Thesis (Ph.D. in Neuroscience) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 84-93). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

A clear definition of the mechanisms involved in synaptic transmission is of paramount importance for the understanding of the processes governing synaptic efficacy. Despite decades of intense study, these mechanisms remain poorly understood. The work contained in this thesis examines several such mechanisms using the hypothalamic-neurohypophysial system (HNS), a classical preparation for the study of Ca2+-dependent neuropeptide release. The first portion of this thesis is comprised of my efforts to define the cellular machinery essential for the exocytosis of secretory granules isolated from peptidergic neurohypophysial terminals of the HNS. Here, using the planar lipid bilayer model system, I have been able to show that syntaxin alone in the target membrane is sufficient to elicit fusion of modified neurohypophysial secretory granules. Surprisingly, SNAP-25 does not appear to be necessary for this process. This suggests that syntaxin may be able to substitute for SNAP-25 to form functional non-cognate fusion complexes. Additionally, the coupling of amperometric detection with the planar lipid bilayer system has allowed me to confirm these results using native, unmodified secretory granules, and also provides some insight into the kinetics of release in this reconstituted system. This model system should provide a convenient means for the study of additional regulatory factors believed to be involved in secretory vesicle exocytosis. The second and third sections of this thesis involve my examination of the role of presynaptic Ca2+ stores in neuropeptide secretion from isolated peptidergic neurohypophysial terminals (NHT). I initially examined the source of recently discovered ryanodine-sensitive Ca2+ stores in this system. Using Immuno-electron microscopy I have found that ryanodine receptor (RyR) labeling appears to co-localize with large dense core granules. Additionally, I have shown that a large conductance cation channel, with similarities to the RyR, found in the membrane of these granules has the same characteristic response to pharmacological agents specific for the RyR. Further, application of RyR agonists modulates basal neuropeptide release from NHT. These results suggest that the large dense core granules of NHT serve as the source of a functional ryanodine-sensitive Ca2+store. Recent work has revealed that spark-like Ca2+ transients, termed syntillas, can be observed in NHT. These syntillas arise from ryanodine-sensitive intracellular stores. In other neuronal preparations, similar Ca2+ transients have been suggested to affect spontaneous transmitter release. However, such a role for syntillas had yet to be examined. To assess if syntillas could directly trigger spontaneous release from NHT, I used simultaneous Ca2+imaging along with amperometric detection of release. Amperometry was adapted to this system via a novel method of false-transmitter loading. Using this approach I have found no apparent correlation between these two events, indicating that syntillas are unable to directly elicit spontaneous transmitter release. As this finding did not rule out an indirect modulatory role of syntillas on release, I additionally present some preliminary studies examining the ability of ryanodine-sensitive Ca2+ release to modulate vesicular priming. Using immunocytochemistry, I have shown that RyR agonist treatment shifts the distribution of neuropeptides toward the plasma membrane in oxytocinergic NHT, but not in vasopressinergic NHT. RyR antagonists have the opposite affect, again only in oxytocinergic NHT. Further, I have found that application of RyR agonists result in a facilitation of elicited release in NHT using membrane capacitance recording. This facilitation appears to be due primarily to an increase in recruitment of vesicles to the readily-releasable pool. These findings suggest that ryanodine-sensitive Ca2+stores may be involved in vesicular priming in NHTs. Taken together, the work presented in this thesis provides some new and interesting insights into the underlying mechanisms and modulation of transmitter release in both the HNS and other CNS terminals.

A key process underlying synapse development and plasticity is stimulus-dependent translation of localized mRNAs. This process entails RNA packaging into translationally silent granules and exporting them over long distances from the nucleus to the synapse. Little is know about (a) where ribonucleoprotein (RNP) complexes are assembled, and if in the nucleus, how do they exit the nucleus; (b) how RNPs are transported to specific synaptic sites. At the Drosophila neuromuscular junction (NMJ), we uncovered a novel RNA export pathway for large RNP (megaRNP) granules assembled in the nucleus, which exit the nucleus by budding through the nuclear envelope. In this process, megaRNPs are enveloped by the inner nuclear membrane (INM), travel through the perinuclear space as membrane-bound granules, and are deenveloped at the outer nuclear membrane. We identified Torsin (an AAA-ATPase that in humans is linked to dystonia), as mediator of INM scission. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the mRNAs fail to localize to postsynaptic sites leading to abnormal NMJ development. We also found that nuclear envelope budding is additionally used for RNP export during Drosophila oogenesis. Our studies also suggested that the nuclear envelope-associated protein, Nesprin1, forms striated F-actin-based filaments or ‘‘railroad tracks,’’ that span from muscle nuclei to postsynaptic sites at the NMJ. Nesprin1 railroad tracks wrap aoround the postsynaptic regions of immature synaptic boutons, and serve to direct RNPs to sites of new synaptic bouton formation. These studies elucidate novel cell biological mechanisms for nuclear RNP export and trafficking during synapse development.

