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Activity Regulates Neuronal Connectivity and Function in the C. elegans Motor Circuit: A DissertationBarbagallo, Belinda 15 July 2014 (has links)
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.
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Astrocyte-Neuron Interactions Regulate Nervous System Assembly and Function: A DissertationMuthukumar, Allie 08 January 2015 (has links)
Astrocytes densely infiltrate the brain and intimately associate with synaptic structures. In the past 20 years, they have emerged as critical regulators of both synapse assembly and synapse function. During development, astrocytes modulate the formation of new synapses, and later, control refinement of synaptic connections in response to activity dependent cues. In a mature nervous system, astrocytes modulate synapse function through a variety of mechanisms. These include ion buffering, neurotransmitter uptake and the release of molecules that activate synaptic receptors. Through such roles, astrocytes shape the structure and function of neuronal circuits. However, how astrocytes and synapses reciprocally communicate during circuit assembly remains an unanswered question in the field. The vast majority of our understanding of astrocyte biology has come from studies conducted in mammals, where it is challenging to dissect molecular mechanisms with cell type specificity. Drosophila melanogaster is a less established model system for studying astrocyteneuron interactions, but its vast array of genetic tools and rapid life cycle promises great potential for precisely targeted manipulations. My thesis work has utilized Drosophila melanogaster to investigate the reciprocal nature of astrocyte-synapse communication. First, I characterized Drosophila late metamorphosis as a developmental stage in which astrocyte-synapse associations can be studied. My work demonstrates that during this time, when the adult Drosophila nervous system is being assembled, synapse formation relies on the coordinated infiltration of astrocyte membranes into the neuropil. Next, I show that in a reciprocal manner, neural activity can shape astrocyte biology during this time as well and impart long lasting effects on neuronal circuit function. In particular expression of the astrocyte GABA transporter (GAT) is modulated in an activity-dependent manner via astrocytic GABABR1/2 receptor signaling. Inhibiting astrocytic GABABR1/2 signaling strongly suppresses hyperexcitability in a Drosophila seizure model, vii arguing this pathway is important for modulating excitatory/inhibitory balance in vivo. Finally, utilizing the ease of the Drosophila system, I performed a reverse genetic screen to identify additional astrocyte factors involved in modulating excitatory-inhibitory neuronal balance.
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Synapse Development: Ribonucleoprotein Transport from the Nucleus to the Synapse: A DissertationJokhi, Vahbiz 09 March 2016 (has links)
A key process underlying synapse development and plasticity is stimulus-dependent translation of localized mRNAs. This process entails RNA packaging into translationally silent granules and exporting them over long distances from the nucleus to the synapse. Little is know about (a) where ribonucleoprotein (RNP) complexes are assembled, and if in the nucleus, how do they exit the nucleus; (b) how RNPs are transported to specific synaptic sites.
At the Drosophila neuromuscular junction (NMJ), we uncovered a novel RNA export pathway for large RNP (megaRNP) granules assembled in the nucleus, which exit the nucleus by budding through the nuclear envelope. In this process, megaRNPs are enveloped by the inner nuclear membrane (INM), travel through the perinuclear space as membrane-bound granules, and are deenveloped at the outer nuclear membrane. We identified Torsin (an AAA-ATPase that in humans is linked to dystonia), as mediator of INM scission. In torsin mutants, megaRNPs accumulate within the perinuclear space, and the mRNAs fail to localize to postsynaptic sites leading to abnormal NMJ development. We also found that nuclear envelope budding is additionally used for RNP export during Drosophila oogenesis.
Our studies also suggested that the nuclear envelope-associated protein, Nesprin1, forms striated F-actin-based filaments or ‘‘railroad tracks,’’ that span from muscle nuclei to postsynaptic sites at the NMJ. Nesprin1 railroad tracks wrap aoround the postsynaptic regions of immature synaptic boutons, and serve to direct RNPs to sites of new synaptic bouton formation. These studies elucidate novel cell biological mechanisms for nuclear RNP export and trafficking during synapse development.
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Role of Astrocytes in Sculpting Neuronal Circuits in the Drosophila CNS: A DissertationTasdemir-Yilmaz, Ozge E. 01 April 2014 (has links)
The nervous system is composed of neurons and glia. Glial cells have been neglected and thought to have only a supportive role in the nervous system, even though ~60% of the mammalian brain is composed of glia. Yet, in recent years, it has been shown that glial cells have several important functions during the development, maintenance and function of the nervous system. Glial cells regulate both pre and post mitotic neuronal survival during normal development and maintenance of the nervous system as well as after injury, are necessary for axon guidance, proper axon fasciculation, and myelination during development, promote synapse formation, regulate ion balance in the extracellular space, are required for normal synaptic function, and have immune functions in the brain. Although glia have crucial roles in nervous system development and function, there are still much unknown about the underlying molecular mechanisms in glial development, function and glial-neuronal communication.
