experiments related to the mechanism of membrane cholesterol on SERT function in the brain has yielded conflicting results and no studies have examined the contribution of cholesterol biosynthetic intermediates in regulating SERT function. To address this knowledge gap, this dissertation examined the neuropharmacological effects of the highly prescribed cholesterol-lowering statin drugs on SERT-dependent 5- HT uptake into neurons. Unexpectedly, statin treatment increased SERT-dependent 5-HT uptake in a neuron cell model, and increased in vivo 5-HT content in synaptosomes. The mechanistic findings demonstrated that (1) statins enhanced activity of SERT rather than altered distribution at the membrane, (2) statins increased 5-HT uptake in a manner that is independent of cholesterol per se but is mediated in part by the cholesterol biosynthetic intermediates of the isoprenylation pathway, namely farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), (3) direct inhibition of the isoprenylation pathway through inhibition of GGPP enzyme geranylgeranyl transferase (GGT) also increased 5-HT uptake in a SERT-dependent manner, and (4) increased 5-HT uptake by statins or GGT inhibition was dependent on Ca2+/calmodulin-dependent protein kinase (CAMKII). Our results provide a novel role for lipid signaling in regulating SERT and a newly identified function of the isoprenylation pathway in the brain. These results also provide a possible explanation for the adverse neurological effects associated with the widely prescribed statin drugs.

This paper provides a fully automated method of finding high-quality seed points in 3D space from a stack of images of neuron cells. These seed points may then be used as initial starting points for automated local tracing algorithms, removing a time consuming required user interaction in current methodologies. Methods to collapse the search space and provide rudimentary topology estimates are also presented. / Master of Science

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Die Aktionspotential (AP) -Aktivität einzelner kortikaler Neuronen kann messbare sensorische Effekte hervorrufen. Es ist jedoch nicht bekannt, wie AP-Sequenzen Parameter und spezifische neuronale Subtypen die hervorgerufenen Sinnesempfindungen beeinflussen. Hier haben wir einen ‘Reverse-Physiology‘ Ansatz angewendet, um die Beziehung zwischen der Aktivität einzelner Neuronen und der Empfindung zu untersuchen. Zunächst wird der Prozess der Nanostimulation, eine von der juxtazellulären Markierungstechnik abgeleiteten Einzelzell-Stimulationsmethode, detailliert beschrieben. Nanostimulation ist einfach anzuwenden und kann auf eine Vielzahl von identifizierbaren Neuronen in narkotisierten und wachen Tieren angewandt werden. Wir beschreiben die Aufnahmetechnik und die elektrische Konfiguration für Nanostimulation. Während eine exakte zeitliche Bestimmung der AP nicht erreicht wurde, konnten Frequenz und Anzahl der AP parametrisch kontrolliert werden. Wir zeigen, dass Nanostimulation auch angewendet werden kann, um sensorische Reaktionen in identifizierbaren Neuronen selektiv zu inhibieren. Als nächstes haben wir untersucht wie sich die Frequenz und Anzahl der AP sowie die Regelmäßigkeit der Pulsfolge auf die Detektion von Einzelzell-Stimulationen im somatosensorischen Kortex von Ratten auswirken. Für mutmaßlichen erregende regular-spiking Neuronen erhöhte sich die Nachweisbarkeit mit abnehmender Frequenz und Anzahl der AP. Die Stimulation einzelner, mutmaßlichen inhibitorischer und schnell feuernder Neuronen führte zu wesentlich stärkeren sensorischen Effekten, die unabhängig von Frequenz und Anzahl der AP waren. Außerdem fanden wir heraus, dass Unregelmäßigkeiten der Pulsfolge die sensorischen Effekte von putativ erregenden Neuronen stark erhöhten. Diese Unregelmäßigkeiten wurden in durchschnittlich 8% der Durchgänge festgestellt. Unsere Daten deuten darauf hin, dass das es auf Verhaltnisebene eine große Sensivität für kortikale AP und deren zeitlichen Abfolge gibt. / The action potential (AP) activity of single cortical neurons can evoke measurable sensory effects, but it is not known how spiking parameters and specific neuronal subtypes affect the evoked sensations. Here we applied a reverse physiology approach to investigate the relationship between single neuron activity and sensation. First, we provide a detailed description of the procedures involved in nanostimulation, a single-cell stimulation method derived from the juxtacellular labeling technique. Nanostimulation is easy to apply and can be directed to a wide variety of identifiable neurons in anesthetized and awake animals. We describe the recording approach and the parameters of the electric configuration underlying nanostimulation. While exact AP timing has not been achieved, AP frequency and AP number can be parametrically controlled. We demonstrate that nanostimulation can also be used to selectively inhibit sensory responses in identifiable neurons. Next, we examined the effects of AP frequency, AP number and spike train regularity on the detectability of single-cell stimulation in rat somatosensory cortex. For putative excitatory, regular spiking neurons detectability increased with decreasing AP frequencies and decreasing AP numbers. Stimulation of single putative inhibitory, fast spiking neurons led to much larger sensory effects that were not dependent on AP frequency and AP number. In addition, we found that spike train irregularity greatly increased the sensory effects of putative excitatory neurons, with irregular spike trains being detected in on average 8% of trials. Our data suggest that the behaving animal is extremely sensitive to cortical APs and their temporal patterning.

