Dissertations / Theses on the topic 'Elegans synapse'

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.

Thesis (Ph. D.)--University of California, San Diego, 2010.
Title from first page of PDF file (viewed June 3, 2010). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (leaves 89-95).

Proper synaptic connectivity is critical for communication between cells and information processing in the brain. Neurons are highly interconnected, forming synapses with multiple partners, and these connections are often refined during the course of development. While decades of research have elucidated many molecular players that regulate these processes, understanding their specific roles can be difficult due to the large number of synapses and complex circuitry in the brain. In this thesis, I investigate mechanisms that establish neural circuits in the simple organism C. elegans, allowing us to address this important problem with single cell resolution in vivo. First, I investigate remodeling of excitatory synapses during development. I show that the immunoglobulin domain protein OIG-1 alters the timing of remodeling, demonstrating that OIG-1 stabilizes synapses in early development but is less critical for the formation of mature synapses. Second, I explore how presynaptic excitatory neurons instruct inhibitory synaptic connectivity. My work shows that disruption of cholinergic neurons alters the pattern of connectivity in partnering GABAergic neurons, and defines a time window during development in which cholinergic signaling appears critical. Lastly, I define novel postsynaptic specializations in GABAergic neurons that bear striking similarity to dendritic spines, and show that presynaptic nrx-1/neurexin is required for the development of spiny synapses. In contrast, cholinergic connectivity with their other postsynaptic partners, muscle cells, does not require nrx-1/neurexin. Thus, distinct molecular signals govern connectivity with these two cell types. Altogether, my findings identify fundamental principles governing synapse development in both the developing and mature nervous system.

Le travail réalisé au cours de cette thèse vise à comprendre par quels processus les récepteurs de neurotransmetteur sont localisés à la synapse en utilisant la jonction neuromusculaire du nématode Caenorhabditis elegans comme modèle. L’agrégation des récepteurs se fait en règle générale par l’intermédiaire de protéines cytoplasmiques. En revanche, nous avons montré que, chez C. Elegans, la localisation des récepteurs de l’acétylcholine (RACh) se faisait par le domaine extracellulaire d’une protéine transmembranaire déjà caractérisée, LEV-10, et d’une nouvelle protéine extracellulaire, LEV-9. LEV-9 est sécrétée par les cellules musculaires et est localisée à la JNM. Les 3 protéines RACh, LEV-9 et LEV-10 sont nécessaires pour former un complexe protéique qui se localise à la synapse. De façon intrigante, LEV-9 contient huit domaines CCP (complement control protein), qui sont habituellement présents dans les protéines impliquées dans la voie du complément chez les mammifères. Comme la voie du complément n’existe pas chez les protostomiens et que de nombreuses protéines à domaines CCP sont exprimées dans le cerveau des mammifères, nous proposons que ces protéines à multiples domaines CCP fonctionnaient ancestralement dans le système nerveux

In the nervous system, billions of neurons undergo a multistep process to establish functional circuits. This entails accurate extension of dendritic and axonal processes and coordinated efforts of pre- and postsynaptic neurons to form synaptic connections. Although many axon guidance molecules and synaptic organizers have been identified, the molecular redundancy and the vast number of synapses in the brain has complicated attempts to define their precise roles. In order to understand the molecular mechanisms that encompass these processes, my studies utilize the genetic strengths and cellular precision available in Caenorhabditis elegans for in vivo investigations of nervous system development. In this work, I unravel cell-specific requirements for the transmembrane receptor integrin in regulating developmental axon guidance of GABAergic motor neurons. Furthermore, I address important questions about mechanisms of synapse formation and maintenance using a novel dendritic spine model in C. elegans. Using high resolution microscopy, I find that the formation of immature presynaptic vesicles and postsynaptic receptors are established prior to the outgrowth of dendritic spines at nascent synapses. During this early period of synapse formation, the kinesin-3 family protein UNC-104/KIF1A transports a transsynaptic adhesion molecule neurexin/NRX-1 to developing active zones, in order to maintain postsynaptic receptors and dendritic spines in the mature circuit. In the absence of nrx-1, spines initially form normally but collapse following their extension. These findings demonstrate that presynaptic NRX-1 is required to maintain postsynaptic structures. Together my work provides new insights into molecular mechanisms that define spatiotemporal characteristics of nervous system development and the maintenance of connectivity.

