Browsing by Department "Neurobiology"

Anxiety is a behavioral state induced by low-threat, uncertain situations in which perceived danger is diffuse. The anxiety state is then accompanied by increased vigilance and risk assessment to one’s surroundings. Recent studies have shown that the brain regions responsible for encoding anxiety are widely located in the frontal cortex and extended limbic system; however, the network architecture responsible for hypervigilance has yet to be elucidated. Here, I propose to employ a data-driven method of using in vivo recordings of electrical activity across multiple brain regions concurrently as mice freely explore classic ethological anxiety-related behavioral assays and are administered pharmacological agents that modulate the anxiety state. Using novel machine-learning techniques, I have generated neural models that reflect the network-level activity engaged during the performance of these tasks. I have then validated the structure of this anxiety network in its ability to generalize to other anxiety-related tasks and models of disease. I anticipate that this strategy will discover an independent network that is correlated with anxiety-related behaviors. Thus, successful completion of the proposed work will lead to a network-level understanding of anxiety. Furthermore, the framework discovered through this study has the potential to facilitate the development of new revolutionary approaches for anxiety disorders.

The development and function of the nervous system relies on complex regulation of gene expression programs. MicroRNAs (miRNAs) are small RNAs that have diverse functions in mammalian development and disease. In concert with the RNA-induced silencing complex, miRNAs repress translation by binding to target mRNAs. The nervous system contains the largest proportion of miRNAs, yet few have been functionally characterized in vivo.

miR-133b is a highly conserved miRNA embedded in the sequence of 7H4, a noncoding RNA that is enriched at the neuromuscular junction (NMJ), a large synapse that is essential for eliciting muscle contraction and movement. I have found that, like 7H4, miR-133b expression is enriched at the NMJ and upregulated postnatally, coinciding with important events in synaptic maturation, including synaptic growth and elimination. Knockdown of miR-133b in postnatal muscle by electroporation of modified antisense oligonucleotides gave rise to abnormally large synapses, indicating a role for miR-133b in synaptic maturation. To specifically remove miR-133b in vivo, I generated a mouse containing a targeted deletion of the miR-133b stemloop. NMJ maturation and synapse elimination proceeded normally in miR-133b knockout mice, suggesting that miR-133b may have other functions at the synapse. The expression of 7H4 and miR-133b is upregulated following nerve transection, consistent with a role in synaptic regeneration. Indeed, NMJ reinnervation is delayed in miR-133b KO mice following nerve crush, but not nerve cut. These data suggest that miR-133b may have a specific protective function at the synapse that could be relevant to disease states, including amyotrophic lateral sclerosis (ALS), where NMJ denervation occurs following motor neuron cell death. However, loss of miR-133b did not affect survival or disease progression in the SOD1(G93A) mouse model, differentiating its role from that of miR-206, another miRNA found in 7H4.

miR-133b has recently been proposed to regulate the development and maintenance of midbrain dopaminergic (mDA) neurons. mDA neurons have critical functions in the control of movement and emotion, and their degeneration leads to motor and cognitive defects in Parkinson's disease. miR-133b is enriched in the midbrain and regulates mDA neuron differentiation in vitro by targeting Pitx3, a transcription factor required for appropriate development of substantia nigra DA neurons. However, the function of miR-133b in the intact midbrain has not been determined. miR-133b KO mice have normal numbers of midbrain dopaminergic neurons during development and aging. Moreover, dopamine neurotransmitter levels are unchanged in the striatum and other brain regions, while expression of dopaminergic genes including Pitx3 is also unaffected. Finally, miR-133b null mice display normal motor coordination and activity, suggesting that miR-133b does not play a significant role in the development or maintenance of the mDA neuron population.

