Circadian rhythms are self-sustained 24-hour period oscillations present in most organisms, from bacteria to human. They can be synchronized to external cues, thus allowing organisms to anticipate environmental variations and optimize their performance in nature. In Drosophila, the molecular pacemaker consists of two interlocked transcriptional feedback loops. CLOCK/CYCLE (CLK/CYC) sits in the center and drives rhythmic transcription of period (per), timeless (tim), vrille (vri) and PAR domain protein 1 (Pdp1). PER and TIM negatively feedback on CLK/CYC transcriptional activity, forming one loop, while VRI and PDP1 form the other by regulating Clk transcription negatively and positively, respectively. Posttranscriptional and posttranslational regulations also contribute to circadian rhythms. Although much has been learned about these feedback loops, we are still far from understanding how stable 24-hour period rhythms are generated. My thesis work was to determine by which molecular mechanisms kayak-α (kay-α) and Ataxin-2 (Atx2) regulate Drosophila circadian behavior. Both genes are required for the precision of circadian rhythms since knocking down either gene in circadian pacemaker neurons results in long period phenotype. The work on kay-α constitutes the first half of my thesis. We found that the transcription factor KAY-α can bind to VRI and inhibit VRI’s repression on the Clk promoter. Interestingly, KAY-α can also repress CLK’s transcriptional activity on its target genes (e.g., per and tim). Therefore, KAY-α is proposed to bring precision and stability to the molecular pacemaker by regulating both transcriptional loops. The second half of my thesis focuses on ATX2, an RNA binding protein whose mammalian homolog has been implicated in neurodegenerative diseases. We found that ATX2 is required for PER accumulation in circadian pacemaker neurons. It forms a complex with TWENTY-FOUR (TYF)—a crucial activator of PER translation—and promotes TYF’s interaction with Poly(A)-binding protein. This work reveals the role of ATX2 in the control of circadian rhythms as an activator of PER translation, in contrast to its well-established role as a repressor of translation. It also further demonstrates the importance of translational regulation on circadian rhythms. Finally, it may help understanding how ATX2 causes neuronal degeneration in human diseases.

A functional central nervous system (CNS) is composed of numerous types of neurons. Neurons are derived from a limited number of multipotent neural stem cells. Previous studies have suggested three major strategies nature uses to diversify neurons: lineage identity specification that gives an individual neural stem cell distinct identity based on its position in the developing CNS; temporal identity specification that gives neurons derived from a neural stem cell distinct identities based on their birth-order within the lineage; and binary cell fate specification that gives different identities to the two sister postmitotic neurons derived from the terminal division of a common precursor. Through the combination of the three strategies, almost unlimited neuron types can be generated. To understand neuronal diversification, we have to understand the underlying molecular mechanisms of each of the three strategies. The fruit fly Drosophila melanogaster, has been an excellent model for studying neuronal diversity, mainly due to its easily traceable nervous system and an impressive collection of genetic tools. Studies in fly have provided us fundamental insights into lineage identity, temporal identity, and binary cell fate specifications. Nevertheless, previous studies mostly centered on the embryonic ventral nerve cord (VNC) because of its simpler organization. Our understanding of the generation of neuronal diversity in the fly brain is still rudimentary. In this thesis work, I focused on the mushroom body (MB) and three antennal lobe neuronal lineages, studying their neuronal diversification during postembryonic brain development. In Chapter I, I reviewed the previous studies that have built our current understanding of the neuronal diversification. In Chapter II, I showed that MB temporal identity changes are instructed by environmental cues. In Chapter III, to search for the potential factors that mediate the environmental control of the MB temporal identity changes, I silenced each of the 18 nuclear receptors (NRs) in the fly genome using RNA interference. Although I did not identify any NR important for the regulation of MB temporal identities, I found that unfulfilled is required for regulating axon guidance and for the MB neurons to acquire all major subtype-specific identities. In Chapter IV, I demonstrated that the Notch pathway and its antagonist Numb mediate binary cell fate determination in the three classical antennal lobe neuronal lineages— anterodorsal projection neuron (adPN), lateral antennal lobe (lAL), and ventral projection neuron (vPN)—in a context-dependent manner. Finally, in Chapter V, I did detailed lineage analysis for the lAL lineage, and identified four classes of local interneurons (LNs) with multiple subtypes innervating only the AL, and 44 types projection neurons (PNs) contributing to olfactory, gustatory, and auditory neural circuits. The PNs and LNs were generated simultaneously but with different tempos of temporal identity specification. I also showed that in the lAL lineage the Notch pathway not only specifies binary cell fates, but is also involved in the temporal identity specification.