Le système nerveux est un réseau complexe qui détecte et analyse les informations. Ces informations sont transmises entre cellules grâce à des connections synaptiques et des jonctions communicantes. Ce réseau n’est pas statique et évolue au cours du développement, de l’apprentissage mais aussi durant le processus de vieillissement – naturels ou pathologiques. Comprendre le système nerveux et son fonctionnement requiert une analyse des connections synaptiques in vivo chez un animal model tel que Caenorhabditis elegans. Cependant les techniques actuellement disponibles pour C. elegans sont laborieuses, ne dépendent pas forcément d’une interaction trans-synaptique ou fixent la synapse. Par conséquent, ces approches ne permettent pas de réaliser des études de populations et dynamiques des modifications synaptiques. Dans ce manuscrit, je décris tout d’abord une nouvelle technique pour visualiser les synapses in vivo chez le vers C. elegans. Cette technique appelé iBLINC (in vivo Biotin Labeling of INtercellular Contacts) qui consiste en la biotinylation d’un peptide par une ligase d’Escherichia coli, BirA. Ces deux molécules sont fusionnées à des protéines trans-membranaires qui forment un complexe à la synapse. La biotinylation est détectée grâce à une streptavidin monomérique taguée avec un fluorophore qui est secrétée dans l’espace extracellulaire. J’ai démontré que cette technique est directionnelle et dynamique. En utilisant iBLINC pour visualiser des synapses faisant partie du circuit sensoriel de C. elegans, une évolution du nombre et de la taille des synapses a pu être observée avec l’âge. Il semblerait que ce changement soit dépendant du segment de la zone synaptique observée. Ces résultats ont été corroborés par l’observation d’une diminution du nombre de vésicules pendant le vieillissement grâce à un marqueur pré-synaptique des synapses GABAergique de la jonction neuromusculaire. Pour conclure, ce manuscrit décrit une nouvelle technique permettant d’observer les synapses chez le vers vivant et démontre une évolution naturelle du nombre de synapses et du nombre de vésicules pré-synaptiques avec l’âge
The nervous system is a complex network that senses and processes information and is essential for the survival of both vertebrates and invertebrates as it is involved in behavior responses. Information within the network is transmitted through specialized cell-cell contacts, including synaptic connections. Importantly, the network is not static and is believed to change during development and learning, as well as during pathological or normal age-related decline. Studying the nervous system in vivo requires the use of animal models such as Caenorhabditis elegans. Understanding of behavior and development requires visualization and analysis of synaptic connectivity. However, existing methods are laborious and may not depend on trans-synaptic interactions, or otherwise ‘trap’ the synapses by fixing the connections, thus precluding dynamic studies. In order to study synaptic modifications, we developed a new transgenic approach for in vivo labeling of specific connections in C. elegans, called iBLINC (in vivo Biotin Labeling of INtercellular Contacts). iBLINC involves the biotinylation of an acceptor peptide (AP) by the Escherichia coli biotin ligase BirA. Both are fused to two interacting post- and pre-synaptic proteins, respectively. The biotinylated acceptor peptide fusion is detected by a monomer streptavidin fused to a fluorescent protein that is transgenically expressed and secreted into the extracellular space. The method is directional, bright and dynamic. Moreover it correlates well with electron micrograph reconstruction. Using this new technique to observe synapses, which are part of the thermosensory circuitry of C. elegans, during aging, we could conclude that the connection pattern varies with age and within a population. Changes of the number and size of the synapses were observed during aging. Some segments of the synaptic region seem to be more affected than others by the aging process. Those results were corroborated by using a GABAergic pre-synaptic marker which allowed us to visualize a decline of the vesicle number with aging. In summary, in this thesis I explained a new in vivo trans-synaptic method to visualize synapses in C. elegans. Then I demonstrated that a natural decline in the number of synapses as well as the number of vesicles occurs during aging