Gut microbes and their host need to eat, but microbes rely on their host to control nutrient intake. Thus, microbes might use their metabolites and molecular patterns to influence the appetite of their host, including the quantity and timing of food intake. But the specific receptors, cells, transmitters, and circuits used by the host to sense and respond to the luminal stimuli of microbial patterns in real time remain unknown. In the small intestine, nutrients elicit fast sensory cues from epithelial neuropod cells that guide appetitive choice. Here, we found that in the mouse colon, microbial flagellin activates toll-like receptor 5 (TLR5) expressed on neuropod cells to reduce food intake. Mice lacking TLR5 in neuropod cells become hyperphagic and overweight. The microbial signal does not act directly on a nerve; instead, it triggers the release of peptide YY by epithelial neuropod cells, which acts on the Y2 receptor expressed in vagal nodose neurons innervating the colon. This feeding behavior change is independent of the common innate immune adaptor MyD88 or metabolic inflammation. Our results reveal a novel sensory modality, distinct from inflammatory responses, that enables an animal host to adjust its appetitive behavior by detecting patterns from its resident microbes in the colon.

Many of the skills we value most as humans, such as speech and learning to play musical instruments, are learned in the absence of external reinforcement. However, the model systems most commonly used to study motor learning employ learning paradigms in which animals perform behaviors in response to external rewards or punishments. Here I use the zebra finch, an Australian songbird that can learn its song as a juvenile in the absence of external reinforcement as well as modify its song in response to external cues as an adult, to study the circuit mechanisms underlying both internally and externally reinforced forms of learning. Using a combination of intersectional genetic and microdialysis techniques, I show that a striatonigral pathway and its downstream effectors, namely D1-type dopamine receptors, are necessary for both internally reinforced juvenile learning and externally reinforced adult learning, as wells as for song modification in response to social cues or to deafening. In addition, I employ optogenetic stimulation during singing to demonstrate that this striatonigral projection is sufficient to drive learning. Interestingly, I find that neither the striatonigral pathway nor D1-type dopamine receptors are necessary for recovery of pitch after externally driven pitch learning. In all, I establish that a common mechanism underlies both internally and externally reinforced vocal learning.

Ever since the initial discovery of general anesthetics almost 170 years ago, how general anesthesia (GA) induces loss of consciousness remains a century-long mystery. In addition, whether diverse anesthetic drugs and sleep share a common neural pathway is hotly debated and largely unknown. Previous studies have established that many GA drugs inhibit neural activity through targeting GABA receptors. Here, by using Fos staining, ex vivo brain slice recording, and eventually in vivo multichannel extracellular electrophysiology, we discovered a core ensemble of hypothalamic neurons in and near the supraoptic nucleus, consisting primarily of peptidergic neuroendocrine cells, which are surprisingly and persistently activated by multiple classes of GA drugs. Strikingly, chemogenetic or optogenetic stimulation of these anesthesia-activated neurons (AANs) strongly potentiated slow-wave sleep and prolonged GA, whereas conditional ablation through diphtheria toxin receptor strategy or inhibition of AANs with optogenetics led to reduced slow-wave oscillation in the brain, significant loss of slow-wave and rapid-eye movement sleep, and shortened durations under GA. Together, these findings identify a previously unknown common neural substrate underlying diverse GA drugs and natural sleep, and further illustrate a crucial role of the neuroendocrine system in regulating global brain states.

The loss of the ability to walk as the result of neurological injury or disease critically impacts the mobility and everyday lifestyle of millions. The World Heath Organization (WHO) estimates that approximately 1% of the world's population needs the use of a wheelchair to assist their personal mobility. Advances in the field of brain-machine interfaces (BMIs) have recently demonstrated the feasibility of using neuroprosthetics to extract motor information from cortical ensembles for more effective control of upper-limb replacements. However, the promise of BMIs has not yet been brought to bear on the challenge of restoring the ability to walk. A future neuroprosthesis designed to restore walking would need two streams of information flowing between the user's brain and the device. First, the motor control signals would have to be extracted from the brain, allowing the robotic prosthesis to behave in the manner intended by the user. Second, and equally important would be the flow of sensory and proprioceptive information back to the user from the neuroprosthesis. Here, I contribute to the foundation of such a bi-directional brain machine interface for the restoration of walking in a series of experiments in two animal models, designed to show the feasibility of (1) extracting locomotor information from neuronal ensemble activity and (2) sending information back into the brain via cortical microstimulation.