Emerging evidence in both vertebrates and invertebrates is redefining glia as active and mobile players in synapse formation, maturation and function. However, the molecular mechanisms through which neurons and glia interact with each other to regulate these processes is not well known. My thesis work begins to understand how glia use secreted factors to modulate synaptic function. We use Drosophila melanogaster, a simple and genetically tractable model system, to understand the molecular mechanisms by which glia communicate with neurons at glutamatergic neuromuscular junctions (NMJs). We previously showed that a specific subtype of glia, subperineurial peripheral glia cells (SPGs), establish dynamic transient interactions with synaptic boutons of the NMJ and is required for synaptic growth. I identified a number of potential functional targets of the glial transcription factor, reverse polarity (repo) using ChIP-chip. I found that one novel target of Repo, Wg, is expressed in SPGs and is regulated by repo in vivo. Wnt/Wg signaling plays a pivotal role during synapse development and plasticity, including the coordinated development of the molecular architecture of the synapse. While previous studies demonstrated that Wg is secreted by motor neurons, herein I provide evidence that a significant amount of Wg at the NMJ is additionally provided by glia. I found that Wg derived from SPGs is required for proper GluR distribution and electrophysiological responses at the NMJ. In summary, my results show that Wg expression is regulated by Repo in SPGs and that glial-derived Wg, together with motor neuron-derived Wg, orchestrate different aspects of synapse development. My thesis work identifies synapse stabilization and/or assembly as a new role for SPGs and demonstrates that glial secreted factors such as Wg regulate synaptic function at the Drosophila NMJ.

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.

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.

Resting-state functional magnetic resonance imaging (rs-fMRI) is a non-invasive neuroimaging technique that utilizes spontaneous low-frequency fluctuations of blood-oxygenation-level dependent (BOLD) signals to examine resting-state functional connectivity in the brain. In the past two decades, this technique has been increasingly utilized to investigate properties of large-scale functional neural networks as well as their alterations in various cognitive and disease states. However, much less is known about large-scale functional neural networks of the rodent brain, particularly in the awake state. Therefore, we attempted to unveil local and global functional connectivity in awake rat through a combination of seed-based analysis, independent component analysis and graph-theory analysis. In the current studies, we revealed elementary local networks and their global organization in the awake rat brain. We further systematically compared the functional neural networks in awake and anesthetized states, revealing that the rat brain was locally reorganized while maintaining global topological properties from awake to anesthetized states. Furthermore, specific neural circuitries of the rat brain were examined using resting-state fMRI. First anticorrelated functional connectivity between infralimbic cortex and amygdala were found to be evident with different preprocessing methods (global signal regression, regression of ventricular and white matter signal and no signal regression). Secondly the thalamocortical connectivity was mapped for individual thalamic groups, revealing group-specific functional cortical connections that were generally consistent with known anatomical connections in rat. In conclusion, large-scale neural networks can be robustly and reliably studied using rs-fMRI in awake rat, and with this technique we established a baseline of local and global neural networks in the awake rat brain as well as their alterations in the anesthetized condition.

Dopamine (DA) is central to movement, reward, learning, sleep, and anxiety. The dopamine transporter (DAT) spatially and temporally controls extracellular dopamine levels by taking DA back up into the presynaptic neuron. Multiple lines of evidence from studies using pharmacological DAT blockade or genetic DAT deletion demonstrate that DAT availability at the plasma membrane is required for maintenance of homeostatic DA levels and DA tone. Therefore, intrinsic mechanisms that regulate the transporter’s availability at the plasma membrane may directly impact downstream DA signaling cascades and DA-dependent behavior. Acute, regulated DAT internalization in response to protein kinase C (PKC) activation has been well documented, however the physiological importance of this mechanism remains untested. Due to DAT’s critical role in regulating DA levels, It is essential to understand mechanisms that acutely regulate DAT function and surface expression, and further, how these mechanisms contribute to DA related behaviors. DAT has intracellular amino and carboxy termini, which contain domains for transporter phosphorylation, recruitment to and from the plasma membrane, and sites for protein-protein interactions. To test whether these domains work synergistically for DAT function and regulated endocytosis I made DAT/SERT chimeras, in which I switched DAT’s amino, carboxy, or both termini with that of SERT, a homologous transporter with highly divergent intracellular domains. I demonstrated that DAT’s amino and carboxy termini synergistically contribute to substrate and select competitive inhibitor affinities. Additionally, I demonstrated that the amino terminus is required for PKC-stimulated DAT endocytosis, and that both N- and C-termini are required for downstream Ack1-dependent regulation of DAT endocytosis. To test the physiological importance of PKC-stimulated DAT endocytosis in vivo, I knocked down Rin, a GTPase required for PKC-stimulated DAT trafficking, in mouse DA neurons. This study was the first to achieve AAV-mediated, conditional, and inducible gene silencing in neurons. Using this AAV approach, I demonstrated a critical role for Rin GTPase signaling and DAT trafficking in both anxiety and locomotor response to cocaine. Taken together, this thesis 1) adds to the understanding of DAT functional and endocytic mechanisms and 2) is the first to report the physiological impact of Rin signaling and DAT endocytosis in DA behavior.

