Browsing by Subject "Synaptic Transmission"
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Item Calcium Triggered Synaptic Vesicle Exocytosis(2007-08-08) Pang, Zhiping; Sudhof, Thomas C.Neurotransmitter release is triggered by the action potential induced influx of Ca 2+ into nerve terminals. One of the central questions in neuroscience is how Ca 2+ promotes synaptic vesicles from rest to fusion leading to release of neurotransmitters. In this thesis, I first addressed if synaptogmin-1/SNARE binding is important for synaptic vesicle release. Using two knock-in mouse lines each with single amino-acid substitution, namely D232N and D238N in synaptotagmin-1, combined with electrophysiology, I found evoked release in D232N mutant neuronal cultures is significantly increased, whereas in D238N cultures release is slightly but significantly decreased. Ca 2+ titration curves indicated the apparent Ca 2+-affinity for vesicle release significantly increased in D232N synapses. These data are consistent with biochemical studies that showed that the D232N substitution in synaptotagmin-1 increases Ca 2+-dependent SNARE bindings but leaves phospholipid binding unchanged, whereas the D238N mutant slightly decreased phospholipid binding but leaves SNARE binding insignificantly changed. Second, I addressed if synaptotamgin-2 is another Ca 2+-sensor for synaptic vesicle release. I and my colleagues used two mouse lines: one contains a single amino acid mutation in synaptotagmin-2 (I377N) and one has synaptotagmin-2 ablated from the genome. By using a combination of biophysical, biochemical and functional techniques, we determined that synaptotagmin-2 is a fast synchronous Ca 2+-sensor. Third, in collaboration with Jianyuan Sun, we explored the biophysical properties of the slow Ca 2+-sensor in the Calyx of Held. Using Ca 2+-uncaging combined with electrophysiology, we mapped increasing Ca 2+ concentrations in relation to neurotransmitter release and built a comprehensive mathematical model for the Ca 2+ control of synaptic vesicle fusion. We found compelling evidence for the existence of two Ca 2+- sensors: one (synaptotagmin-2 in the Calyx of Held) is responsible for fast synchronous release, and the other one is responsible for slow delayed synaptic release. Surprisingly, we found the two Ca 2+-sensors have similar apparent Ca 2+ affinities. This study showed clearly that synaptotagmin-2 is a fast Ca 2+-sensor, and gave us a prediction that narrows down the potential candidate for the slow Ca 2+-sensor.Item Changes in Brain Functional Connectivity Following Donepezil Treatment in Alzheimer's Disease(2006-05-16) Zaidel, Liam; Allen, GregThis study used resting state functional connectivity magnetic resonance imaging (fcMRI) to explore changes in brain connectivity and their behavior correlates in nine regions of interest (ROIs) in eleven patients with mild Alzheimer's disease (AD) following treatment with the cholinesterase inhibitor, donepezil. The ROIs were selected on the basis of their association with cholinergic neurotransmission, AD neuropathology, and neurocognitive deficits in AD. These ROIs included the medial septal nuclei, left and right hippocampi, left Broca's area and its right hemisphere homologue, left and right dorsolateral prefrontal cortices, and left and right primary visual cortices. Changes in connectivity were also related to changes in performance on neurocognitive tests of verbal fluency and episodic memory. Among the ROIs, the effects of the drug were selective. Only the connection between left and right DLPFC increased significantly after treatment. However, ten of the eighteen connections measured showed significant relationships between connectivity and behavior. The significant correlations centered around left hippocampus, left Broca's area, and dorsolateral prefrontal cortex bilaterally. Connections originating in the left hippocampus showed mostly inverse relationships with behavior. Predictions of selective increases in connectivity in networks associated with the neurochemical, the neuropathological, and neurocognitive profiles of AD were generally not supported. A separate, whole-brain, exploratory, analysis measured changes in connectivity throughout the brain with each of the nine regions of interest (ROIs). There were increases in connectivity among bilateral frontal areas in language circuits, including the left IFG, left superior temporal gyrus, and left supramarginal gyrus, and in the sensory-motor integrative network. Further connections were noted between the left inferior frontal gyrus and caudate nucleus. The data suggest that the drug had selective effects on executive networks of attention.Item MeCP2 and the Epigenetic Regulation of Excitatory Synaptic Transmission(2007-08-08) Nelson, Erika Dawn; Bezprozvanny, IlyaAccurate regulation of gene expression is critical for normal brain function. Many human neurodevelopmental and neurodegenerative disorders are associated with mutations in genes important for controlling transcription. Mutations in