Browsing by Subject "Synapses"
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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 membrane potential and imput resistance of fore- and hindlimb motoneurons as a result of post-brachial spinal cord coldblock(Texas Tech University, 1978-05) Schadt, James CNot availableItem Cholinergic interneurons and synaptic reorganization within the nucleus accumbens shell and core: potential neural substrates underlying drug addiction(2006) Berlanga, Monica Lisa; Alcantara, Adriana A.Drug abuse and dependence are among the most challenging public health issues facing America today. The acute treatment of drugs of abuse such as psychostimulants (Trantham-Davidson and Lavin, 2004) and opiates (Harris and Williams, 1991) produce transient changes in cellular activity and synaptic signaling. Repeated drug treatment, however, results in persistent cellular and behavioral changes, such as altered dendritic morphology and behavioral sensitization (Robinson and Kolb, 1999b). Synaptic changes in the brain are posited to underlie a repertoire of drug-induced persistent behaviors, including sensitization, psychosis and relapse. Direct evidence of drug-induced synaptic plasticity, however, has not been demonstrated. The present studies were designed to examine cholinergic neurons and synaptic rewiring as potential neural substrates involved in acute and chronic drug exposure. The proposed studies tested the hypotheses that 1) cholinergic interneurons within the nucleus accumbens (NAcc) are activated by the acute self-administration of cocaine, 2) dopamine (DA) D5 and D2 receptors localized on cholinergic interneurons potentially undergo cocaine-induced neuroadaptation, and 3) repeated administration of cocaine leads to an increase, while repeated administration of morphine leads to a decrease, in the number of synapses within the NAcc, whereas an increase in the number of synapses occurs in the NAcc core of animals exhibiting behavioral sensitization. These studies revealed that accumbal cholinergic interneurons are activated by acute cocaine self-administration and elucidate the specific localization of DA receptor subtypes, D5 and D2, on these cells, suggesting their potential role in mediating druginduced DA changes within the NAcc. The final study provided the first ultrastructural evidence that an increase in the number of excitatory synapses in the NAcc shell occurs following 4-weeks of cocaine and morphine treatment followed by 3 weeks abstinence and that cocaine sensitization is associated with an increase in the number of excitatory synapses in the NAcc core. These findings provide the groundwork for future studies examining the precise cellular and synaptic substrates underlying a repertoire of druginduced behaviors that contribute to the persistence of addiction. Improved pharmacotherapeutic and behavioral treatments can then target the specific cellular and synaptic microcircuitry critically involved in the different stages of drug abuse and dependence.Item Competition Between Synaptotagmin 1 and Complexin for SNARE Complex Binding, Controls Fast Synaptic Vesicle Exocytosis(2007-05-23) Tang, Jiong; Sudhof, Thomas C.Calcium binding to synaptotagmin 1 triggers fast exocytosis of synaptic vesicles that were primed for release by SNARE complex assembly. Besides synaptotagmin 1, fast Ca2+- triggered exocytosis requires complexins. Synaptotagmin 1 and complexins both bind to assembled SNARE complexes, but it is unclear how their functions are coupled. To clarify previous debates on calcium dependent and independent binding between synaptotagmin 1 and SNARE proteins, I systematically examined the interactions between synaptotagmin 1 and purified SNARE monomer, heterodimer and core complex separately. This would avoid the problem of doing binding assays in an undefined protein mixture. We found the calcium dependency of synaptotagmin 1 and SNARE interactions relied on the accurate binding conditions that include protein concentration and ionic strength. In addition, at physiological conditions, calcium dependent binding is favored. Based on this system, I discovered the competition between complexin and synaptotagmin 1 for SNARE complex binding. Although in hydrophilic environment, complexin shows much higher affinity for SNARE complex than synaptotagmin 1, synaptotagmin 1 can more efficiently replace complexin from membrane embedded SNARE complex in a strictly calcium