Drosophila offers great opportunity to study glial biology, with its simple yet sophisticated and stereotypic nervous system. Glial cells in flies show great complexity similar to the mammalian nervous system, and many cellular and molecular functions are conserved between flies and mammals. In this study, I use Drosophila as a model organism to study the function of one subtype of glia: astrocytes. The role of astrocytes in synapse formation, function and maintenance has been a focus of study. However, their role in engulfment and clearance of neuronal debris during development remains unexplored.
I generated a driver line that enables the study of astrocytes in Drosophila.In chapter two of this thesis, I characterize astrocytes during metamorphosis, when extensive neuronal remodeling takes place. I found that astrocytes turn into phagocytes in a cell-autonomous, steroid-dependent manner, by upregulating the phagocytic receptor Draper and forming acidic phagolysosomal structures. I show that astrocytes clear neuronal debris during nervous system remodeling and that this is a novel function for astrocytes during the development of nervous system. I analyzed two different neuronal populations: MB γ neurons that prune their neurites and vCrz+ neurons that undergo apoptosis. I discovered that MB γ axons are engulfed by astrocytes using the Draper and Crk/Mbc/dCed-12 pathways in a partially redundant way. Interestingly, Draper is required for clearance of vCrz+ cell bodies, while Crk/Mbc/dCed-12, but not Draper, are required for clearance of vCrz+ neurites. Surprisingly, I also found that loss of Draper delayed vCrz+ neurite degeneration, suggesting that glia facilitate neurite destruction through engulfment signaling.
Taken together, my work identifies a novel function for astrocytes in the clearance of synaptic and neuronal debris during developmental remodeling of the nervous system. Additionally, I show that Crk/Mbc/dCed-12 act as a new glial signaling pathway required for pruning, and surprisingly, that glia use different engulfment pathways to clear neuronal debris generated by cell death versus local pruning.
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Increased Body Weight in Adulthood Following a Peripubertal Stressor and Proposed Mechanism for Effects of Increased Adiposity on Estrogen-dependent BehaviorsGagliardi, Christina F 07 November 2014 (has links)
Exposure to certain stressors during a sensitive period around puberty can lead to enduring effects on an animal’s response to estradiol. In estradiol-influenced behaviors, such as sexual receptivity, hippocampal-dependent learning and memory, depression-like behavior, and anxiety-like behaviors, exposure to a peripubertal stressor such as shipping stress or an injection of lipopolysaccharide (LPS) can eliminate or even reverse the normal response to estradiol. In addition to regulating these behaviors, estradiol play a role in the regulation of body weight. While some of the previous studies touched on short-term effects on body weight, no systemic long-term study of the effects of a peripubertal stressor on body weight, particularly without interruption by ovariectomy, have been undertaken. This paper introduces a hypothesis that proposes that increased adiposity following exposure to a peripubertal stressor leads to the changes to estrogen-dependent behaviors through altered levels of estrogens and changes to estrogen receptors. The first chapter examines body weight data collected during studies with other aims, and then proposes an experiment to test whether either of two peripubertal stressors results in increased weight gain and body weight. The following chapter proposes further experiments designed to determine the proximate mechanisms leading to weight gain following peripubertal stressors and the role of diet on weight gain. The final chapter proposes experiments to test the effects of adiposity on peripheral levels of testosterone, aromatase, estradiol, and estrone; central levels of estradiol and estrone; and estrogen receptors in the brain.
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Effects of a circadian mutation on adult neurogenesisBahiru, Michael 01 February 2021 (has links)
Rotating shift work, irregular sleep patterns and jetlag disrupt circadian rhythms, induce or aggravate disease, and produce deficits in cognitive function. Internal misalignment, a state in which abnormal phase relationships prevail between and within organs, is widely proposed to account for these adverse effects of circadian disruption. This hypothesis has been difficult to test because phase shifts of the entraining environmental cycle lead to transient desynchrony. Thus, it remains possible that phase shifts, regardless of internal desynchrony, account for adverse effects of circadian disruption. I have used the duper mutant hamster, whose locomotor activity rhythms re-entrain 5-fold faster than wild types after a phase shift of 8 hours, to test whether internal desynchrony can account for adverse effects of jet lag on adult neurogenesis. I subjected wild type and duper female hamsters to alternating 8h phase advances and delays of the LD cycle at 16-day intervals. I injected 5-Bromo-2’-deoxyuridine (BrdU, a thymidine analogue) after the 4th shift and collected brains after the 8th shift. As expected, mutants re-entrained activity rhythms more rapidly than did wild types. On the other hand, estrous cycles, as assessed by vaginal smears, were rarely disrupted by repeated phase shifts in either genotype.
