<p>Learning and memory is one of the critical components of the human experience. In one model of memory, hippocampal LTP, it is believed that the trafficking of AMPA receptors to the synapse is a fundamental process, yet the spatiotemporal kinetics of the process remain under dispute. In this work, we imaged the trafficking of AMPA receptors by combining two-photon glutamate uncaging on single spines with a fluorescent reporter for surface AMPA receptors. We found that AMPA receptors are trafficked to the spine at the same time as the spine size is increasing. Using a bleaching protocol, we found that the receptors that reach the spine come from a combination of the surface and endosomal pools. Imaging exocytosis in real time, we found that the exocytosis rate increases briefly (~1 min.), both in the spine and neighbouring dendrite. Finally, we performed pharmacological and genetic manipulations of signaling pathways, and found that the Ras-ERK signaling pathway is necessary for AMPAR exocytosis.</p> <p>In a set of related experiments, we also investigated the capacity of single spines to undergo potentiation multiple times. By stimulating spines twice using glutamate uncaging, we found that there is a refractory period for synaptic plasticity in spines during which they cannot further be potentiated. We furthermore found that inducing plasticity in a given spine inhibits plasticity at nearby spines.</p> / Dissertation

Thesis (Ph. D.)--Case Western Reserve University, 2004. / [School of Medicine] Department of Neurosciences. Includes bibliographical references. Available online via OhioLINK's ETD Center.

Thesis (Ph. D.)--University of Missouri--Columbia, 2002. / Typescript. Vita. Includes bibliographical references (leaves 121-148). Also issued on the Internet.

Immune molecules, including complement proteins, C1q and C3, have emerged as critical mediators of synaptic refinement and plasticity. Complement proteins localize to synapses and refine the developing retinogeniculate system via C3-dependent microglial phagocytosis of synapses. Retinal ganglion cells (RGCs) express C1q, the initiating protein of the classical complement cascade, during retinogeniculate refinement; however, the signals controlling C1q expression and function remain elusive. RGCs grown in the presence of astrocytes significantly upregulated C1q compared to controls, implicating an astrocyte-derived factor in neuronal C1q expression. A major goal of my dissertation research was to identify the signals that regulate C1q expression and function in the developing visual system. In this study, I have identified transforming growth factor beta \((TGF-\beta)\), an astrocyte-secreted cytokine, as both necessary and sufficient for C1q expression in RGCs through an activity-dependent mechanism. Specific disruption of retinal \(TGF-\beta\) signaling resulted in a significant reduction in the deposition of C1q and downstream C3 at retinogeniculate synapses and significant synaptic refinement defects in the retinogeniculate system. Microglia engulfment of RGC inputs in the lateral geniculate nucleus (LGN) was also significantly reduced in retinal \(TGF\beta\)RII KOs, phenocopying the engulfment defects observed in C1q KOs, C3 KOs, and CR3 KOs. Interestingly, in C1q KOs and retinal \(TGF\beta\)RII KOs, microglia also failed to preferentially engulf less active inputs when retinal activity was manipulated, suggesting that retinal activity and \(TGF-\beta\) signaling cooperatively regulate complement mediated synaptic refinement. In support of this hypothesis, blocking spontaneous activity in RGC cultures significantly reduced C1q upregulation by \(TGF-\beta\). Moreover, manipulating spontaneous retinal activity in vivo modulated C1q expression levels in RGCs and C1q deposition in the LGN. Together these findings support a model in which retinal activity and \(TGF-\beta\) signaling control expression and local release of C1q in the LGN to regulate microglia-mediated, complement-dependent synaptic pruning. These results provide mechanistic insight into synaptic refinement and, potentially, pathological synapse loss which occurs in the early stages of neurodegenerative diseases concurrently with aberrant complement expression and reactive gliosis.