In a set of experiments designed to investigate the extraction of locomotor parameters, I chronically recorded from ensembles of neurons in primary motor (M1) and primary somatosensory (S1) cortices in two adult female rhesus macaques as they walked bipedally, at various speeds, both forward and backward on a custom treadmill. For these experiments, rhesus monkeys were suitable because of their ability to walk bipedally in a naturalistic manner with training. I demonstrate that the kinematics of bipedal walking in rhesus macaques can be extracted from neuronal ensemble recordings, both offline and in real-time. The activity of hundreds of neurons was processed by a series of linear decoders to extract accurate predictions of leg joints in three dimensional space, as well as leg muscle electromyograms (EMGs). Using a multi-layered switching model allowed us to achieve increased extraction accuracy by segregating different behavioral modes of walking.

In a second set of experiments designed to investigate the usage of microstimulation as a potential artificial sensory channel, I instructed two adult female Aotus trivirgatus (owl monkeys) about the location of a hidden food reward using a series of cortical microstimulation patterns delivered to primary somatosensory (S1) cortex. The owl monkeys discriminated these microstimulation patterns and used them to guide reaching movements to one of two targets. Here, owl monkeys were used which were previously implanted with electrode arrays of high longevity and stability. These monkeys were previously trained on a somatosensory cued task, which allowed a quick transition to microstimulation cueing. The owl monkeys learned to interpret microstimulation patterns, and their skill and speed of learning new patterns improved over several months. Additionally, neuronal activity recorded on non-stimulated electrodes in motor (M1), premotor (PMD) and posterior parietal (PP) cortices allowed us to examine the immediate neural responses to single biphasic stimulation pulses as well as overall responses to the spatiotemporal pattern. Using this recorded neuronal activity, I showed the efficacy of several linear classification algorithms during microstimulation.

These results demonstrate that locomotor kinematic parameters can be accurately decoded from the activity of neuronal ensembles, that multichannel microstimulation is a viable information channel for sensorized prosthetics, and that the technical limitations of combining these techniques can be overcome. I propose that bi-directional BMIs integrating these techniques will one day restore the ability to walk to severely paralyzed patients.

It has long been appreciated that the process of learning might invoke a physical change in the brain, establishing a lasting trace of experience. Recent evidence has revealed that this change manifests, at least in part, by the formation of new connections between neurons, as well as the modification of preexisting ones. This so-called structural plasticity of neural circuits – their ability to physically change in response to experience – has remained fixed as a primary point of focus in the field of neuroscience.

A large portion of this effort has been directed towards the study of dendritic spines, small protrusions emanating from neuronal dendrites that constitute the majority of recipient sites of excitatory neuronal connections. The unique, mushroom-like morphology of these tiny structures has earned them considerable attention, with even the earliest observers suggesting that their unique shape affords important functional advantages that would not be possible if synapses were to directly contact dendrites. Importantly, dendritic spines can be formed, eliminated, or structurally modified in response to both neural activity as well as learning, suggesting that their organization reflects the experience of the neural network. As such, elucidating how these structures undergo such rearrangements is of critical importance to understanding both learning and memory.

As dendritic spines are principally composed of the cytoskeletal protein actin, their formation, elimination, and modification requires biochemical signaling networks that can remodel the actin cytoskeleton. As a result, significant effort has been placed into identifying and characterizing such signaling networks and how they are controlled during synaptic activity and learning. Such efforts have highlighted Rho family GTPases – binary signaling proteins central in controlling the dynamics of the actin cytoskeleton – as attractive targets for understanding how the structural modification of spines might be controlled by synaptic activity. While much has been revealed regarding the importance of the Rho GTPases for these processes, the specific spatial and temporal features of their signals that impart such structural changes remains unclear.