I next compared cell proliferation and neurogenesis in the subgranular zone of the hippocampus between Duper mutants and wild type siblings using the S-phase marker BrdU and the neuronal marker NeuN. I assessed the total number of BrdU cells in the subgranular zone of the hippocampus, as the proportion that expressed NeuN. Duper mutants had more BrdU-ir cells, and more BrdU+/NeuN+ cells than did wild types, whether or not they experienced phase shifts, revealing an unexpected increase in neurogenesis. Surprisingly, repeated phase shifts increased neurogenesis in WT but not duper hamsters. Despite the increase in neurogenesis, phase shifts reduced the number of adult-born non-neuronal (BrdU+/NeuN-) cells in WT hamsters but had no such effect on duper mutants. In addition, the duper mutation increases hippocampal neurogenesis regardless of circadian. Our results suggest that adult-born non-neuronal cells are most vulnerable to circadian disruption, and that internal desynchrony promotes their demise. disruption.
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Modeling Down Syndrome Neurodevelopment with Dosage CompensationCzerminski, Jan T. 11 July 2019 (has links)
Due to their underlying genetic complexity, chromosomal disorders such as Down syndrome (DS), which is caused by trisomy 21, have long been understudied and continue to lack effective treatments. With over 200 genes on the extra chromosome, even the specific cell pathologies and pathways impacted in DS are not known, and it has not been considered a viable target for the burgeoning field of gene therapy. Recently, our lab demonstrated that the natural mechanism of dosage compensation can be harnessed to silence the trisomic chromosome in pluripotent cells. Using an inducible XIST transgene allows us to study the effects of trisomy in a tightly controlled system by comparing the same cells with either two or three active copies of chromosome 21. In addition, it raises the prospect that insertion of a single gene into a trisomic chromosome could potentially be developed in the future for “chromosome therapy”.
This thesis aims to utilize this inducible system for dosage compensation to study the neurodevelopmental effects of trisomy 21 in vitro, and to answer basic epigenetic questions critical to the viability of chromosome silencing as a therapeutic approach. Foremost, for XIST to have any prospect as a therapeutic, and to strengthen its experimental utility, it must be able to initiate chromosome silencing beyond its natural context of pluripotency. Here I demonstrate that, contrary to the current literature, XIST is capable of initiating chromosome silencing in differentiated cells and producing fully dosage compensated DS neurons. Additionally, I show that silencing of the trisomic chromosome in neural stem cells enhances their terminal differentiation to neurons, and transcriptome analysis provides evidence of a specific pathway involved. Separate experiments utilize novel three-dimensional organoid technology and transcriptome analysis to model DS neurodevelopment in relation to isogenic euploid cells. Overall, this work demonstrates that dosage compensation provides a powerful experimental tool to examine early DS neurodevelopment, and establishes that XIST function does not require pluripotency, thereby overcoming a perceived obstacle to the potential of XIST as a therapeutic strategy for trisomy.
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Early Rearing Experience, Hypothalamic-Pituitary-Adrenal (HPA) Activity, and Serotonin Transporter Genotype: Influences on the Development of Anxiety in Infant Rhesus Monkeys (Macaca mulatta)Dettmer, Amanda 01 May 2009 (has links)
A gene x environment interaction exists in the expression of anxiety for both human and nonhuman primates, such that individuals who are carriers of the (s) allele of the serotonin transporter genotype ( 5-HTT LPR) and exposed to early life stress are more at risk for exhibiting anxiety. The hypothalamic-pituitary-adrenal (HPA) axis has also been implicated in anxiety disorders but the relationship between early life/genotype, HPA activity, and anxiety is not well understood. Further, studies linking the HPA axis to anxiety have relied on "point" samples (blood and salivary cortisol) which reflect moments in time rather than long-term activity. The purpose of this dissertation was three fold: (1) to examine anxious behavior in monkeys with different 5-HTT LPR genotypes and rearing environments across the first two years of life, (2) to compare long-term HPA activity (as measured with hair cortisol) with acute HPA activity (as measured with salivary cortisol) in the same period, and (3) to determine which measure of HPA activity predicts anxiety and/or mediates the rearing/genotype influences on anxious behavior. Infant rhesus monkeys ( Macaca mulatta , N=61) were mother-peer-reared (MPR, n=21), peer-reared (PR, n=20), or surrogate-peer-reared (SPR, n=20) for 8 months, then all relocated into a large social housing situation for the next 18 months. Monkeys were genotyped for 5-HTT LPR and hair and saliva samples were collected for cortisol analysis at months 6, 12, 18, and 24. Behavior was recorded twice per week per subject from 2-24 months and analyzed for the duration of anxiety, social play, and grooming. Regression analysis established predictors of these behaviors. Rearing condition and sex were significant predictors of anxiety across the two years, and HPA activity added significant predictive power in the first six months only. Mediation of the rearing/anxiety relationship by the HPA axis was not evident. Interestingly, hair (but not salivary) cortisol early in life was positively correlated with later anxious behavior. These findings demonstrate the detrimental effects of adverse early life experience on behavioral development and shed light on the interplay between environment, adrenocortical activity, and anxiety. They further demonstrate the usefulness of a long-term measure of HPA activity in predicting later behavior.
