Perception is the result of more than just the unbiased processing of sensory stimuli. At each moment in time, sensory inputs enter a circuit already impacted by signals of arousal, attention, and memory. This thesis aims to understand the impact of such internal states on the processing of sensory stimuli. To do so, computational models meant to replicate known biological circuitry and activity were built and analyzed. Part one aims to replicate the neural activity changes observed in auditory cortex when an animal is passively versus actively listening. In part two, the impact of selective visual attention on performance is probed in two models: a large-scale abstract model of the visual system and a smaller, more biologically-realistic one. Finally in part three, a simplified model of Hebbian learning is used to explore how task context comes to impact prefrontal cortical activity. While the models used in this thesis range in scale and represent diverse brain areas, they are all designed to capture the physical processes by which internal brain states come to impact sensory processing.

How neurons choose appropriate synaptic partners to form functional neural circuits is not well understood. Two subfamilies of Drosophila immunoglobulin superfamily (IgSF) cell surface proteins, Dprs (defective proboscis response) and DIPs (Dpr interacting proteins) are broadly expressed in the nervous system and involved in the development of neural circuits. A qualitative interactome developed from high-throughput experiments has shown that each DIP interacts with a unique set of Dpr proteins. Neurons with distinct synaptic specificities express distinct combinations of Dprs, while a subset of their synaptic partners express the complementary DIPs. These findings are consistent with the idea that the specificity of interactions between Dprs and DIPs help to define the synaptic connectivity of the neurons in which they are expressed. Thus, it is essential to fully understand interactions between members of these two protein families. Using surface plasmon resonance (SPR), we have generated a quantitative Dpr and DIP interactome, which contained several novel features. We determined the binding affinities of the majority of Dpr-DIP interactions, revealing binding groups that span a range of affinities and reflect DIP and Dpr phylogeny. Crystal structures of Dpr-DIP heterocomplexes were determined and used to design site-specific mutants that, along with SPR experiments, reveal the major determinants of Dpr-DIP binding specificity. Using analytical ultracentrifugation (AUC), we show that some Dpr and DIP family members form homophilic dimers as well. Multiple crystal structures of DIP homodimers reveal the molecular determinants of homophilic binding and structure-guided mutants along with AUC experiments further validated their mechanism of interaction. The existence of DIP and Dpr homodimers suggests the possibility of still-unknown mechanisms of Dprs and DIPs in neural circuit formation. Based on information derived from our crystal structures and biophysical experiments, we designed, produced, and tested Dpr and DIP proteins with altered binding properties. Many of the structural and biophysical studies described in this thesis were undertaken to produce tools to probe Dpr and DIP function in an in vivo setting. Parallel studies utilizing many of the mutant proteins described here (and other reagents that are not described here) are underway in the Zipursky lab, and are not described herein.

Intra-axonal protein synthesis is important for the proper wiring of the nervous system and can have restorative or pathogenic effects in response to nerve injury and neurodegenerative stimuli. The set of axonally translated transcripts, the axonal translatome, is regulated through the control of mRNA localization, stability, and translation. Targeting the axonal translatome could result in the development of novel therapies for the treatment of neurological disorders. Yet, there are gaps in our understanding of the selective mechanism regulating the specific localization of mRNAs into axons. Currently, axonal localization of transcripts is understood to be controlled by the presence of sequence elements that direct axonal transport. In an attempt to identify novel localization motifs, I found that a well-known motif corresponding to the Pumilio Binding Element (PBE) is significantly depleted in axonally enriched mRNAs. Moreover, I found this element to be highly informative of axonal mRNA localization and translation across different neuronal types and developmental stages suggesting that it is a highly conserved regulatory motif. I found Pum2 neuronal expression and subcellular localization to be highly consistent with the way the PBE predicts mRNA regulation. I then demonstrated that interfering with Pum2 function results in increased axonal localization of PBE containing mRNAs. Finally, Pum2 downregulation was associated with gross defects in axonal outgrowth, branching, and regeneration. Altogether, this data suggests that Pum2 regulates axonal mRNA localization through an exclusion mechanism that is important during neuronal development.

