Thesis (Ph.D.)--Indiana University, 2009. / Title from screen (viewed on December 1, 2009). Department of Anatomy and Cell Biology, Indiana University-Purdue University Indianapolis (IUPUI). Advisor(s): Zao C. Xu, Feng C. Zhou, Charles R. Yang, Theodore R. Cummins. Includes vitae. Includes bibliographical references (leaves 115-135).

The ability of a neuron to alter its synaptic connections during development is essential to circuit assembly. Synapse remodeling or refinement has been observed in many species and many neuronal circuits, yet the mechanisms defining which neurons undergo remodeling are unclear. Moreover, the molecules that execute the process of remodeling are also obscure. To address this issue, we sought to identify targets of the transcription factor unc-55 COUP-TF, which acts as a cell-specific repressor of synapse remodeling in C. elegans. unc-55 COUP-TF is expressed in VD neurons, where it prevents synapse remodeling. DD neurons can remodel synapses because they do not express unc-55 COUP-TF. Ectopic expression of unc-55 COUP-TF in DD neurons prevents remodeling. We identified the transcription factor Hunchback-like hbl-1 as a target of UNC-55 COUP-TF repression. Differential expression of hbl-1 explains the cell-type specificity of remodeling. hbl-1 is expressed in the DD neurons that are capable of remodeling, and is not expressed in the VD neurons that do not remodel. In unc-55 mutants, hbl-1 expression increases in VD neurons where it promotes ectopic remodeling. Moreover, hbl-1 expression levels bidirectionally regulate the timing of DD remodeling, as increases in hbl-1 cause precocious remodeling while decreases in hbl-1 cause remodeling delays. Finally, hbl-1 coordinates heterochronic microRNA and neuronal activity pathways to regulate the timing of remodeling. Increases or decreases in circuit activity cause increases or decreases in hbl-1 expression, and consequently early or delayed remodeling. Thus, convergent regulation of hbl-1 expression defines a genetic mechanism that patterns activity-dependent synaptic remodeling across cell types and across developmental time. We identified other targets of UNC-55 COUP-TF regulation using gene expression profiling, and implicate some of these factors in the regulation of remodeling using functional genomic screens. Our work suggests roles for conserved networks of transcription factors in the regulation of remodeling. We propose a model in which hbl-1 and other targets of unc-55 COUP-TF transcriptional repression are responsible for regulating synapse remodeling in C. elegans.

Presynaptic terminals of neurons are optimized for neurotransmitter release, which is tightly controlled by presynaptic calcium. Here, we evaluate the role of calcium influx through voltage-gated calcium channels (VGCCs) in regulating the initial vesicular release probability (p) and the number of vesicles available for release by action potentials (effective RRP) at the calyx of Held synapse in mice. Two established methods of estimating effective RRP size and p reveal that both are calcium dependent. Reducing calcium influx by blocking R-type (VGCCs) or P/Q-type VGCCs also reduces EPSC amplitude via p and effective RRP size. Furthermore, activation of gamma-aminobutryic acid class B (GABAB) receptors, which reduces presynaptic calcium by regulating VGCCs without other significant effects on release, also reduces the effective RRP size and p. These findings suggest that the calcium dependence of RRP size may influence the manner in which certain neuromodulators affect neurotransmitter release.

The retinogeniculate synapse, the connection between retinal ganglion cells (RGCs) and thalamic relay neurons, undergoes extensive remodeling and refinement in the first few postnatal weeks. While many studies have focused on this process, little is known about the factors that influence excitatory transmission during this dynamic period. A major goal of my dissertation research was to identify mechanisms that regulate glutamate release and clearance at the developing synapse. First, we investigated the role of glutamate transporters and metabotropic glutamate receptors (mGluRs) in shaping excitatory transmission. Early in development, we found presynaptic group II/III mGluRs are present and are activated by glutamate released from RGCs following optic tract stimulation at natural frequencies. This response was found to diminish with age, but glutamate transporters continued to shape synaptic currents throughout development. The finding that glutamate is able to escape the synaptic cleft and bind extrasynaptic high-affinity mGluRs led us to speculate that glutamate might also diffuse to neighboring synapses and bind ionotropic glutamate receptors opposing quiescent release sites. Excitatory currents recorded from immature, but not mature, retinogeniculate synapses display a prolonged decay timecourse. We found evidence that both asynchronous release of glutamate as well as spillover of glutamate between neighboring synapses contributes to these slowly decaying synaptic currents. Furthermore, we uncovered and characterized a novel, purely spillover-mediated current from immature relay neurons, which strongly supports the presence of glutamate spillover between boutons of different RGCs. The results of my studies indicate that far more RGCs contribute to relay neuron firing than would be predicted by the anatomy alone. Finally, in an ongoing study, we investigated the functional role of the neuronal glutamate transporter GLT-1 at the immature retinogeniculate synapse. While GLT-1 has been found in both neurons and glia, excitatory currents at the retinogeniculate synapse were largely unaffected in mice lacking neuronal GLT-1, suggesting non-neuronal glutamate transporters are responsible for the majority of glutamate removal from the developing synapse. Taken together, these results provide insight into the synaptic environment of the developing retinogeniculate synapse and identify a number of mechanisms that shape excitatory transmission during this period of synaptic maturation and refinement.