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Working Memory Impairments in Chromosome 22q11.2 Deletion Syndrome: The Roles of Anxiety and Stress PhysiologySanders, Ashley F. P. 13 May 2016 (has links)
Stress and anxiety negatively impact the working memory system by competing for executive resources. Broad memory deficits have been reported in individuals with chromosome 22q11.2 deletion syndrome (22q11.2DS). We investigated anxiety and physiological stress reactivity in relation to visuospatial working memory impairments in 20 children with 22q11.2DS and 32 typically developing children (M = 11.10 years, SD = 2.95). Results indicate reduced post-stress RSA recovery and overall increased levels of cortisol in children with 22q11.2DS. Additionally, anxiety mediated the relationship between 22q11.2DS and visuospatial working memory impairment. However, there was no indication that stress response physiology mediated this association. Results suggest that anxiety exacerbates impaired working memory in children with 22q11.2DS. Thus, treatment and intervention methods for children with 22q11.2DS should address anxiety related symptomology.
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ROLE OF SOX11 DURING VERTEBRATE OCULAR MORPHOGENESIS AND RETINAL NEUROGENESISPillai, Lakshmi Shashidharan 01 January 2015 (has links)
Microphthalmia, anophthalmia, and coloboma (MAC) are distinct abnormalities demonstrating a continuum of developmental eye defects that contribute to 15-20% of blindness and severe vision deficiencies in children worldwide. The genetic etiology of MAC is large, complex and encompasses the whole developmental biology of the eye. Understanding how the eye develops will aid in identifying genes and developmental pathways involved in MAC. Although investigation of the genetic architecture of congenital anomalies is growing exponentially, much work remains to be accomplished to understand the complex, genetically heterogeneous congenital anomalies, which significantly impact childhood vision.
With an interest in elucidating the mechanisms that are involved in eye morphogenesis, I have characterized a SRY-Box transcription factor, Sox11, during zebrafish ocular development. The SRY (sex determining region Y)-box 11 (sox11) gene, codes for a transcription factor which functions as a regulator of cell fate, survival, and differentiation in the embryonic and adult nervous system. By titrating the levels of sox11 gene function in developing zebrafish embryos I have investigated the role of Sox11 during ocular morphogenesis and retinal neurogenesis. Chapter 1 of this dissertation provides a review of vertebrate eye development with a focus on emerging roles of SoxC proteins during vertebrate ocular morphogenesis. Chapter 2 presents data demonstrating that knockdown of both paralogs of sox11 in zebrafish results in microphthalmia, coloboma, as well as a specific deficit in mature rod photoreceptors. Additionally, we demonstrate for the first time that Sox11 regulates early ocular and photoreceptor development in part by maintaining proper levels of Hedgehog (Hh) signaling. Deficiency of Sox11 results in elevated Sonic Hedgehog a (Shha) transcript levels, which in turn leads to improper patterning of the optic vesicle into the proxio-distal territories. Furthermore, the data indicate that alterations in SOX11 gene dosage or mutation within the SOX11 coding region are potentially disease causing and contribute to human ocular defects like MAC and rod dysfunction. Chapter 3 presents data indicating that sox11 gene function is required during the critical period of neurulation (4-10 hours post fertilization) to guide choroid fissure closure and proper ocular morphogenesis to occur in the developing zebrafish. Chapter 4 is a technical report on the progress towards generating stable sox11a/b knockout zebrafish lines using the CRISPR/Cas9 genome editing approach. Transient F0 injected embryos and F0 adults carry mutations in the sox11a/b target site in addition to displaying ocular abnormalities similar to those previously reported in sox11 morphants. F1 juveniles are ready to be screened for establishment of mutagenesis efficiency and germ line transmission. Finally, in Chapter 5 I discuss how the results of each chapter demonstrate the functional requirement of Sox11 for ocular development. Furthermore, I discuss the implications of this work in the field of developmental biology and how the current data will shape future investigations. My dissertation incorporates human genetics, biochemical analyses, and zebrafish reverse genetic analyses, and will help in better understanding the exact role of Sox11 during eye development at the cellular and molecular level. Moreover, by identifying Sox11 targets belonging to the Hh pathway, as well as novel targets of Sox11 regulation, these studies may also contribute to our understanding of the function of Sox11in development and disease pathogenesis.
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