Addiction is thought to arise, in part, from a maladaptive learning process in which enduring memories of drug-related experiences are formed, resulting in persistent and uncontrollable drug-seeking behavior. However, it is well known that both acute and chronic alcohol (ethanol) exposures impair various types of learning and memory in both humans and animals. Consistent with these observations, both acute and chronic exposures to ethanol suppress synaptic plasticity, the major neural substrate for learning and memory, in multiple brain areas. Therefore, it remains unclear how powerful memories associated with alcohol experience are formed during the development of alcoholism. The mesolimbic dopaminergic system is critically involved in the learning of information related to rewards, including drugs of abuse. Both natural and drug rewards, such as ethanol, cause release of dopamine in the nucleus accumbens and other limbic structures, which is thought to drive learning by enhancing synaptic plasticity. Accumulating evidence indicates that plasticity of glutamatergic transmission onto dopamine neurons may play an important role in the development of addiction. Plasticity of NMDA receptor (NMDAR)-mediated transmission may be of particular interest, as NMDAR activation is necessary for dopamine neuron burst firing and phasic dopamine release in projection areas that occurs in response to rewards or reward-predicting stimuli. NMDAR plasticity may, therefore, drive the learning of stimuli associated with rewards, including drugs of abuse. This dissertation finds that repeated in vivo ethanol exposure induces a metaplasticity of NMDAR-mediated transmission in mesolimbic dopamine neurons, expressed as an increased susceptibility to the induction of NMDAR LTP. Enhancement of NMDAR plasticity results from an increase in the potency of inositol 1,4,5- trisphosphate (IP3) in producing the facilitation of action potential-evoked Ca2+ signals critical for LTP induction. Interestingly, amphetamine exposure produces a similar enhancement of IP3R function, suggesting this neuroadaptation may be a common response to exposure to multiple drugs of abuse. Additionally, ethanol-treated mice display enhanced learning of cues associated with cocaine exposure. These findings suggest that metaplasticity of NMDAR LTP may contribute to the formation of powerful memories related to drug experiences and provide an important insight into the learning component of addiction. / text

Drosophila melanogaster is an excellent model system to identify genes involved in synaptic growth and function. In Drosophila, the Serrate (Ser) gene encodes a transmembrane protein that is a ligand for Notch receptor. Several previous studies implicated a role for Serrate in normal wing development and patterning. In this study, I demonstrate that Serrate is required for normal synaptic growth and function. I characterized the phenotype of a Serrate mutation (serB936) that was identified by an EMS-induced genetic screen aimed at identifying novel genes that play a role in synaptic growth and function. Co-localization studies show that Serrate protein is expressed at both the pre- and postsynaptic side of larval neuromuscular junctions (NMJs). Mutations in ser impair synaptic transmission at larval NMJs. This defect is entirely presynaptic, as nerve-evoked excitatory junction potentials (EJP) and quantal content (QC) of neurotransmitter release are significantly reduced when compared to wild-type control. Further, mutations in ser also alter the growth of the NMJ and the underlying muscle. Mutations in ser significantly reduce the size of larval body wall muscles (length and surface area) as well as the number and size of synaptic boutons, and the number of secondary axonal branches. Ubiquitous or muscle-specific expression of normal Serrate in serB936 mutants restores a normal muscle size but not a normal size and structure of the innervating NMJ. However, expression of normal Serrate in the motor axon restores a normal number of synaptic boutons and secondary branches at serB936 mutant NMJs. In addition, it restores normal neurotransmitter release. These data suggest that Serrate protein is required presynaptically for normal synaptic growth and function. Interestingly, overexpression of Serrate in a wild type background resulted in similar phenotypes than to those of loss-of-function mutants. In conclusion, these data suggest a new functional role for Serrate in synaptic growth and function.

Synaptic transmission occurs as a result of either a spontaneous release of presynaptic vesicles or a batch release of presynaptic vesicles driven by action potentials. The physiological consequence of synaptic transmission driven by different patterns and frequencies of presynaptic stimulation has been extensively investigated. However, the physiological nature, mechanism as well as relevance of prolonged presynaptic stimulation have been poorly characterized. In this dissertation, I present three projects in which prolonged stimulation of synaptic transmission in different forms and different brain regions was studied for its effect on synaptic transmission, mechanisms and physiological relevance. In the first project, prolonged electrical stimulation (100 sec) at high frequency induced a deep synaptic depression in acute hippocampal slices, followed by a recovery of synaptic transmission after ~15 min. The deep synaptic depression was attributed to a complete depletion of presynaptic vesicle pools. In the second project, attempts were made to characterize the mechanism of nuclear activation of gene transcription induced by prolonged electrical stimulation (100 sec). Our results demonstrated that reduced inactivation of non-L-type calcium channels failed to provide calcium required for gene transcription, leaving the activation of gene transcription a selective function for L-type calcium channels. In the third project, we sought to study the physiological relevance of enhanced miniature events of inhibitory synapses induced by prolonged chemical stimulation. We showed that prolonged application (2 min) of nicotine to the striatal slice enhanced the frequency of miniature inhibitory currents that was accompanied with a reduction in the amplitude of evoked response. This reduction in the amplitude of evoked responses was ascribed to a compromised action potential invasion of presynaptic terminals possibly due to inactivation of sodium channels resulting from nicotine-induced depolarization. To summarize, prolonged stimulation of presynaptic vesicle release imposes significant influence upon neuron-to-neuron communication, with distinct mechanisms in different brain regions.