Proper chromosome segregation at meiosis I depends on the initial alignment of homologous chromosomes, the establishment of the synaptonemal complex (SC), and the formation of chiasma between homologs. Previous studies have demonstrated that HIM-3, a component of the meiotic chromosome axis, is required for these processes through the recruitment of the autosomal pairing center proteins (ZIMs) and of SC components like SYP-1. The him-3(vv6) mutation results in the substitution of a highly conserved residue in the HORMA domain, believed to mediate protein-protein interactions. him-3(vv6) mutant germlines display immature and discontinuous axes at early meiosis and the autosomal pairing center (PC) protein ZIM-3 fails to localize properly to the pairing centers, despite the fact that HIM-3vv6 is appropriately expressed and localized; mutants exhibit severe defects in homolog alignment, extensive nonhomologous synapsis and defects in chiasma formation, resulting in a high embryonic lethality (emb) and a high incidence of male (him) phenotype as consequences of chromosome missegregation. To identify proteins that interact with HIM-3 in early meiotic processes and other proteins that function in the HIM-3 pathway, an EMS-based suppressor screen was performed using the vv6 allele and a strong extragenic suppressor, vv39 was isolated. vv39 was identified as a novel allele of cct-4, which encodes the delta subunit of type II chaperonin complex. In wild-type germlines, CCT-4 is localized to the cytoplasm and to the nucleus, indicating that it has a nuclear role. In germlines depleted for cct-4 or other subunits of the CCT complex, axis assembly is delayed, ZIM-3 fails to be recruited to the PCs and SYP-1 fails to localize to the axes, suggesting that the CCT complex is required for morphogenesis of meiotic chromosome axes competent for meiotic processes. In him-3(vv6); cct-4(vv39) mutants, the timely morphogenesis of chromosome axes is appears to be restored and is accompanied by ZIM-3 recruitment to PCs, improved homologue alignment and reduced nonhomologous synapsis. These results collectively suggest that CCT-4 mediates timely axes morphogenesis through CCT-mediated folding of axis component HIM-3 or its interactors. This study is the first to provide insight on the function of molecular chaperonin in mediating meiotic processes and reveals an unprecedented nuclear function for the complex. It would be interesting in the future to further study the nuclear CCT chaperonin complex and its clients to learn more about their roles in meiotic processes.
La ségrégation des chromosomes lors de la méiose I dépend de l'alignement initial de chromosomes homologues, la mise en place du complexe synaptonémal (SC), et la formation de chiasmas entre les homologues. Des études antérieures ont démontré que HIM-3, une composante de l'axe du chromosome méiotique, est requis pour ces processus par le recrutement des protéines autosomique centre de liaison (ZIMS) et des composants SC comme SYP-1. Le him-3(vv6) mutation se traduit par la substitution d'un résidu hautement conservé dans le domaine HORMA, considérés comme des médiateurs des interactions protéine-protéine. him-3(vv6) germlines mutant affichage des axes immature et discontinue à la méiose précoce et le centre autosomique appariement (PC) protéine ZIM-3 ne parvient pas à localiser correctement vers les centres de liaison, malgré le fait que HIM-3vv6 est correctement exprimé et localisées; mutants présentent des défauts graves dans homologue alignement, non homologue vaste synapse et des défauts dans la formation des chiasmas, entraînant une mortalité élevée embryonnaires (emb) et une incidence élevée de sexe masculin (him) comme des conséquences de phénotype missegregation chromosome. Pour identifier les protéines qui interagissent avec HIM-3 au début du processus de la méiose et d'autres protéines qui fonctionnent dans la voie HIM-3, un crible EMS à base de suppresseur a été réalisée avec l'allèle vv6 et un suppresseur de forte extragénique, vv39 a été isolé. vv39 a été identifié comme un nouvel allèle de cct-4, qui code pour la sous-unité delta du complexe chaperonine type II. En germlines de type sauvage, cct-4 est localisée dans le cytoplasme et le noyau, ce qui indique qu'il a un rôle nucléaire. En germlines appauvri pour cct-4 ou d'autres sous-unités du complexe du CCT, le montage axe est retardée, ZIM-3 ne parvient pas à être recrutés pour les PC et SYP-1ne parvient pas à localiser les axes, ce qui suggère que le complexe CCT est nécessaire pour la morphogenèse des axes chromosome méiotique compétente pour les processus de la méiose. En him-3(vv6); cct-4 (vv39) mutants, la morphogenèse en temps opportun des axes chromosome est semble être restauré et il est accompagné par ZIM-3 de recrutement pour les PC, l'alignement homologue accrue et une réduction non homologue synapsis. Ces résultats suggèrent collectivement que CCT-4 est un médiateur de axes morphogenèse par pliage de l'axe composante HIM-3 ou des interacteurs du HIM-3. Cette étude est la première à donner un aperçu sur la fonction de chaperonine moléculaire dans la médiation des processus méiotique. Il serait intéressant à l'avenir d'approfondir l'étude du complexe nucléaire chaperonine CCT et de ses clients à en apprendre davantage sur leur rôle dans les processus de la méiose.