one such gene, the transcriptional repressor methyl-CpG-binding protein 2 (MeCP2), lead to a form of mental retardation called Rett Syndrome (RTT). Though the MeCP2 protein is expressed ubiquitously, symptoms of RTT patients are primarily neurological, which include reduced mental capacity, autistic-like behavior and autonomic dysfunction. In addition, a mouse model with reduced MeCP2 expression specifically in postnatal, forebrain neurons recapitulates many of the phenotypes seen in human patients. These findings, among others, lead to interest in MeCP2's function in the brain. Our research has focused on the transcriptional repression activity of MeCP2 and its role in the regulation of synapse function. Using mainly electrophysiological techniques, we found that the loss of MeCP2 in hippocampal neurons results in deficits in both spontaneous and evoked excitatory synaptic transmission. Using pharmacological manipulations, we were able to attribute these deficits to the loss of transcriptional repression by MeCP2. By utilizing a conditional knockout approach, we found that these effects were not due to the loss of MeCP2 during neurodevelopment and that they were primarily due to a deficiency in presynaptic vesicle release. We further extended these findings by looking at two mechanisms for controlling the repression of gene expression, DNA methylation and histone deacetylation, both of which are important for MeCP2's function as a transcriptional repressor. Using inhibitors of DNA methyltransferases, we discovered that synaptic activity-dependent decreases in DNA methylation occur in post-mitotic neurons, and that these changes in DNA methylation can regulate spontaneous synaptic transmission. We were also able to rescue the MeCP2-dependent decrease in spontaneous activity by treating neurons with the methyl donor, S-adenosyl-L-methionine. Finally, we addressed the role of histone deacetylation in synapse function by conditionally deleting histone deacetylases (HDACs) 1 and 2 from mature hippocampal neurons. HDAC1 and 2 are present in the transcriptional repressor complex containing MeCP2. After acute knockdown of HDAC1 or HDAC2, we found deficits in excitatory synaptic transmission that mimicked the defects seen after the constitutive loss of MeCP2. In summary, we have discovered a role for the transcriptional repressor, MeCP2, and two components of its repressor complex, DNA methylation and HDACs, in the control of excitatory synaptic transmission between hippocampal neurons.Item Membrane Lipids and Synaptic Vesicle Trafficking in the CNS(2009-01-14) Wasser, Catherine Rebecca; Kavalali, Ege TMost vesicles within a synapse are dormant. The rest participate in synaptic neurotransmission, with a portion of these preferentially fusing first. Moreover, all synapses experience spontaneous neurotransmitter release which may originate from the random exocytosis of vesicles prepared to fuse immediately upon calcium influx; however, spontaneously fusing vesicles may be independent because they prefer spontaneous fusion. The functional separation argues that the compositions the synaptic vesicle membranes are somehow unique between pools. The first three chapters explore the role of cholesterol in synaptic transmission. We treated hippocampal cultures with methyl-beta-cyclodextrin, which reversibly binds cholesterol, or mevastatin, an inhibitor of cholesterol biosynthesis, to deplete cholesterol. We also used hippocampal cultures from Niemann-Pick type C1-deficient mice defective in intracellular cholesterol trafficking. These conditions revealed augmented spontaneous neurotransmission. In contrast, the same treatments severely impaired responses evoked by action potentials and hypertonicity. These results suggest that synaptic cholesterol balances evoked and spontaneous neurotransmission by hindering spontaneous synaptic vesicle turnover and sustaining evoked exo-endocytosis. Chapter five examines the role of sphingosine on neurotransmitter release. By adding sphingosine to hippocampal cultures, we found that sphingosine enhances neurotransmission in a synaptobrevin-2-dependent manner. Chapter six investigates the stability of actively recycling synaptic vesicles. We employed several approaches (fluorescent and ultrastructural imaging) to monitor not only the fate recycling vesicles, but also the origin and reuse of spontaneously fusing vesicles. We conclude that at rest, the total recycling pool remains active and resists spontaneous fusion up to at least six hours; while spontaneous fusion of spontaneously fusing vesicles is much faster. This argues that vesicles fusing spontaneously do not originate from the recycling pool. In chapter seven, we observe how modifying synaptic vesicle membranes might affect neurotransmitter release. By the uptake of horseradish peroxidase into vesicles followed by hydrogen peroxide perfusion, we induced free radical modification of vesicle membranes and found that modifying recycling pool vesicles increased spontaneous fusion and attenuated evoked