Microscopic analysis of biological structures can be significantly enhanced by representing the object of study as a three-dimensional entity. To assist neurobiologists investigate the molecular mechanisms involved in neurite formation requires an adequate visual model or at least some measurable data. Reconstruction helps analysis of biological structures by representing the object of study as a three-dimensional entity. It helps gain insight into the morphological variation observed in each class of neurons and for simulations of neuronal behavior. To perform the reconstruction, biologists today have to rely on time-consuming manual or semi-manual methods which either doesn't exhibit robustness against noise of microscopy images or fail to capture precise dendritic structures, thus necessitating the need of fully-automated reconstruction methods for neuronal structures. In our work, we designed a framework with the goal of enabling automation and yet produce flexible outputs to ensure a high quality reconstruction with minimal user intervention. Our framework is also not bound by varying contrast, size or resolution of data, thus capable of working on data obtained from wide variety of acquisition methods and neuronal structures.

Poster exhibited at GPSC Student Showcase, February 24th, 2016, University of Arizona. Recipient of the 2016 Katheryne B. Willock Library Research Award. / The nervous system is composed of two types of cells: neurons and glia. In neuronal circuits, neurons communicate through synapses and glia play a crucial modulatory role. To modulate chemical reuptake, glia send processes close to synapses and many glia directly appose or ensheathe a synapse. This structural motif is one of the elements often included in describing a vertebrate tripartite synapse, which includes a bidirectional functional neuron-glia relationship. The exact nature of this neuron-glia communication is not well understood. In the invertebrate fruit fly, we have also found that particular neurons and glia also have a bidirectional functional relationship. This allows us to ask new questions about glial morphology. Throughout multiple images, I identified particular neuronal synapses and surrounding glia. After creating a 3D reconstruction, I measured the distance between a particular neuronal synapse and its closest glial process. Interestingly, the neuronal synapses were not directly apposed or ensheathed by glia, and the distance to the closest glial process varied one-hundred-fold. With variable distance, functional communication is consistently present. These findings provide important insight into invertebrate neuron-glia communication, and offer new avenues to investigate the structural neuron-glia relationships that are required for reciprocal signaling between the two cell classes.

Polyglutamine expansion disorders are progressive neurodegenerative diseasesthat are caused by the pathological expansion of polyglutamine repeats. Huntington'sdisease (HD) is a polyglutamine disorder caused by the expansion of an existingpolyglutamine tract in a novel protein, Huntingtin (Htt). Oxidative stress has beenimplicated in the neural dysfunction observed in multiple neurodegenerative conditionsincluding HD. The tumor suppressor p53 is a multifunctional protein that has roles inthe cell cycle, apoptosis and neurodevelopment. The role of p53 in HD-associatedneurodegeneration has been studied but not fully elucidated, nor has the role of p53 inoxidative stress toxicity been fully elucidated.Here I present work that demonstrates polyglutamine expansion inducedalterations to p53 stability, localization, and activity. The transcriptional activity of p53was found to have a role in oxidative stress mediated as well as polyglutaminemediated neurotoxicity in vitro. The expression of p53 was also altered in vivo in amouse model of HD as well as in HD brain.Taken together, these data demonstrate a role for p53 in polyglutamine and oxidativestress toxicity.

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No description available.