化学シナプスにおいて、シナプス小胞内に蓄えられた神経伝達物質の開口放出によってシナプス伝達が起こる。開口放出後、シナプス小胞はエンドサイトーシスによって細胞内へ回収されて再利用される。膜容量測定法とpHイメージングを併用して、開口放出とエンドサイトーシスに伴う小胞膜および小胞タンパク質の動態を同時測定した。Ca2+-カルモジュリン-Munc13シグナリングが小胞タンパク質の回収に関与していることが示唆された。 / Neurotransmitter is released at synapses by fusion of synaptic vesicles with the plasma membrane. To sustain synaptic transmission, membranes and vesicular proteins has to be retrieved for reuse. I have combined capacitance measurements and pH-imaging via a pH-sensitive vesicular protein marker, and compared the retrieval kinetics of membranes and synaptotagmin2 (Syt2) at the calyx of Held presynaptic terminal. Data in this thesis identifies a novel mechanism of stimulus- and Ca2+-dependent regulation of coordinated endocytosis of synaptic membranes and Syt2. / 博士(理学) / Doctor of Philosophy in Science / 同志社大学 / Doshisha University

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Antecedendo movimentos voluntariamente gerados, existe atividade neuronal encefálica que se inicia alguns segundos antes da execução deste movimento. Esta atividade preparatória é responsável pela elaboração de um plano de execução que alcança a via final comum para realização de um ato motor voluntário, os motoneurônios. Entretanto, na última década, evidências apontam para a participação de circuitos neuronais na medula espinhal apresentando padrão de atividade similar aos padrões observados em áreas encefálicas e que, possivelmente, estaria relacionado a uma atividade preparatória para o movimento voluntariamente gerado. Por este motivo, o presente trabalho teve por objetivo verificar a atividade de circuitos neuronais na medula espinhal durante diferentes instantes de proximidade da ação voluntariamente gerada em paradigma de tarefa motora com período de instrução. Para isso, inicialmente, 15 sujeitos saudáveis, sem histórico de doença neuromuscular foram submetidos ao protocolo experimental. O protocolo experimental constituiu-se do processo de recrutamento dos sujeitos, sua preparação para o ensaio dentro do ambiente experimental, bem como as orientações necessárias para execução dos procedimentos e paradigmas. Os procedimentos referem-se às etapas realizadas para captação do reflexo H, bem como desta captação sob a influência de técnica de condicionamento por inibição pré-sináptica. Essa captação ocorreu em janelas de aquisição em que o sujeito encontrava-se em repouso e em três instantes de expectativa para a execução de ação voluntária, estando o músculo sóleo atuando como agonista (flexão plantar) ou antagonista (dorsiflexão), em paradigma de tarefa motora voluntária com período de instrução. Após os registros, por meio de processamento dos sinais coletados, foi possível se calcular a amplitude pico-a-pico do reflexo H nas diferentes condições experimentais de proximidade da execução (1000, 600 e 200 milissegundos) e de atuação do músculo sóleo (agonista e antagonista) que foi usado para: (1) análise da variação da excitabilidade reflexa, em porcentagem da onda M máxima, (2) análise da ocorrência de inibição pré-sináptica e (3) análise da variação da inibição pré-sináptica, em porcentagem de inibição. Os resultados mostram que a porcentagem da onda M máxima aumentou significativamente nos três instantes de proximidade com os sujeitos estando em expectativa da execução da tarefa motora quando o músculo sóleo atuaria como agonista da contração, quando comparados com os registros obtidos nas mesmas condições em repouso. Contudo, somente a 200 ms da execução é que foi observado aumento da porcentagem da onda M máxima quando o músculo sóleo atuaria como antagonista. Inibição pré-sináptica ocorreu em todas as condições experimentais, contudo aumento significativo da porcentagem de inibição pré-sináptica foi somente observado a 200 ms da execução da tarefa motora em que o músculo sóleo atuaria como antagonista. Diferenças entre agonista e antagonista com relação ao padrão de excitabilidade reflexa foi somente observado a 600 ms de proximidade da execução da tarefa e essas diferenças com relação à porcentagem de inibição pré-sináptica foi somente detectada a 200 ms. Nossos resultados nos permitem concluir que circuitos neuronais na medula espinhal apresentam atividade no período preparatório para a execução de tarefa motora voluntária que podem estar relacionadas ao comportamento de expectativa da realização de uma ação motora eminente, bem como relacionada ao planejamento motor para a ação a longa proximidade da execução de movimentos. / There is brain activity preceding voluntary movements a few seconds before the execution of the movement. This preparatory activity is responsible for the execution plan that reaches the final common pathway, i.e., the motoneurons. In the last decade, there have been reports indicating the involvement of spinal cord circuits in the preparatory activity for movement. The present work has the objective of verifying the activity of spinal cord neuronal circuits at different times preceding a voluntary action, under an instructed delay period paradigm. Fifteen healthy subjects participated in the study. The protocol included an explanation of the experimental tasks. Electrophysiological recordings of the H reflex with and without presynaptic inhibition conditioning were employed. The epochs of H reflex recording were associated either with a resting period or with one of three pre-action periods. The subject received a cue at an appropriate time about the type of contraction: plantarflexion or dorsiflexion. Peak to peak H reflex values were computed in the control resting period and at 1000 ms, 600 ms and 200 ms before the action. Percent values of H amplitude with respect to maximum M values were computed as well as the level of presynaptic inhibition. The results have shown that the relative H reflex value increased significantly at the three premovement times for the soleus under an agonist contraction (i.e., plantarflexion) when compared to control. However, when the soleus was an antagonist to the contraction (i.e., dorsiflexion) there was a statistical difference in the H amplitude only at 200 ms before movement. Presynaptic inhibition occurred in all experimental conditions, however only at 200 ms before contraction there was a significant increase. Differences in reflex excitability between agonist and antagonist activity were only observed at 600 ms before action. On the other hand, differences in presynaptic inhibition were only found at 200 ms before contraction. The results indicated that spinal cord neuronal circuits are activated during the preparatory period preceding a voluntary action. These may be correlated with an expectancy behavior for the execution of an imminent motor action and also with the planning of a motor action at larger times preceding movement execution.