release. Taken together, the results of each chapter appear to suggest that the fusion of action potential-dependent and-independent vesicles are regulated by different mechanisms, supporting the theory that some vesicles may be unique within a synapse.Item Regulation of Excitatory Neurotransmission, Synaptic Plasticity, and learning by Cyclin-Dependent Kinase 5(2009-06-17) Hawasli, Ammar Hamami; Bibb, James A.Cyclin-dependent kinase 5 has been implicated in many physiological and pathological processes in the central nervous system. To better understand Cyclin-dependent kinase 5's roles in the adult brain, we developed and studied several conditional Cyclin-dependent kinase 5 knockout model systems. Soon after conditional loss of Cyclin-dependent kinase 5, mice displayed improved hippocampal learning and enhanced synaptic plasticity in the hippocampal Schaffer collateral pathway. The genetically enhanced mice displayed increased N-methyl-D-aspartate receptor-mediated currents and elevated levels of the NR2B N-methyl-D-aspartate receptor subunit. The enhancement in synaptic plasticity was directly attributed to the increased current through NR2B-containing receptors. NR2B levels were elevated in Cyclin-dependent kinase 5 knockout mice due to an impairment in the calpain-mediated degradation of NR2B. Consistently, Cyclin-dependent kinase 5 directly facilitated the degradation of NR2B cytoplasmic-tail in vitro. Cyclin-dependent kinase 5, NR2B, and calpain coimmunoprecipitated in vivo and directly bound one another in vitro. NR2B inhibited Cyclin-dependent kinase 5 activity in vitro, indicating a potential feedback mechanism. These findings suggested that Cyclin-dependent kinase 5 interacts directly with NR2B and calpain to facilitate the degradation of NR2B, thereby attenuating synaptic plasticity. In addition to regulating functional plasticity, Cyclin-dependent kinase 5 also plays roles in structural plasticity and presynaptic function. Cyclin-dependent kinase 5 facilitated the calpain-mediated degradation of spectrin in vitro. Spectrin degradation and depolymerized actin levels were decreased in conditional Cyclin-dependent kinase 5 knockout hippocampus. These results implicate Cyclin-dependent kinase 5 dendritic in spine dynamics which is critical for synaptic plasticity. Loss of Cyclin-dependent kinase 5 also led to a presynaptic enhancement in post-tetanic potentiation and a deficit in paired-pulse facilitation, which are consistent with an increase in probability of synaptic vesicle release, due to increased numbers of vesicles in the readily releasable pool or altered sensitivity to presynaptic calcium. Finally, chronic Cyclin-dependent kinase 5 loss produced increases in behavioral and neuronal excitability followed by electrographic abnormalities in vivo and reduced brain weight. These findings suggest that the enhancement in excitatory neurotransmission which initially led to improvements in learning and plasticity preceded excessive excitability and subsequent neuropathology. Consequently, Cyclin-dependent kinase 5 regulates excitatory neurotransmission, synaptic plasticity.Item Regulation of SNARE-Mediated Synaptic Vesicle Release by Synaptotagmins and Complexins(2010-11-02T18:13:03Z) Kaeser-Woo, Yea Jin; Südhof, Thomas C.In the brain, neurons communicate with each other by synaptic transmission. This process includes release of neurotransmitter from vesicles in the presynaptic neuron into the synaptic cleft, and sensing of these neurotransmitters by the postsynaptic neuron with specific receptors. Long-lasting changes in the strength of synaptic contacts between neurons in the human brain, a process that is referred to as long-term synaptic plasticity, are the cellular correlates that underlie learning and memory. Synaptic transmission is initiated when an action potential arrives at the presynaptic terminal, and induces Ca2+ influx through voltage-gated Ca2+ channels. The SNARE (Soluble N-ethylmaleimide-sensitive-factor Attachment Protein Receptors) complex is the core component of the fusion machinery in the presynaptic terminal, as it forms a physical bridge between the vesicular membrane and the presynaptic target membrane that delivers the force to fuse the two membranes. Additional presynaptic proteins are required to activate or suppress neurotransmitter release which allows the presynaptic neuron to tightly control and regulate the process of neurotransmitter release. Among these proteins are synaptotagmins and complexins, two protein families that directly interact with the SNARE complex, and that are interdependent to each other in regulating SNARE-mediated synaptic vesicle release: complexin clamps neurotransmitter release until synaptotagmin is recruited by Ca2+ influx, and then it activates SNARE-mediated fusion process together with synaptotagmin. Here I describe the prospective in vivo function of synaptotagmin 12, a novel isoform of synaptotagmin, which lacks the typical Ca2+ binding residues of synaptotagmin, but instead contains a unique sequence motif