Les récepteurs nicotiniques de l’acétylcholine (RAChs) appartiennent à une famille de protéines transmembranaires très conservée et impliquée dans de nombreux processus physiologiques et pathologiques chez l'homme. Le contrôle de l'expression et de la localisation des RAChs est fondamental pour la formation, la maintenance et la plasticité synaptique. J'ai utilisé une stratégie génétique chez le nématode Caenorhabditis elegans afin d’identifier de nouveaux gènes requis pour la biosynthèse et la localisation synaptique des RAChs. J'ai initialement réalisé un crible visant à identifier des gènes dont l'invalidation altère la localisation des RAChs à la jonction neuromusculaire (JNM). Après inactivation par ARN interférence de plus d'un millier de gènes évolutivement conservés, j'ai validé 18 candidats requis pour le fonctionnement de la JNM cholinergique. J'ai ensuite réalisé la caractérisation du gène emc 6 dont un allèle mutant avait été isolé au cours d'un crible de résistance au lévamisole, un agoniste cholinergique. EMC-6 est une petite protéine très conservée localisée dans le réticulum endoplasmique (RE). En l'absence d'EMC-6, les sous-unités du RACh en cours d'assemblage sont dégradées. EMC-6 appartient à un complexe évolutivement conservé appelé EMC (ER protein Membrane Complex). Chez C. Elegans, l'inactivation de l'EMC par ARNi provoque des défauts développementaux, une diminution de l'expression des RAChs, et l'activation de rapporteurs du stress du RE. Ces résultats suggèrent que l'EMC protège les RAChs de la dégradation pendant leur maturation et est requis pour le maintien de l'homéostasie du RE en conditions physiologiques
Nicotinic acetylcholine receptors (AChRs) belong to a widely conserved family of transmembrane protein implicated in numerous physiological and pathological processes in human. Control of AChR expression and localisation is essential for the synapse formation, stability and plasticity. I used a genetic approach in the nematode Caenorhabditis elegans to identify novel genes required for biosynthesis and synaptic localization of AChRs. First, I performed a visual screen for genes required for proper localization of AChRs at neuromuscular junction (NMJ). After inactivation of more than 1000 evolutionarily conserved genes by RNA interference, I validated 18 candidates required for cholinergic NMJ function. Second, I realized the functional and molecular characterization of the novel gene emc-6. Emc-6 mutant was identified in a screen for mutants partially resistant to levamisole, a cholinergic agonist. EMC-6 is a small protein widely conserved from plant to human and localized in the endoplasmic reticulum (ER). When EMC-6 is absent, AChR subunits are targeted to degradation. EMC-6 belongs to the conserved ER Membrane protein Complex (EMC). In C. Elegans, RNAi against EMC members causes developmental defects, decreased AChR expression and activation of ER stress reporters. These results suggest that EMC protects AChR subunit from degradation and is required for ER homeostasis in metazoans in physiological conditions

Activity plays diverse roles in shaping neuronal development and function. These roles range from aiding in synaptic refinement to triggering cell death during traumatic brain injury. Though the importance of activity-dependent mechanisms is widely recognized, the genetic underpinnings of these processes have not been fully described. In this thesis, I use the motor circuit of Caenorhabditis elegans as a model system to explore the functional and morphological consequences of modulating neuronal activity. First, I used a gain-of-function ionotropic receptor to hyperactivate motor neurons and asked how increased excitation affects neuronal function. Through this work, I identified a cell death pathway triggered by excess activation of motor neurons. I also showed that suppression of cell body death failed to block motor axon destabilization, providing evidence that death of the cell body and of motor axons can be genetically separated. Secondly, I removed excitatory drive from a simple neural circuit and asked how loss of excitatory activity alters circuit development and function. I identified excitatory motor neurons as master regulators of inhibitory synaptic connectivity. Additionally, I was able to identify previously undescribed activity-dependent mechanisms for regulating inhibitory synapses in both developing and mature neural circuits. Finally, I show data to implicate the highly conserved genes neurexin and neuroligin in determining inhibitory synapse connectivity. Collectively this work has lent insight into activity-dependent mechanisms in place to regulate neuronal development and function, a core function of neurobiology that is relevant to the study of a wide range of neurological disorders.