The central hypotheses of the following research dissertation are as follows: first, that synaptic activity rapidly initiates Rho GTPase signaling within single dendritic spines, serving as the core mechanism of dendritic spine structural plasticity. Next, that each of the Rho GTPases subsequently expresses a spatially distinct pattern of activation, with some signals remaining highly localized, and some becoming diffuse across a region of the nearby dendrite. The diffusive signals modify the plasticity induction threshold of nearby dendritic spines, and the spatially restricted signals serve to keep the expression of plasticity specific to those spines that receive synaptic input. This combination of differentially spatially regulated signals thus equips the neuronal dendrite with the ability to perform local biochemical computations, potentially establishing an organizational preference for the arrangement of dendritic spines along a dendrite. Finally, the consequences of the differential signal patterns also help to explain several seemingly disparate properties of one of the primary upstream activators of these proteins: brain-derived neurotrophic factor (BDNF).

The first section of this dissertation describes the characterization of the activity patterns of one of the Rho family GTPases, Rac1. Using a novel Förster Resonance Energy Transfer (FRET)- based biosensor in combination with two-photon fluorescence lifetime imaging (2pFLIM) and single-spine stimulation by two-photon glutamate uncaging, the activation profile and kinetics of Rac1 during synaptic stimulation were characterized. These experiments revealed that Rac1 conveys signals to both activated spines as well as nearby, unstimulated spines that are in close proximity to the target spine. Despite the diffusion of this structural signal, however, the structural modification associated with synaptic stimulation remained restricted to the stimulated spine. Thus, Rac1 activation is not sufficient to enlarge spines, but nonetheless likely confers some heretofore-unknown function to nearby synapses.

The next set of experiments set out to detail the upstream molecular mechanisms controlling Rac1 activation. First, it was found that Rac1 activation during sLTP depends on calcium through NMDA receptors and subsequent activation of CaMKII, suggesting that Rac1 activation in this context agrees with substantial evidence linking NMDAR-CaMKII signaling to LTP in the hippocampus. Next, in light of recent evidence linking structural plasticity to another potential upstream signaling complex, BDNF-TrkB, we explored the possibility that BDNF-TrkB signaling functioned in structural plasticity via Rac1 activation. To this end, we first explored the release kinetics of BDNF and the activation kinetics of TrkB using novel biosensors in conjunction with 2p glutamate uncaging. It was found that release of BDNF from single dendritic spines during sLTP induction activates TrkB on that same spine in an autocrine manner, and that this autocrine system was necessary for both sLTP and Rac1 activation. It was also found that BDNF-TrkB signaling controls the activity of another Rho GTPase, Cdc42, suggesting that this autocrine loop conveys both synapse-specific signals (through Cdc42) and heterosynaptic signals (through Rac1).

The next set of experiments detail one the potential consequences of heterosynaptic Rac1 signaling. The spread of Rac1 activity out of the stimulated spine was found to be necessary for lowering the plasticity threshold at nearby spines, a process known as synaptic crosstalk. This was also true for the Rho family GTPase, RhoA, which shows a similar diffusive activity pattern. Conversely, the activity of Cdc42, a Rho GTPase protein whose activity is highly restricted to stimulated spines, was required only for input-specific plasticity induction. Thus, the spreading of a subset of Rho GTPase signaling into nearby spines modifies the plasticity induction threshold of these spines, increasing the likelihood that synaptic activity at these sites will induce structural plasticity. Importantly, these data suggest that the autocrine BDNF-TrkB loop described above simultaneously exerts control over both homo- and heterosynaptic structural plasticity.