which can be phosphorylated by cAMP-dependent protein kinase A. By using gene targeting method, I directly examined whether phosphorylation of synaptotagmin 12 is involved in presynaptic forms of long-term plasticity. In parallel, I developed a structure-function approach to functionally dissect how individual domains of complexin contribute to its dichotomic functions of clamping and activating neurotransmitter release. With this approach, we focused on how the accessory alpha-helix of complexin participates in SNARE-mediated synaptic fusion.Item Regulation of Synaptic Vesicle Trafficking at Central Synapses(2009-09-04) Chung, Chihye; Kavalali, Ege T.Synapses are where electrical information is converted to chemical signaling, allowing for careful regulation of inter-neuronal communication in the brain. At presynaptic terminals, synaptic vesicles fuse with plasma membrane in response to electrical stimulation, followed by rapid retrieval to the terminal and re-organization for reuse. Thus, synaptic vesicle trafficking is of interest as to where presynaptic regulations of synaptic transmission begins to occur. The first two chapters explored a novel secretagogue, lanthanum (La3+), and its potential usage as a probe to study vesicle recycling at central synapses. Chapter two describes the characteristics of La3+ -evoked transmission at hippocampal synapses. La3+ has two separate actions on transmission, with a different time course and underlying mechanism of action. This newly characterized rapid action of La3+ is intracellular Ca2+ -independent, in contrast to its delayed action, yet requires functional SNARE complex formation. Therefore, chapter three took advantage of La3+-evoked transmission as a tool to investigate the coupling between exo- and endocytosis in SNARE-dependent fusion. Using multifaceted approaches, I propose that La3+ induces transmitter release via narrow fusion pore opening and closure, or a 'kiss-and-run' mode of exo- and endocytosis. Chapter four investigates the molecular requirement for the synaptic vesicle recycling pathway. I analyzed the impact of one of main players in endocytosis, dynamin in different forms of release. Acute inhibition of dynamin in central synapses impairs activity-dependent synaptic vesicle recycling while leaves spontaneous recycling intact, suggesting the operation of two parallel recycling pathways in central synapses as well as proposing the molecular signature between spontaneously and activity-dependently recycling pathways. In chapter five, I further investigated the origins of spontaneously recycling synaptic vesicles by simultaneous monitoring of spectrally separable FM dyes, as chapter suggested four that they are originated from an isolated pool. This chapter includes comprehensive analysis of the endocytic pathway operating at rest and its molecular participants -specifically dynamin, which was implicated to play a role in the endocytic pathway from observations I made in chapter four. Chapter six expands the investigation as to how presynaptic signaling regulates synaptic vesicle trafficking in glutamatergic synapses. I focused on the impact of ambient glutamate concentration on vesicle recycling as a feedback signal to rapid synaptic reuse to impact short-term synaptic plasticity. Taken together, these results suggest that synaptic vesicle trafficking is an actively regulated process, impacting various aspects of information cascades between neurons.Item A Structural/Behavioral Analysis of the Regulation of Dopamine Signaling by Striatal RGS Proteins(2005-08-11) Waugh, Jeffrey Lynn; Gold, Stephen J.The regulators of G-protein signaling (RGS) proteins negatively modulate heterotrimeric G protein signaling by acting as GTPase activating proteins for Galpha subunits. In the striatum and nucleus accumbens, brain regions critical for control of movement, motivation and reward, overlapping RGS expression profiles suggested that functional specificity could not be explained by anatomical localization alone. We set out to assess striatal specificity within two distinct RGS pools, the R7 RGS subfamily and RGS10. The highly striatal-specific splice form RGS9-2 is a negative modulator of dopamine D2 receptor signaling, and has been shown to inhibit drug stimulated (cocaine or direct dopamine receptor agonists) locomotor activity. RGS9-2 is a member of the R7 subfamily, comprised of RGS6, -7, -9, and -11, which share highly similar subdomain structure. We analyzed the specificity of R7 modulation of dopamine receptor signaling using a novel behavioral assay. R7 RGS proteins were virally-overexpressed in rat or mouse accumbens via a stereotaxic injection of an engineered HSV virus. Following this surgery, drug-stimulated locomotor responses were assayed. We found that in rats and RGS9 KO mice, overexpression of R7 RGS proteins produces distinct locomotor and drug sensitization phenotypes, each of which occurs only during the period of RGS overexpression. Moreover, studies using truncation and chimeric RGS mutants demonstrated