Alternative splicing of pre-mRNA is a critical mechanism for enabling genetic diversity, and is a carefully regulated process in neuronal differentiation. RNA binding proteins (RBPs) are developmentally expressed and physically interact with RNA to drive specific splicing changes. This work tests the hypothesis that RBP-RNA interactions are critical for regulating timed and coordinated alternative splicing changes during neurodevelopment and that these splicing changes are in turn part of major regulatory mechanisms that underlie morphological and functional maturation of neurons. I describe our efforts to identify functional RBP-RNA interactions, including the identification of previously unobserved splicing events, and explore the combinatorial roles of multiple brain-specific RBPs during development. Using integrative modeling that combines multiple sources of data, we find hundreds of regulated splicing events for each of RBFOX, NOVA, PTBP, and MBNL. In the neurodevelopmental context, we find that the proteins control different sets of exons, with RBFOX, NOVA, and PTBP regulating early splicing changes and MBNL largely regulating later splicing changes. These findings additionally led to the observation that CNS and sensory neurons express a variety of different RBP programs, with many sensory neurons expressing a less mature splicing pattern than CNS neurons. We also establish a foundation for further exploration of neurodevelopmental splicing, by investigating the regulation of previously unobserved splicing events.

Despite their small brains, insects are capable of incredible navigational feats. Even Drosophila melanogaster (the common fruit fly) uses visual cues to remember locations in the environment. Investigating sophisticated navigation behaviors, like visual place learning, in a genetic model organism enables targeted studies of the neural circuits that give rise to these behaviors. Recent work has shown that the ellipsoid body, a midline structure deep within the fly brain, is critical for certain navigation behaviors. However, nearly all aspects of visual place learning remain mysterious. What visual features are used to encode place? What is the site of learning? How do the learned actions integrate with the core navigation circuits? To begin to address these questions I have established an experimental platform where I can measure neural activity using a genetically encoded calcium indicator in head-fixed behaving Drosophila. I further developed a virtual reality paradigm where flies are conditioned to prefer certain orientations within a virtual environment. In dendrites of ellipsoid body neurons, I observe a range of specific visual responses that are modified by this training. Remarkably, I find that distinct calcium responses are observed during presentation of preferred visual features. These studies reveal learning-associated neural activity changes in the inputs to a navigation center of the insect brain.

Myelination of axons in the CNS by oligodendrocytes (OLs) is critical for the rapid and reliable conduction of action potentials down neuronal axons, as evidenced by the severe disabilities associated with myelin loss in multiple sclerosis and other diseases of myelin. The specification, differentiation, and maturation of OLs along with myelin formation by OLs have been thoroughly characterized. How myelin is turned over, however remains unclear. It is unsurprising that little is known about myelin turnover considering that for decades following their discovery, myelin and OLs were considered static elements in the adult nervous system. Recent evidence, however, shows that myelin in the CNS is actually plastic. Moreover, myelin remodeling in humans has been suggested to be mediated by mature OLs. As mature OLs have limited capacity to generate new myelin sheaths, we must ask whether mature OLs can remodel the myelin at preexisting myelin sheaths. One intriguing but unproven possibility is that myelin at individual internodes may be remodeled cell-autonomously by mature OLs to modulate neuronal circuit function. Macroautophagy (MA) is responsible for the lysosome-mediated elimination of cytosolic proteins, lipids, and organelles. MA achieves this by capturing cargo in bulk or selectively in a transient, multilamellar structure known as an autophagosome (AP). In this study, we used a combination of in vivo and cellular approaches to test the hypothesis that MA in OLs may be important for myelin remodeling in the adult CNS. We establish that myelin of individual internodes is remodeled, and does so through the coordinated efforts of endocytosis and MA. We found that autophagy protein Atg7 is essential for myelin remodeling in vivo: loss of Atg7 in OLs leads to an age-dependent increase of myelin at the internode and the formation of aberrant myelin structures, most notably myelin outfoldings. In addition, we find that MA has the potential to occur throughout the mature OL, and examination of OLs in culture suggests that formation of a mature AP structure, the amphisome, is required to facilitate the efficient degradation of myelin-containing endocytic structures. Together, we propose that myelin is a dynamic structure that is regularly remodeled through the cooperative efforts of MA and endocytosis. These findings raise the possibility that myelin remodeling is involved in neural plasticity and the tuning of neural circuits.