The final set of experiments reveals that the spreading of GTPase activity from stimulated spines helps to overcome the high activation thresholds of these proteins to facilitate nearby plasticity. Both Rac1 and RhoA, the activity of which spread into nearby spines, showed high activation thresholds, making weak stimuli incapable of activating them. Thus, signal spreading from a strongly stimulated spine can lower the plasticity threshold at nearby spines in part by supplementing the activation of high-threshold Rho GTPases at these sites. In contrast, the highly compartmentalized Rho GTPase Cdc42 showed a very low activation threshold, and thus did not require signal spreading to achieve high levels of activity to even a weak stimulus. As a result, synaptic crosstalk elicits cooperativity of nearby synaptic events by first priming a local region of the dendrite with several (but not all) of the factors required for structural plasticity, which then allows even weak inputs to achieve plasticity by means of localized Cdc42 activation.

Taken together, these data reveal a molecular pattern whereby BDNF-dependent structural plasticity can simultaneously maintain input-specificity while also relaying heterosynaptic signals along a local stretch of dendrite via coordination of differential spatial signaling profiles of the Rho GTPase proteins. The combination of this division of spatial signaling patterns and different activation thresholds reveals a unique heterosynaptic coincidence detection mechanism that allows for cooperative expression of structural plasticity when spines are close together, which in turn provides a putative mechanism for how neurons arrange structural modifications during learning.

Ube3a is a HECT domain E3 ubiquitin ligase originally recognized for its role in degrading p53 in the presence of the human papilloma virus protein E6. Loss of maternal Ube3a expression causes Angelman syndrome, a severe neurodevelopmental disorder characterized by mental retardation, ataxia, epilepsy, lack of speech, and a unique behavioral phenotype that includes a happy demeanor and frequent laughing. However, characterization of the endogenous properties and cellular role for Ube3a has been limited. Over the last few years, an interesting cohort of Ube3a interacting partners and putative substrates were named, though the consequences of these interactions were not thoroughly investigated. These include two Golgi localized proteins - PIST and Golgin-160 - as well as several proteins that can regulate trafficking of proteins at the Golgi apparatus: Src family kinases, ubiquilin, and tuberin. Therefore, we decided to focus on whether Ube3a could regulate Golgi structure or function.

In this dissertation, I will describe a new role for Ube3a at the Golgi apparatus in the regulation of intralumenal ion homeostasis. First, I characterized the expression pattern of endogenous Ube3a and overexpressed Ube3a isoforms by immunostaining and fractionation and demonstrated that although Ube3a has diffuse nuclear/cytoplasmic localization, it also associates with membrane fractions. I also confirmed that Ube3a interacts endogenously with both PIST and Golgin-160. Next, I demonstrated that Golgi morphology is perturbed in a cell line with stable knockdown of Ube3a. I found that the Golgi apparatus in Ube3a knockdown cells is under-acidifed, and that this is the primary defect underlying the disrupted Golgi morphology. Finally, I extended these findings in vivo and examined the morphology of the Golgi apparatus in the brains of Angelman syndrome model mice. The Golgi structures in the visual cortex of these mice appeared disorganized by immunohistochemistry and individual cisternae were significantly distended by electron microscopy, consistent with a defect in ion homeostasis at the Golgi apparatus. These findings define new cellular role for Ube3a at the Golgi apparatus and provide insight into the pathogenesis of Angelman syndrome.

Hypoxia is a profound stressor of the central nervous system implicated in numerous neurodegenerative diseases. While it is increasingly evident that the early effects of hypoxia cause impairment at the level of the axon, the precise mechanisms through which hypoxia compromises axonal structure and function remain unclear. However, links between hypoxia-induced axonopathic disease and the amyloid cascade, as well as the upregulation of amyloid precursor protein (APP) and amyloid beta (Aβ) by hypoxic stress, give rise to the hypothesis that proteolytic cleavage of APP into Aβ may be specifically responsible for axonopathy under conditions of hypoxia.