Activity plays diverse roles in shaping neuronal development and function. These roles range from aiding in synaptic refinement to triggering cell death during traumatic brain injury. Though the importance of activity-dependent mechanisms is widely recognized, the genetic underpinnings of these processes have not been fully described. In this thesis, I use the motor circuit of Caenorhabditis elegans as a model system to explore the functional and morphological consequences of modulating neuronal activity. First, I used a gain-of-function ionotropic receptor to hyperactivate motor neurons and asked how increased excitation affects neuronal function. Through this work, I identified a cell death pathway triggered by excess activation of motor neurons. I also showed that suppression of cell body death failed to block motor axon destabilization, providing evidence that death of the cell body and of motor axons can be genetically separated. Secondly, I removed excitatory drive from a simple neural circuit and asked how loss of excitatory activity alters circuit development and function. I identified excitatory motor neurons as master regulators of inhibitory synaptic connectivity. Additionally, I was able to identify previously undescribed activity-dependent mechanisms for regulating inhibitory synapses in both developing and mature neural circuits. Finally, I show data to implicate the highly conserved genes neurexin and neuroligin in determining inhibitory synapse connectivity. Collectively this work has lent insight into activity-dependent mechanisms in place to regulate neuronal development and function, a core function of neurobiology that is relevant to the study of a wide range of neurological disorders.

The brain network is a multiscale hierarchical organization from neurons and local circuits to macroscopic brain areas. The precise synaptic transmission at each synapse is therefore crucial for neural communication and the generation of orchestrated behaviors. Activation of presynaptic voltage-gated calcium channels (CaV2) initiates synaptic vesicle release and plays a key role in neurotransmission. In this dissertation, I have aimed to uncover how CaV2 activity affects synaptic transmission, circuit function and behavioral outcomes using Caenorhabditis elegans as a model. The C. elegans genome encodes an ensemble of highly conserved neurotransmission machinery, providing an opportunity to study the molecular mechanisms of synaptic function in a powerful genetic system. I identified a novel gain of function CaV2α1 mutation that causes CaV2 channels to activate at a lower membrane potential and slow the inactivation. Cell-specific expression of these gain-of-function CaV2 channels is sufficient to hyper-activate neurons of interest, offering a way to study their roles in a given circuit. CaV2(gf) mutants display behavioral hyperactivity and an excitation-dominant synaptic transmission. Imbalanced excitation and inhibition of the nervous system have been associated with several neurological disorders, including Familial Hemiplegic Migraine type 1 (FHM1) which is caused by gain- of-function mutations in the human CaV2.1α1 gene. I showed that animals carrying C. elegans CaV2α1 transgenes with corresponding human FHM1 mutations recapitulate the hyperactive behavioral phenotype exhibited by CaV2(gf) mutants, strongly suggesting the molecular function of CaV2 channels is highly conserved from C. elegans to human. Through performing a genome-wide forward genetic screen looking for CaV2α(gf) suppressors, we isolated new alleles of genes that required for CaV2 trafficking, localization and function. These regulators include subunits of CaV2 channel complex, components of synaptic and dense core vesicle release machinery as well as predicted extracellular proteins. Taken together, this work advances the understanding of CaV2 malfunction at both cellular and circuit levels, and provides a genetically amenable model for neurological disorders associated with excitation-inhibition imbalance. Additionally, through identifying regulators of CaV2, this research provides new avenues for understanding the CaV2 channel mediated neurotransmission and potential pharmacological targets for the treatments of calcium channelopathies.