that while all tested subdomains were necessary for activity, only the C-terminus of RGS9-2 was sufficient to convey activity to RGS7. Lastly, RGS overexpression leads to distinct acute changes in weight: loss (RGS9-2) or gain (RGS7, RGS11). To elucidate RGS10 function in the brain, we mapped RGS10 protein in rodent brain using light microscopic and electron microscopic immunohistochemical techniques. Light microscopic analyses showed that RGS10 immunoreactivity labels all subcompartments of neurons and microglia, including their nuclei. Electron microscopy confirmed the presence of dense RGS10 immunoreactivity in euchromatin and resolved dense staining on terminals at symmetric synapses onto pyramidal cell somata. Dual-labeling histochemistry showed that RGS10 is expressed in specific neuronal cell types and circuits. Taken together, these data support a role for RGS10 in diverse processes including modulation of pre- and postsynaptic G-protein signaling and a potential role in modulating gene expression.Item Study of Presynaptic Calcium Channels and a Novel Calcium Channel Calhm1(2010-11-02T18:19:16Z) Ozkan, Emin D.; Bezprozvanny, IlyaCalcium signaling is essential for all cellular processes. In brain these processes include basic synaptic transmission, modification of established synapses, elimination of synapses as well as elimination of whole neurons by way of apoptosis. In this thesis, we have studied two of these processes. One process is synaptic targeting of presynaptic calcium channels. We have tested the hypothesis that synaptic targeting of presynaptic calcium channels depends on carboxy terminal interactions with mint and CASK proteins. To this end we have used mice mutant for P/Q-type calcium channel. Our experiments show that a P/Q-type calcium channel without its carboxy terminal can partially contribute to synaptic transmission. Secondly we have studied the role of Calhm1 gene in modulating calcium signaling pathways. For this purpose we have used overexpression of Calhm1 gene in HEK293 cells as well as in dissociated neuronal cultures. Our experiments show that Calhm1 modulates calcium signaling in HEK293 cell and in certain neurons. Calhm1 overexpression also modulates spontaneous synaptic transmission.Item Study of the Mechanisms Underlying Hippocampal Neuron Synaptogenesis: The Roles of Neurotrophin Signaling and Micrornas(2010-11-02T18:20:38Z) Zhang, Wei; Parada, LuisSynapse formation requires contacts between dendrites and axons. Although this process is often viewed as axon mediated, dendritic filopodia may be actively involved in mediating synaptogenic contacts. Brain-derived neurotrophic factor (BDNF) increases the density of dendritic filopodia and the conditional deletion of tyrosine receptor kinase B (TrkB) reduces synapse density in vivo (Luikart et al., 2005). Here, we report that TrkB associates with dendritic growth cones and filopodia, mediates filopodial motility, and does so via the phosphoinositide 3 kinase (PI3K) pathway. We used genetic and pharmacological manipulations of mouse hippocampal neurons to assess signaling downstream of TrkB. Conditional knock-out of two downstream negative regulators of TrkB signaling, Pten (phosphatase with tensin homolog) and Nf1 (neurofibromatosis type 1), enhanced filopodial motility. This effect was PI3K-dependent and correlated with synapse density. Phosphatidylinositol 3,4,5- trisphosphate (PIP3) was preferentially localized in filopodia and this distribution was enhanced by BDNF application. Thus, intracellular control of filopodial dynamics converged on PI3K activation and PIP3 accumulation, a cellular paradigm conserved for chemotaxis in other cell types. Our results suggest that filopodial movement is not random, but responsive to synaptic guidance cues. In order to further elucidate the mechanisms of BDNF-TrkB-PI3K pathway downstream signaling involved in regulating dendritic filopodial motility, we used a pharmacological approach as well as a gene expression approach to show that Rac1 and RhoA may play a role in this pathway. Rac1 positively regulated dendritic filopodial motility while RhoA had a negative effect. Our data suggest that BDNF-TrkB signaling might function to regulate the balance between Rac1 and RhoA, thus controlling dendritic filopodial motility. The developing nervous system is shaped by highly orchestrated programs of gene expression. This tight regulation is regulated by various transcriptional and post-transcriptional events that control individual gene expression. The recent discovery of small, non-coding RNAs has greatly expanded our understanding of the mechanisms that regulate gene expression at the post-transcriptional level. Here, I characterized the expression pattern of one neuronal microRNA, miR-381, and used in vitro cultured hippocampal neurons to show that miR-381 regulates neurite growth, as overexpression of miR-381 promotes neuronal dendritic branching. The effect of miR-381 on neuronal dendritic branching might be through a net regulation of multiple target genes.