The goal of this dissertation was thus to understand dependence of hypoxia-induced axonal morphological and functional impairment on APP cleavage and the production of Aβ. I have developed a model of hypoxia-induced axonopathy in retinal explants. Using this model, I have experimentally addressed the core hypothesis that APP cleavage, and in particular the formation of Aβ, is necessary and sufficient to mediate morphological and functional axonopathy caused by hypoxia. I have found that there is a dissociation between the mechanisms responsible for hypoxia-induced morphological and functional impairment of the axon in the explanted retina, with the former being dependent on APP-to-Aβ processing and the latter likely being dependent on cleavage of a non-APP substrate by the enzyme BACE1. These findings shed light on mechanisms of hypoxia-induced axonopathy.

The ability to learn and to modify complex vocal sequences requires extensive practice coupled with performance evaluation through auditory feedback. An efficient solution to the challenge of vocal learning, stemming from reinforcement learning theory, proposes that an “actor” learns correct vocal behavior through the instructive guidance of an auditory “critic.” However, the neural circuit mechanisms supporting performance evaluation and even how “actor” and “critic” circuits are instantiated in biological brains are fundamental mysteries. Here, I use a songbird model to dissociate “actor” and “critic” circuits and uncover biological mechanisms for vocal learning.

First, I employ closed-loop optogenetic methods in singing birds to identify two inputs to midbrain dopamine neurons that operate in an opponent fashion to guide vocal learning. Next, I employ electrophysiological methods to establish a microcircuit architecture underlying this opponent mechanism. Notably, I show that disrupting activity in these midbrain dopamine inputs precisely when auditory feedback is processed impairs learning, showing that they function as “critics.” Conversely, I show that disrupting activity in a downstream premotor region prior to vocal production prevents learning, consistent with an “actor” role. Taken together, these experiments dissociate discrete “actor” and “critic” circuits in the songbird’s brain and elucidate neural circuit and microcircuit mechanisms by which “actors” and “critics” working cooperatively enable vocal learning.

Memory is a fundamentally important process that guides our future behavior based on past experience. Its importance is underscored by the fact that a major feature of many neurodegenerative disorders is memory loss, which is disabling to an increasing portion of the aging population. However, the underlying electrophysiological processes underlying memory formation and retrieval in humans remains very poorly understood, and in turn, limits our abilities to provide effective therapy for patients suffering from these disorders. Here, we endeavored to investigate the underpinnings of human memory through intracranial recordings in human epilepsy patients undergoing routine monitoring for potential resective surgery. This unprecedented access to the human brain during awake behavior allowed us to make several inroads into understanding human memory. First, we investigated fast frequency oscillations in the brain, termed ripples, and their relevance during human episodic memory. We found that during a paired associates verbal memory task, ripples coupled between the medial temporal lobe (MTL) memory system and the temporal association cortex, and this coupling preceded the reinstatement of memory representations from the memory encoding period. Next, we measured single unit spiking activity from anterior temporal lobe in order to examine if temporal patterns of activity may serve as a general neural code that is replayed during memory retrieval. We found that verbal memories corresponded to item-specific sequences of cortical spiking activity, these sequences replayed during memory retrieval, and replay was preceded by ripples in the MTL. Finally, to develop a more mechanistic understanding of our findings, we used a randomly connected recurrent leaky integrate and fire neural network model to investigate the characteristics needed for significant spike sequence generation. We found that randomly connected networks can generate sequences under many parameter regimes with just white noise inputs, the specific output sequence was inherently related to the connectivity of the network, and these models could make quantitative predictions about dynamic excitatory and inhibitory balance during spiking sequences in the human data. Taken together, our results demonstrate a flexible mode of communication between the MTL and cortex in the service of episodic memory, and we provide a theoretical framework for understanding the generation of these neural patterns in the human cortex.