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Nervenzellen sind hochspezialisierte Zellen, die an Synapsen miteinander verbunden sind, was die Übertragung von neuronalen Informationen erlaubt. Die Entwicklung von Synapsen und die Informationsverarbeitung und Gedächtnisbildung bei reifen Synapsen erfordert eine dynamische Umorganisation von neuronalen Netzwerken. Das beinhaltet die Bildung und Entfernung von Synapsen, Umsatz von synaptischen Proteinen und die Veränderung und Anpassung von synaptischer Erregungsübertragung. U. a. Ubiquitinierung, als regulatorische, posttranslationale Modifikation von Proteinen, könnte eine entscheidende Rolle für solche komplexe, synaptische Umorganisationen spielen. Nedd4-1, eine HECT-Typ E3 Ubiquitin Ligase, reguliert und fördert die Entwicklung von Nervenzellfortsätzen durch die Ubiquitinierung von Rap2. Um die Bedeutung von Nedd4-abhänginger Ubiquitinierung im entwickelten Gehirn zu untersuchen, wurden Mausmodelle generiert und analysiert, in denen Nedd4-1 und dessen nächstes Homolog Nedd4-2, speziell in Nervenzellen ausgeschaltet wurde. Ich habe herausgefunden, dass Nedd4-1 und Nedd4-2 wichtige regulatorische Proteine für die neuronale Morphogenese und die synaptische Plastizität, insbesondere die Aufrechterhaltung von LTP, darstellen. Desweiteren habe ich festgestellt, dass Synaptopodin (SYNPO), ein Prolin-reiches, Aktin-assoziiertes Protein, von Nedd4-1 und Nedd4-2 in vitro ubiquitiniert wird. Dieses Ergebnis deutet daraufhin, dass SYNPO in dem Mechanismus eine Rolle spielt, durch den Nedd4-1 und Nedd4-2 LTP aufrechterhalten. Diese Studie wirft ein neues Licht auf die funktionelle Rolle von Nedd4-abhänginger Ubiquitinierung bei höheren Funktionen des Gehirns von Säugetieren sowie der neuronalen Entwicklung.

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No description available.

The aim of this thesis is to develop a novel technique for the estimation of firing rate dynamics from single-unit recordings of neural pulse trains. This method applies an offline digital filtering technique to extract information transmitted by a neuron in teens of a rate code. While there is increasing evidence that the traditional rate coding cannot account for all the information transmitted by a cell, and that information may also be contained in the precise timing of spikes, the firing rate signal remains the benchmark by which the vast majority of electrophysiological studies relating neural activity to functional behaviour have been interpreted. Nevertheless, there does not seem to be an agreement on a single definition of a rate code let alone a consensus on an optimal estimation method. This study raises significant concerns about the validity of some of the most common methods in systems neuroscience, and proposes a simple yet more robust alternative. This latter is based on the convolution of the spike train with an optimally designed Kaiser window. Using computer-simulated as well as experimental data obtained from single-unit recordings of vestibular canal afferents, the proposed technique is shown to consistently outperform the current methods and even to permit robust estimations under time-varying conditions. These results suggest that estimates acquired with the conventional methods are biased and hence models of neural dynamics based on these latter may not be reliable.

The effects of alpha1A subunit mutations on presynaptic Ca2+ channel activity and functional development of synaptic properties remain elusive. The calyx of Held/medial nucleus of the trapezoid body synapse is an ideal model for studying the developmental effects of presynaptic voltage-gated Ca2+ channel (VGCC) impairment on synaptic function since simultaneous voltage-clamp recordings can be made directly from the pre- and postsynapse. The alpha1A subunit leaner (tgla/la) mutation induced a profound reduction in synaptic transmission after hearing onset (> postnatal day 12; P12), with relatively preserved relationship between presynaptic Ca2+ current (Pre-ICa) and release and G-protein-mediated inhibition. Some synaptic properties were more reflective of an immature state, while other properties displayed a delay in maturation after P12. Direct presynaptic recordings from P15/16 tgla/la nerve terminals revealed a decrease in the density of Pre-ICa, elevated activation threshold and slowing in the kinetics of VGCCs, all of which contribute to the deficit in transmitter release. Fractional contribution of P/Q-type channels to total Pre-ICa and their role in vesicle release was markedly reduced. N-type Ca2+ channels and close association of VGCCs to release sites was not sufficient to fully compensate for impaired P/Q-type channel function. The extent to which compensatory mechanisms preserve synaptic transmission at tgla/la synapses was further constrained by the developmental narrowing of the action potential waveform. Activation of the cAMP pathway by forskolin or direct modulation of VGCCs by cdk inhibitors rescued deficits in transmitter release at P15/16 tgla/la synapses. The major effect of roscovitine was a slowing of presynaptic VGCC deactivation kinetics accompanied by a leftward shift in the activation curve. Activation of the cAMP pathway or direct modulation of presynaptic VGCCs may serve as two potential pathways to facilitate release and improve neuronal communication at synapses normally compromised by impaired P/Q-type channel function. While significant for the tgla/la mutant, these studies provide an important advancement in our understanding of the crucial developmental and functional roles of P/Q-type Ca2+ channels in driving the maturation of synaptic properties at central synapses. These findings may improve our understanding of the pathophysiology of presynaptic VGCCs and elucidate essential mechanisms underlying the tgla/la phenotype.