dependent manner. Expression of synaptic vesicle targeted complexin (by fusion to synaptobrevin 2) in cultured cortical neurons severely blocks fast synchronous release, but not asynchronous release, which is very similar to that of synaptotagmin 1 knockout mice. Based on electrophysiological data and biochemical confirmation of competition, we suggest that the phenotype could result from the replacement of synaptotagmin 1 from SNARE complex by local high concentration of fused complexin. We propose our model as: complexin binding promotes the assembly of SNARE complex and further stabilizes it. As a result, vesicles are activated into a "superprimed" metastable state, and are clamped at the same time waiting for triggering signals. Synaptotagmin 1 replaces complexin and releases this clamp through SNARE complex binding upon calcium entry. The simultaneous binding of synaptotagmin 1 with SNARE complex and phospholipids finally triggers membrane fusion and vesicle release.Item Depression of spinal reflexes following changes in osmolality(Texas Tech University, 1978-05) Lake, David AllenNot availableItem Evidence for muscle-dependent neuromuscular synaptic site determination in mammals(2007-12) Vock, Vita Marie, 1963-; Rimer, Mendell; Thompson, Wesley J.Recent evidence has challenged the prevalent view that neural factors induce the formation of a de novo postsynaptic apparatus during development of the vertebrate neuromuscular junction. The latest experiments suggest an alternative, muscle-dependent model in which the muscle induces the nascent postsynaptic apparatus and sets the location of the future synapse. Once contacted by the incoming axons, these sites, laid out in a pre-pattern in the central area of developing muscle fibers, mature into synapses by the combined action of neural factors such as agrin and ACh. In this study, I sought to provide a test in mammals for these two models of neuromuscular synaptogenesis. Previously, our laboratory showed that continuous muscle expression of constitutively active ErbB2 (CAErbB2) during embryogenesis leads to synaptic loss, exuberant axonal sprouting and lethality at birth. Here, I transiently induced CAErbB2 during midgestation and examined the process of synapse restoration after inducer withdrawal. Centrallyenriched AChR transcription and AChR clustering were abolished as a result of transient CAErbB2 induction. After inducer withdrawal, synapses were restored but were distributed widely over the entire surface of the diaphragm. Under the nerve-dependent model, this distribution would have been explained by the wide pattern of axonal sprouting triggered by CAErbB2 expression. Yet, in the absence of the nerve, introduced in our transgenic animals by mating to Hb9+/- mice, a very similar, wide distribution of aneural AChR clusters was generated. Thus, even in a case where the central pre-pattern of AChR transcription and clustering is missing, it is the muscle, and not the nerve, that seems to set the site for synapse formation. My results support a muscle-dependent model for the induction of neuromuscular synaptogenesis in mammals.Item Facilitation by the locus coeruleus of lumbar cord activities in cats(Texas Tech University, 1980-05) Fung, Simon JimNot availableItem Fragile X Mental Retardation Protein Induces Synapse Loss Through Acute Postsynaptic Translational Regulation(2009-01-14) Pfeiffer, Brad Erich; Huber, KimberlyFragile X Syndrome (FXS) is the most common form of inherited mental retardation. The root cause of FXS is loss of the function of a single protein: the Fragile X Mental Retardation Protein (FMRP). FMRP is an RNA-binding protein that plays a complex role in translational regulation. FMRP may be an important regulator of dendritic protein synthesis, which occurs at or near synapses in response to synaptic activity. Many types of long-term synaptic change require local protein synthesis for their induction and/or maintenance, and several protein synthesis-dependent forms of synaptic plasticity are altered in the absence of FMRP. Both human FXS patients and mice lacking FMRP (Fmr1-KO mice) display increased numbers of dendritic spines, the primary sites of excitatory synaptic connections. In addition to increased numbers, the spines of FXS patients and Fmr1-KO mice appear morphologically immature. It was unknown whether FMRP plays a direct, cell-autonomous role in the regulation of synapse number or function. Moreover, the mechanisms