Early life stress (ELS) leads to alterations in anatomy and function of the adult hippocampal dentate gyrus (DG), but the mechanisms by which these lasting changes occur have not been fully elucidated. We tested the hypothesis that the immediate decrease in cell proliferation and neurogenesis induced by stress is the key mediator of the negative long-term outcomes of ELS. First, we tested whether inhibition of cell proliferation during early life is sufficient to reproduce the ELS-induced reduction in adult DG neurogenesis. We demonstrate that targeting dividing stem cells for elimination during the first or third postnatal weeks leads to diminished adult neurogenesis and reduction of the stem cell pool. Also, we hypothesized that ELS leads to more persistent effects on DG function than stress later in life because of the stress-induced elimination of specific birth cohorts of DG granule cells (GCs) that have distinct functions. We tested whether different birth cohorts of DG GCs differ in function by assessing behavioral and stress response outcomes of pharmacogenetic elimination or optogenetic activation of adult GCs born during the first or third postnatal week. We demonstrate that dorsal GCs born during the first or third postnatal week may be involved in modulating exploratory and anxiety behavior, but that only third postnatal week born GCs stimulate HPA activity. These results suggest that mature DG GCs may differ in specific functions with birth date determining their functional role. Third, we directly assessed the effect of ELS on DG development to better understand the immediate effects of ELS on the DG and to identify other potential mediators of the long-term effects. We demonstrate that ELS using the limited bedding/nesting paradigm leads to developmental delay of the DG. The work presented in this dissertation contributes to our understanding of the mechanisms by which ELS produces lasting impairments in DG function and also to our knowledge of how DG GC function is specified.

I defend the view that mental states have degrees of strength. Our pains are more or less intense, our mental imagery is more or less vivid, our visual perceptions are more or less striking, and our desires and thoughts are more or less gripping. Mental strength is a phenomenal magnitude shared by all conscious experiences that determines their degree of felt intensity. Mental strength, however, has been largely ignored over other aspects of mental states such as their representational contents, phenomenology, or type. Considering mental strength is crucial for illuminating philosophical discussions related to representationalism, the transparency of experiences, cognitive phenomenology, attention, and the structure and function of consciousness. I use mental strength to develop in detail a neuropsychologically plausible theory of introspection and its limits that is inspired by a signal detection theoretic model of perception. In the second half of the dissertation, I look into methodological issues concerning the neural correlates of consciousness such as controlling for performance capacity and stimulus strength, and what these methodological concerns reveal about our theories of consciousness and its function.

Synapses play a critical role in neural circuits, and their highly specialized structures and biochemical characteristics have been widely studied in learning and memory. Along with their role in signal transmission, synapses also serve as adhesion structures, yet their mechanical characteristics have not received much attention. Given the important role of mechanics in cell adhesion, mechanical studies of synapses could offer insights into synaptic development, maintenance, and function. Here, I investigated synaptic elasticity in cultured rat hippocampal neurons and suggest that mechanical elasticity may be related to synaptic plasticity. I used torsional harmonic atomic force microscopy (TH-AFM) to measure the nanomechanical properties of functional mature excitatory synapses, whose identity and activity was verified by fluorescence microscopy. I combined TH-AFM with transmission electron microscopy and found that high stiffness of synapses originated from postsynaptic spines, not presynaptic boutons. I observed that spines at functional mature excitatory synapses were on average 10 times stiffer than dendritic shafts and that the distribution of spine stiffness exhibited a lognormal-like pattern. Importantly, I found that spine stiffness was correlated with spine size, and it is well established that spine size is correlated with synaptic strength. Based on the stiffness measurements and theoretical modelling of cell adhesion stability, I suggest that stiffness not only helps maintain spine morphology in the presence of synapse adhesion, but also helps stabilize synaptic adhesion. I propose a mechanical synaptic plasticity model. According to this model, mechanical strength leads to functional strength, which could provide a potential causal link between structural plasticity and functional plasticity of synapses.

A key feature of sleep is reduced responsiveness to the environment, which puts animals in a particularly vulnerable state; yet, sleep has been conserved throughout evolution, indicating that it fulfills a vital purpose. A core function of sleep across species has not been identified, but substantial advances in sleep research have been made in recent years using the genetically tractable model organism, Drosophila melanogaster. While a standard approach in sleep research is to study the effects of short-term sleep deprivation on an animal, tools are now available to genetically manipulate sleep amount in the fruit fly. In particular, a number of short-sleeping Drosophila mutants have been identified that model the long-term sleep restriction that is widespread in modern society. This thesis describes a body of work in which short-sleeping Drosophila mutants, as well as other genetic and pharmacological tools, were used to shed light on the function and regulation of sleep.