The brain network is a multiscale hierarchical organization from neurons and local circuits to macroscopic brain areas. The precise synaptic transmission at each synapse is therefore crucial for neural communication and the generation of orchestrated behaviors. Activation of presynaptic voltage-gated calcium channels (CaV2) initiates synaptic vesicle release and plays a key role in neurotransmission. In this dissertation, I have aimed to uncover how CaV2 activity affects synaptic transmission, circuit function and behavioral outcomes using Caenorhabditis elegans as a model. The C. elegans genome encodes an ensemble of highly conserved neurotransmission machinery, providing an opportunity to study the molecular mechanisms of synaptic function in a powerful genetic system. I identified a novel gain of function CaV2α1 mutation that causes CaV2 channels to activate at a lower membrane potential and slow the inactivation. Cell-specific expression of these gain-of-function CaV2 channels is sufficient to hyper-activate neurons of interest, offering a way to study their roles in a given circuit. CaV2(gf) mutants display behavioral hyperactivity and an excitation-dominant synaptic transmission. Imbalanced excitation and inhibition of the nervous system have been associated with several neurological disorders, including Familial Hemiplegic Migraine type 1 (FHM1) which is caused by gain- of-function mutations in the human CaV2.1α1 gene. I showed that animals carrying C. elegans CaV2α1 transgenes with corresponding human FHM1 mutations recapitulate the hyperactive behavioral phenotype exhibited by CaV2(gf) mutants, strongly suggesting the molecular function of CaV2 channels is highly conserved from C. elegans to human. Through performing a genome-wide forward genetic screen looking for CaV2α(gf) suppressors, we isolated new alleles of genes that required for CaV2 trafficking, localization and function. These regulators include subunits of CaV2 channel complex, components of synaptic and dense core vesicle release machinery as well as predicted extracellular proteins. Taken together, this work advances the understanding of CaV2 malfunction at both cellular and circuit levels, and provides a genetically amenable model for neurological disorders associated with excitation-inhibition imbalance. Additionally, through identifying regulators of CaV2, this research provides new avenues for understanding the CaV2 channel mediated neurotransmission and potential pharmacological targets for the treatments of calcium channelopathies.

Synapses are specialized sub-cellular junctions that transmit signals between neurons and their targets. In Caenorhabditis elegans (C. elegans) the F-box protein FSN-1 and the PHR family member RPM-1 form the SCFFSN-1 E3 ubiquitin ligase, which plays an important role in regulating synaptic growth factors. This SCF complex is evolutionarily conserved across species, and regulates many cellular processes including axon outgrowth, apoptosis and synaptogenesis. This thesis focuses on identifying targets of SCFFSN-1 that contribute to synaptogenesis. Forward genetics was employed to screens and isolate mutants that exhibit genetic interactions with fsn-1. I have identified an allele of the MAPK pmk-3(hp246) and three alleles of the MAPKKK dlk-1(hp180, hp192, hp195) that suppress fsn-1 defects. In addition, I have isolated five fsn-1 suppressing alleles and evidence suggests that these suppressors are likely novel fsn-1 suppressors.

Synaptogenesis entails the development and establishment of functional synapses, which form the fundamental unit of communication in the nervous system. Initially identified in Caenorhabditis elegans (C. elegans), the FSN-1, F-box protein family has emerged as evolutionarily conserved binding partners of PHR family proteins, which regulate synaptogenesis. Previously, we have shown that FSN-1 and RPM-1 form a SCF/FSN-1/RPM-1 ubiquitin ligase complex that negatively regulates synapse growth by downregulating presynaptic targets, like the MAP kinase pathway. For my master’s thesis, I used a combination of both candidate and forward genetic approaches to identify additional components of signaling pathways that are regulated by FSN-1 during synaptogenesis. Our studies are among the first to suggest diverging roles for these partners and provide the first evidence for a mechanism through which the F-box protein regulates synaptogenesis via retrograde insulin/IGF/FOXO signaling and glucosaminidase/O-GlcNAc modifications.

The nematode C.elegans, with its 302 neurons and abundance of genetic, laser ablation, electrophysiological and imaging tools, is a compact and attractive system for neural circuit analysis. An understanding of the functional dynamics of neural computation requires physiological analyses. We undertook the first characterization of transfer at central synapses in C.elegans. To achieve this we employed optical stimulation techniques using channelrhodopsin-2, and combined this with whole-cell patch clamp electrophysiological recording techniques. We show that the synapse between AFD and AIY, the first stage in the thermotactic circuit, exhibits excitatory, tonic and graded release. The gain at the synapse was low (<0.1), and release was frequency independent, showing no signs of facilitation or depression. The AFD-AIY synapse thus seems designed for robust and reliable transmission of a scaled-down temperature signal from AFD to AIY, enabling AIY to continuously monitor temperature information and integrate it with other incoming sensory information. We also investigated the synapse between ASER, a chemosensory neuron, and AIY, and found that the synaptic response was small and inconsistent. The combination of optical stimulation tools with neural recording techniques is a powerful way to analyze neural circuitry, and will be a significant aid in achieving the goal of understanding how information is processed in the compact yet densely interconnected nervous system of the worm.