through which FMRP might govern neuronal function or number were unclear. I report that acute postsynaptic expression of FMRP in Fmr1-KO neurons results in a decrease in the number of functional and structural synapses without an effect on their synaptic strength or maturational state. Similarly, wild-type neurons endogenously expressing FMRP have fewer synapses than neighboring Fmr1-KO neurons, indicating a clear role for FMRP in the regulation of synapse number. An intact K homology 2 (KH2) RNA-binding domain and dephosphorylation of FMRP at S500 are required for the effects of FMRP on synapse number, indicating that FMRP-dependent translation of mRNA targets of FMRP leads to synapse loss. Furthermore, I demonstrate novel phenotypic interactions of FMRP with the transcription factor MEF2. MEF2 activity in wild-type neurons induces robust synapse loss; however, MEF2 fails to decrease synapse number in Fmr1-KO neurons. A dominant-negative form of MEF2 increases synapse number in WT, but not Fmr1-KO neurons. Finally, when co-expressed with a dominant negative form of MEF2, FMRP fails to induce synapse loss in Fmr1-KO neurons. These data represent novel mechanisms through which FMRP regulates neuronal function and suggest novel therapeutic targets and strategies for FXS treatment.Item Neurologin function in excitatory and inhibitory synapses(2008-09-19) Zang, Tong; Sudhof, Thomas C.Neuroligins (NLs) are postsynaptic cell adhesion molecules which by binding to presynaptic neurexins (NRXs) are thought to mediate synapse formation and function. Both NLs and NRXs are discussed in the genetic correlation to Autism. Over-expression of NLs could induce the formation of synaptic contacts with axons in non-neuronal cells and increase the synaptic density and response in cultured neurons, through binding and recruiting NRXs; however, little is known about NL signaling though NRXs or inside the cell. First, we hypothesized that NLs signal through their cytoplasmic region. Over-expression of NL1 with cytoplasmic tail truncation abolished the increase of synaptic density by NL1 full length. By yeast two hybrid screening using NL2 cytoplasmic region, we identified potential interaction partners, of which Necab2 and NRP/B (also named as ectodermal cortex 1, EC1) are two promising candidates and the interactions were confirmed. NL1 or NL2 c-tail truncations partially abolished the change in miniature IPSC, but not the evoked responses. NL c-tail binding partners?ver-expression does not show any change in evoked responses. It suggested that NL cytoplasmic region is important for some neuronal changes but does not contribute to the major phenotype of NLs. And we investigated the contribution of NL-NRX binding by using NL extracellular NRX binding mutants. The mutants abolished the change of the evoked and miniature inhibitory responses from the NL2 wild type, which suggested the inhibitory responses triggered by NL2 go through NRXs. And the slight change of the paired pulse ratio suggested the change of presynaptic calcium through binding. The study suggested that NL2 facilitate the inhibitory synaptic transmission through extracellular region via neurexin binding, possibly by the increase in presynaptic calcium. We also found Brain-specific Angiogenesis Inhibitors (BAIs), a family of G-protein coupled receptors (GPCRs), will bind to NLs extracellularly and may serve as signaling modules binding to NLs. Over-expression of BAIs do not change evoked IPSCs, but Bai1 decreased evoked EPSCs and increased the burst duration in the spontaneous responses, possibly because of some secondary responses. Therefore, we found NL-NRX though NL extracellular region is important for NL2 function in synaptic transmission, and BAIs may be potential signaling molecules of NLsItem Rapid Protein Translation Governs Persistent Changes in AMPAR Trafficking in a Form of Long-term Synaptic Plasticity(2010-05-14) Waung, Maggie Wai-Ming; Huber, KimActivation of group 1 metabotropic glutamate receptors (mGluRs) induces long-term depression of glutamatergic synapses (mGluR-LTD). Postsynaptic endocytosis of ionotropic α-amino-5-hydroxy-3-methyl-4-isoxazole propionic acid receptors (AMPARs) accompanies mGluR-LTD, and long-term decreases in AMPAR surface expression most likely mediate this form of synaptic plasticity. In support of this idea, both mGluR-LTD and decreases in AMPA receptors require rapid protein synthesis in dendrites. To understand how