The minimal element of the nervous system, the synapse, is a plastic structure that has the ability to change in response to various internal and external factors. This property of the synapse underlies complex behaviors such as learning and memory. However, the exact molecular and cellular mechanisms involved in this process are not fully understood. To understand the mechanisms that regulate synapse development and plasticity I took advantage of a powerful model system, the Drosophila larval neuromuscular junction (NMJ). In this system, both anterograde and retrograde signaling pathways critical for coordinated synapse development and plasticity have been documented. An anterograde WNT/Wingless (Wg) signaling pathway plays a crucial role in both developmental and activity-dependent synaptic plasticity at the NMJ. Presynaptic motor neuron terminals secrete highly hydrophobic Wg, which travels to relatively distant postsynaptic sites where it activates a signal transduction pathway required for postsynaptic development. In the first half of my thesis I unraveled a previously unrecognized cellular mechanism by which Wg is shuttled to postsynaptic sites. In this mechanism Wg rides on secreted microvesicles or exosomes that contain a dedicated WNT secretion factor, the WNT-binding transmembrane protein, Evenness Interrupted/Wntless/Sprinter (Evi/Wls/Srt). To our knowledge, this was the first in vivo study demonstrating that neurons release exosomes, which are involved in trans-synaptic communication. Moreover, this was the first study showing that hydrophobic WNT signals are transported to the extracellular space on exosomes to reach WNT-receptor containing target cells. Retrograde signals are also critical during development and plasticity of synaptic connections. These signals function to adjust the activity of presynaptic cells according to postsynaptic cell outputs, to maintain synaptic function within a dynamic range. However, the mechanisms that trigger the release of retrograde signals and the role of presynaptic cells in this signaling event are not clear. In the second half of my thesis, I provided evidence that a crucial component of retrograde signaling at the fly NMJ, Synaptotagmin-4 (Syt4), is transmitted to the postsynaptic cell through anterograde delivery of Syt4 via exosomes. Drosophila Syt4 is known to reside on postsynaptic vesicles at the NMJ and function as a calcium sensor to release a retrograde signal upon synaptic activity. This event is required for coordinated maturation of the presynaptic terminal. We demonstrated that retrograde Syt4 function in postsynaptic muscle is required for activity-dependent presynaptic growth. However, surprisingly, Syt4 protein was not synthesized in postsynaptic muscles. Instead, Syt4 was produced in motorneurons and transferred to postsynaptic muscle cells via exosome secretion by presynaptic cells. The above study provided evidence for a presynaptic control of postsynaptic retrograde signaling through exosomal transfer of an essential retrograde signaling component. In summary, this body of work reveals a novel mechanism of trans-synaptic communication through exosomes. While intercellular communication through exosomes had been demonstrated during antigen presentation in the immune system, our studies were the first to substantiate this mode of communication in the nervous system. Thus, these studies provide a significantly deeper and novel understanding of the mechanisms underlying synapse development and plasticity.

What selective forces and molecular mechanisms govern the integration of worker body size and morphology, brain architecture, and behavior in insect societies? Workers of the remarkably polyphenic and socially complex fungus-growing leafcutter ant Atta cephalotes exhibit a striking agricultural division of labor. The number of morphologically distinct and behaviorally differentiated worker groups, adaptive mosaic neural phenotypes, and brain transcriptomes have not been examined and the influences of socioecological challenges on behavioral performance, cognition, and brain evolution are unclear. We quantified worker morphological and behavioral variation to assess the number of worker size classes and characterized their social roles. We discriminated multiple worker size groups using a Gaussian mixture model: mid-sized workers (“medias”) had the most diverse task repertories and serve dominant roles in leaf harvesting, whereas workers of other size classes performed fewer, more specialized behaviors. We used variation among tasks in sensorimotor functions and task performance frequencies to create an estimate of sensory integration and processing demands across worker size groups. This metric predicted that medias require the greatest neural investment due to the high diversity of sensory inputs and motor functions associated with their task set. We quantified the volumes of key neuropils in brains of workers of different sizes and determined their allometries, finding that our estimate corresponded to proportional investment in the mushroom bodies, a brain compartment responsible for learning, memory, and sensory integration, and identifying allometric scaling patterns in other brain centers. Additionally, we measured whole-brain gene expression and identified significant differences in expression levels for numerous genes likely to underpin behavior. Differences were most pronounced between the smallest (fungal gardener “minims”) and largest (defensive “majors”), although not all expression differences were driven by worker size. Overrepresented gene functional categories included those related to sensory processing (enriched in genes upregulated in medias and minims) and metabolism (enriched in genes upregulated in majors). These results identify the nature of selective forces favoring differentiation along morphological, neuroanatomical, behavioral, and molecular axes among A. cephalotes workers and the impact of advanced division of labor on brain evolution. / 2023-03-01T00:00:00Z

Circadian rhythms are endogenous cycles that control physiological and behavioral changes that can be affected by environmental factors which allow most eukaryotic organisms to synchronize their daily activities with the 24-hour day. Parasteatoda tepidariorum,the common house spider, demonstrates a short-period circadian clock averaging 21.6 hours when left in constant darkness, yet they are able to entrain to a 24-hour light cycle. We tested whether these spiders were able to use non-photic Zeitgebers to entrain to the 24-hour day. Periodic presentation of food and disturbance were not found to be effective cues for the spiders’ entrainment. A few individuals were clearly able to entrain to an 8 oC amplitude temperature cycle, while most did not.