Smooth pursuit eye movements are movements of the eyes that are used to foveate moving objects. Their precision and adaptation is believed to depend on a constellation of sites across the cerebellum, but only one region’s contribution is well characterized, the floccular complex. Here, I characterize the response properties of neurons in the oculomotor vermis, another major division of the oculomotor cerebellum whose role in pursuit remains unknown. I recorded Purkinje cells, the output neurons of this region, in two monkeys as they executed pursuit eye movements in response to step ramp target motion. The responses of these Purkinje cells in the oculomotor vermis were very different from responses that have been documented in the floccular complex. The simple spikes of these cells encoded movement direction in retinal, as opposed to muscle coordinates. They were less related to movement kinematics, and had smaller values of trial-by-trial correlations with pursuit speed, latency, and direction than their floccular complex counterparts. Unlike Purkinje cells in the floccular complex, simple spike firing rates in the oculomotor vermis remained unchanged over the course of pursuit adaptation, likely excluding the oculomotor vermis as a site of directional plasticity. Complex spikes of these Purkinje cells were only partially responsive to target motion, and did not fall into any clear opponent directional organization with simple spikes, as has been found in the floccular complex. In general, Purkinje cells in the oculomotor vermis were responsive to both pursuit and to saccadic eye movements, but maintained tuning for the direction of these movements along separate directions at a population level. Predictions of caudal fastigial nucleus activity, generated on the basis of our population of oculomotor vermal Purkinje cells, faithfully tracked moment-by-movement changes in pursuit kinematics. By contrast, these responses did not faithfully track moment-by-moments changes in saccade kinematics. These results suggest that the oculomotor vermis is likely to play a smaller role in influencing pursuit eye movements by comparison to the floccular complex.

AbstractSCA7 is an autosomal dominant CAG/polyglutamine trinucleotide repeat expansion disease that accounts for ~4% of all spinocerebellar ataxias in the USA. In addition to atrophy of the cerebellar cortex and brainstem, an important feature of SCA7, that allows it to be distinguished from the 40+ other SCAs, is a cone-rod dystrophy form of retinal degeneration. Recent studies of ALS and CAG-polyglutamine trinucleotide repeat expansion neurodegenerative diseases indicate that nuclear membrane dysfunction and impaired nucleocytoplasmic transport could play pivotal roles in SCA7 disease pathogenesis; hence, here we will examine the nuclear membrane and nucleocytoplasmic transport in the retina and cerebellum of SCA7 mice and in neuronal models of SCA7 disease to determine if alteration of these cellular processes is contributing to SCA7 retinal and cerebellar degeneration.