newly synthesized proteins maintain decreases in AMPAR surface expression, we examined how mGluRs persistently alter AMPAR trafficking. Using biochemical and immunocytochemical methods in dissociated rat hippocampal cultures, we find that brief activation of mGluRs by the group 1 mGluR selective agonist, DHPG, results in a rapid (10 min) increase in AMPAR endocytosis rate that persists for at least one hour after the removal of agonist. This persistent increase in endocytosis rate is blocked by the protein synthesis inhibitor anisomycin, suggesting that components of the endocytosis machinery are synthesized and necessary for mGluR-LTD. In contrast, treatment of cultures with NMDA, which induces NMDA receptor-dependent LTD causes a long-term (60 min) decrease in AMPAR surface expression, but does not persistently increase endocytosis rate. Recent work has implicated activity-regulated cytoskeletal associated protein (Arc) in the regulation of AMPAR endocytosis through its interactions with endophilin and dynamin, and Arc mRNA is induced in hippocampal CA1 dendrites following behavioral activity. However, little is known about how Arc is locally synthesized at synapses or whether its local synthesis contributes to synaptic plasticity. We find that DHPG induces rapid increases in local and synaptic dendritic Arc protein expression within 10 minutes in hippocampal neurons. Knockdown of Arc by lentiviral delivery of short-hairpin RNA increases basal surface AMPAR expression and synaptic transmission as measured by mEPSC amplitude. Arc knockdown blocks mGluR-induced decreases in surface AMPARs, AMPAR endocytosis as well as mGluR-LTD. Acute inhibition of new Arc translation with antisense nucleotides also blocks mGluR-induced persistent changes in AMPAR trafficking and mGluR-LTD. The involvement of rapid Arc synthesis in mGluR regulation of synaptic function provides a link between behavior-driven neuronal activity and plasticity at the synapse.Item Role of Neuroligin in Synapse Formation and Autism(2006-08-11) Chubykin, Alexander Anatoly; Sudhof, Thomas C.Neuroligins mediate synaptogenesis through formation of a trans-synaptic complex with presynaptic neurexins. Interaction of neuroligin 1 with neurexins is regulated by alternative splicing of both neuroligin 1 (at splice site B) and of neurexins (at splice site #4). Full-length neuroligin 1 that binds only beta -neurexin more potently promotes synapse formation in hippocampal neurons, whereas neuroligin 1 lacking splice site B, which binds both alpha - and beta -neurexins, is more efficient at synapse expansion. Mutations in two surface loops of neuroligin 1 abolished neuroligin binding to neurexin 1beta and blocked synapse formation. Neuroligin mutation found in autism spectrum disorders impairs cell-surface transport but does not completely abolish synaptogenic activity. In hippocampal neurons, overexpressed neuroligin 1 enhances excitatory but not inhibitory synaptic responses, and increases the ratio of NMDA to AMPA receptor-mediated synaptic currents. In contrast, genetic deletion of neuroligin 1 in mice decreases NMDA receptor-mediated synaptic currents and the NMDA/AMPA receptor ratio. Contrary to neuroligin 1, neuroligin 2 potentiates inhibitory but not excitatory synaptic responses. The synaptic actions of neuroligin 1 are suppressed by chronic blockade of NMDA receptors or of CaM-kinase II. Neuroligin 1 with an autistic-spectrum syndrome mutation decreases excitatory synaptic responses, consistent with a role for endogenous neuroligin 1 in synapse development. Taken together, our data suggest that neuroligin-neurexin interaction regulated by their alternative splicing promotes formation of specific synapses; synaptogenic function of neuroligin is regulated by NMDA receptor and Cam-kinase II activation, suggesting a critical role for neuroligins in synaptic plasticity and modulation of neural circuits.Item Roles of Class II Histone Deacetylases in Muscle and Brain(2010-01-12T18:54:04Z) Kim, Mi-Sung; Olson, Eric N.A defining feature of brain and muscle is their ability to remodel their phenotypes in response to extracellular stimuli to maintain the balance between physiological demand and functional capacity. Successful adaptation to the environment is essential for the survival and this plasticity is achieved by activation of intracellular signaling pathways and subsequent activation of gene expression, the so-called “extrinsic genetic programs”. Although