NCA-1 (putative nematode calcium channel) and NCA-2 are two cation channel-like proteins in Caenorhabditis elegans that function redundantly to regulate locomotion through unknown mechanisms. A recent study from our lab showed that in vivo Ca2+ imaging analyses of egg-laying neurons in nca-1 loss- and gain-of-function mutants implicate that NCA channels regulate Ca2+ flux at synapses, without affecting Ca2+ dynamics in neuron somas. Furthermore, we observed that NCA-1 localizes to non-synaptic region along axons, strongly suggesting that NCA channels propagate electrical signals from cell bodies to synapses. To identify molecular components that function in the nca-1 genetic pathway, I performed a genetic suppressor screen that led to the identification of behavioral suppressors of nca-1 gain-of-function mutant. Possible NCA auxiliary subunits, UNC-79 (uncoordinated) and UNC-80, were identified from this screen. Molecular characterization of other suppressors will help to identify other regulators and downstream signaling components through which NCA channels transmit electrical signals.

The LIM-Kinase family of proteins (LIMK) plays an important role in actin dynamics through its regulation of ADF/cofilin. A subtype of LIMK, LIMK1, is mostly expressed in neuronal tissues with high levels in the mature synapse. Previous studies from the Zhen Ping Jia laboratory have shown that LIMK1-/- mice exhibit abnormal spine morphology as well as altered hippocampal synaptic plasticity. LIMK1 has been shown to interact with CREB during neuronal development (Yang et al., 2004). We propose that LIMK1 is able to phosphorylate CREB in response to a synaptic activity. We hypothesize that if LIMK1 activates CREB in mature neurons, then LIMK1 knockout mice will have decreased L-LTP and deficits in long-term memory. My results show that LIMK1 and CREB exist in a complex and are bound to each other in mature neurons. LIMK1-/- mice exhibit deficits in the late phase of long-term potentiation and specific deficits in long-term memory while short-term memory remains unaltered. Pharmacological activation of CREB attenuates the observed deficits in synaptic plasticity and long-term memory. These results show a potentially novel mechanism of CREB activation in response to synaptic activity. Moreover, using peptides to manipulate actin dynamics in LIMK1 lacking animals only has effects on early LTP and is not able to rescue the late phase LTP deficits found in LIMK1 -/- mice. These results indicate a specific role of LIMK1 long-term memory and synaptic plasticity through regulation of CREB and not through regulation of the actin cytoskeleton.

The LIM-Kinase family of proteins (LIMK) plays an important role in actin dynamics through its regulation of ADF/cofilin. A subtype of LIMK, LIMK1, is mostly expressed in neuronal tissues with high levels in the mature synapse. Previous studies from the Zhen Ping Jia laboratory have shown that LIMK1-/- mice exhibit abnormal spine morphology as well as altered hippocampal synaptic plasticity. LIMK1 has been shown to interact with CREB during neuronal development (Yang et al., 2004). We propose that LIMK1 is able to phosphorylate CREB in response to a synaptic activity. We hypothesize that if LIMK1 activates CREB in mature neurons, then LIMK1 knockout mice will have decreased L-LTP and deficits in long-term memory. My results show that LIMK1 and CREB exist in a complex and are bound to each other in mature neurons. LIMK1-/- mice exhibit deficits in the late phase of long-term potentiation and specific deficits in long-term memory while short-term memory remains unaltered. Pharmacological activation of CREB attenuates the observed deficits in synaptic plasticity and long-term memory. These results show a potentially novel mechanism of CREB activation in response to synaptic activity. Moreover, using peptides to manipulate actin dynamics in LIMK1 lacking animals only has effects on early LTP and is not able to rescue the late phase LTP deficits found in LIMK1 -/- mice. These results indicate a specific role of LIMK1 long-term memory and synaptic plasticity through regulation of CREB and not through regulation of the actin cytoskeleton.