A new, biologically plausible model of task-set learning that reproduces effects from both rule-learning experiments and task-switching experiments.<br>

Background: Light-adaptation is a multifaceted process in the retina that helps adjust the visual system to changing illumination levels. Many studies are focused on the photochemical mechanism of light-adaptation. Neural network adaptation mechanisms at the photoreceptor synapse are largely unknown. We find that large, spontaneous Excitatory Amino Acid Transporter (EAATs) activity in cone terminals may contribute to cone synaptic adaptation, specifically with respect to how these signals change in differing conditions of light. EAATs in neurons quickly transport glutamate from the synaptic cleft, and also elicit large thermodynamically uncoupled Cl- currents when activated. We recorded synaptic EAAT currents from cones to study glutamate-uptake events elicited by glutamate release from the local cone, and from adjacent photoreceptors. We find that cones are synaptically connected via EAATs in dark ; this synaptic connection is diminished in light-adapted cones. Methods: Whole-cell patch-clamp was performed on dark- and transiently light-adapted tiger salamander cones. Endogenous EAAT currents were recorded in cones with a short depolarization to -10mV/2ms, while spontaneous transporter currents from network cones were observed while a local cone holding at -70mV constantly. DHKA, a specific transporter inhibitor, was used to identify EAAT2 currents in the cone terminals, while TBOA identified other EAAT subtypes. GABAergic and glycinergic network inputs were always blocked with picrotoxin and strychnine. Results: Spontaneous EAAT currents were observed in cones held constantly at -70mV in dark, indicating that the cones received glutamate inputs from adjacent photoreceptors. These spontaneous EAAT currents disappeared in presence of a strong light, possibly because the light suppressed glutamate releases from the adjacent photoreceptors. The spontaneous EAAT currents were blocked with TBOA, but not DHKA, an inhibitor for EAAT2 subtype, suggesting that a / non-EAAT 2 subtype may reside in a basal or perisynaptic area of cones, with a specialized ability to bind exocytosed glutamate from adjacent cones in dark. Furthermore, these results could be artificially replicated by dual-electrode recordings from two adjacent cones. When glutamate release was elicited from one cone, the TBOA-sensitive EAAT currents were observed from the other cone. Conclusions: Cones appear to act like a meshwork, synaptically connected via glutamate transporters. Light attenuates glutamate release and diminishes the cone-cone synaptic connections. This process may act as an important network mechanism for cone light adaptation. / by Matthew JM Rowan. / Thesis (Ph.D.)--Florida Atlantic University, 2011. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2011. Mode of access: World Wide Web.

Diss. (sammanfattning) Umeå : Umeå universitet, 2009. / Härtill 4 uppsatser. Även tryckt utgåva.