it has been well known that calcium-dependent signaling is critical to regulate this extrinsic genetic program, little is known about how calcium-dependent signaling is propagated to the nucleus to induce the transcription of specific genes responsible for tissue plasticity. Furthermore, physiological and behavioral consequences of failure of plasticity are still poorly known. Here, I demonstrate that class II HDACs and MEF2 transcription factors are essential for tissue plasticity, and that defects of this signaling pathway in muscle and brain cause muscle-fatigue susceptibility and a pronounced neurological deficit. Protein kinase D1, a potent class II HDAC kinase, in skeletal muscle promotes transformation to type I myofibers through activation of MEF2. Conversely, genetic deletion of PKD1 in type I myofibers increases susceptibility to fatigue, suggesting that PKD1 is a key regulator of skeletal muscle plasticity. Deletion of the class II HDAC4 in neurons impairs memory formation and synaptic plasticity in mice, while mice lacking class II HDAC5 exhibit normal memory formation. Furthermore, deletion of both HDAC4 and HDAC5 in neurons produced a more pronounced neurological deficit, including severe seizure activity, suggesting distinct and redundant roles for HDAC4 and HDAC5 in memory formation and in brain homeostasis. While deletion of MEF2C in brain causes impairments in memory formation, mice lacking MEF2A and MEF2D exhibit no such deficits. Furthermore, deletion of MEF2A, MEF2C, and MEF2D results in decreased REM sleep, brief spontaneous seizures, and postnatal lethality accompanied by increased apoptosis, suggesting distinct and redundant roles for MEF2A, MEF2C, and MEF2D in brain homeostasis. Taken together, these series of studies provide important clues to understanding the mechanism by which extrinsic genetic programs are regulated in vivo, especially focusing on the regulation of muscle and brain functions. [Keywords: histone deacetylases; muscle remodeling; learning and memory; myocyte enhancer factor 2; synaptic plasticity; protein kinase D]Item Schwann cell processes guide axons reinnervating the neuromuscular junction(2004) Kang, Hyuno; Thompson, Wesley J.Item Synaptic Cell Adhesion And Functional Architecture Of CNS Synapses(2007-08-08) Atasoy, Deniz; Kavalali, Ege T.Synapses are specialized intercellular junctions through which neurons communicate. The two sides of a synapse are held together with adhesion molecules. We investigated the role of synaptic adhesion molecules neuroligins, neurexins, SynCAM and dystroglycan using overexpression and knock-out mice analysis approaches. We showed that neuroligins mediate validation of synapses in an activity dependent manner and different isoforms of neuroligins mediate different types of synapse validation. We also showed that binding partners of neuroligins, i.e. neurexins, have a cell autonomous effect on inhibitory synapses, independent of neuroligins. Analysis of dystroglycans failed to reveal a significant phenotype in dissociated cultures, whereas SynCAM had a robust synapse inducing role in developing networks. Furthermore we have shown that this effect of SynCAM is mediated through its cytoplasmic interactions. We further investigated cytoplasmic downstream effectors of neurexins and SynCAM by analyzing CASK and Mint proteins using knock-out approaches. We have shown that both CASK and Mints are essential for survival and synaptic function. Our results indicate that synaptic cell adhesion molecules are not merely passive structural elements but actively participate in information transfer and signaling between neurons by interacting with each other and with their intracellular effectors. We have also investigated homeostatic properties of vesicle recycling and we have demonstrated that activity levels of a neuronal network determines the pathways through which synapses replenish the neurotransmitter vesicles during high frequency stimulation. Finally, we explored the relationship between evoked and spontaneous vesicle fusion and their postsynaptic targets using NMDA receptors. We uncovered previously unexpected segregation of receptor pools that respond to spontaneous or evoked vesicle fusion events. Our experiments also revealed that synaptic cleft is not a mere empty space but rather a complex structure filled with various elements can effect diffusion of neurotransmitter and hence information transfer between neurons.