Synapse formation and elimination are two developmental processes that concurrently take place in the neonatal brain. Dysregulation of these two processes have been implicated in the etiology and progression of neurodevelopmental and neurodegenerative diseases. Previous work has found that in mice, the first three postnatal weeks are highly active periods of synapse remodeling throughout the entire brain. Glial cells called astrocytes are highly complex neural derived cells that are born and mature during this period. As they mature, astrocytes instruct the formation of synapses through contact with synaptic components and through the secretion of various synaptogenic factors. Microglia by contrast are the tissue resident macrophages of the central nervous system (CNS). During the first three postnatal weeks, microglia sculpt developing synaptic circuits by engulfment of synaptic components through various phagocytic mechanisms. While the field has steadily grown our understanding of the importance of these two cell types in synapse formation and elimination separately, few studies have addressed the possibility of communication between these two cell types to regulate their respective functions at synapses. Here I used the developing visual thalamocortical circuit as a model system to investigate the molecular cross talk between astrocytes and microglia. To address the impact of this communication on synapse development and function, I focused on one factor called Hevin/Sparcl1 which has previously been shown to be necessary and sufficient for thalamocortical synapse formation and plasticity. Previous studies have shown that Hevin induces thalamocortical synapse formation during the second postnatal week in mouse visual cortex. Hevin orchestrates this process by bridging pre-and post-synaptic cell adhesion molecules, Nrxn1α and Nlgn1B. Curiously, I found that despite high levels of Hevin in the maturing primary visual cortex, thalamocortical synapse numbers decrease even during the time when Hevin expression is at its peak. This refinement process, I determined, was dependent on microglia. Using super resolution microscopy, I found that only a subset thalamocortical synapses have Hevin at their cleft and that loss of Hevin aberrantly enhances microglia phagocytic activity. These initial findings suggested that Hevin likely functioned to spare only specific synapses from microglia mediated elimination. To interrogate this possibility, I used an in vitro microglia culture system to assess the transcriptional responses of microglia to Hevin treatment. Surprisingly, this treatment led to robust transcriptional changes in microglia that were distinct from well described immunological stimulation. This screen implicated Toll-like receptors (TLR) 2 and 4 in this transcriptional response. Further studies using our in vitro culture system showed that proteolytic cleavage of Hevin was required to upregulate TLR2 expression in microglia and that its C-terminus alone was sufficient to upregulate TLR2. Moving in vivo, I found that TLR2 expression is strongly developmentally regulated and highly heterogeneously expressed by microglia in the mouse primary visual cortex. Using overexpression studies in vivo, I also found that microglia strongly upregulate TLR2 in response to Hevin or Hevin’s C-terminus and that these TLR2 high microglia have enhanced phagocytic activity both in normal development and after Hevin/Hevin C-terminal overexpression. These findings indicate that Hevin function is regulated by proteolytic cleavage and suggest that Hevin is a dual signal in synaptic development: both to stimulate synapse formation by neurons and enhance synapse elimination by microglia. I next sought to test the functional relevance of the microglia specific response to Hevin. To do this, I used co-immunoprecipitation studies to identify candidate receptors for Hevin on microglia. I found that Hevin and its C-terminus interacted with both TLR2 and TLR4 but seemed to have a stronger affinity for TLR4. Therefore, I used TLR4 KO mice to test if microglia could still be stimulated by Hevin in vivo. I found that TLR4 KO microglia were no longer responsive to Hevin overexpression and had reduced phagocytic capacity compared to WT microglia. Ultimately, I found that TLR4 KO mice had impaired thalamocortical synapse refinement and impaired circuit plasticity. Taken together, my results identify astrocyte-derived Hevin as a synaptogenic molecule that links thalamocortical synapse formation with synaptic refinement mediated by microglia.

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.

During development, cell-cell recognition events mediate crucial steps in the formation of organized cellular patterns critical for tissue function. In the nervous system, cell recognition cues guide migrating neurons during development to appropriate terminal locations and sculpt their characteristic sizes, shapes, and circuit connectivity. The retina contains a multitude of neuron types; however, neurons of the same cell type (homotypic) are patterned into evenly spaced arrangements known as “mosaics” across the retina surface. Disrupting mosaic formation impairs visual function, so it is important to understand the precise cellular and molecular mechanisms that allow homotypic neurons to recognize and adjust their proximity to neighbors. To understand this process, we studied two populations of interneurons, the OFF and ON starbursts amacrine cells (SACs), which require the cell-surface receptor MEGF10 to establish their mosaics. We find that SACs in Megf10 mutants still make lateral movements in the plane of the retina, but fail to recognize their proximity to homotypic neighbors. Using transgenic tools to visualize SACs early in development, we identify a transient developmental phase where SAC dendrite territories are bounded by homotypic somata, a relationship which is lost in SACs lacking MEGF10. Further, we determine that MEGF10 utilizes distinct signal transduction pathways in neurons from those identified in non-neuronal cells. Lastly, we demonstrate that specific amino acids within the intracellular domain of MEGF10 are required to recapitulate a cellular recognition-like event in a heterologous cell system. These findings support a model whereby MEGF10 signals in SACs by a distinct mechanism to mediate dendrite-soma interactions necessary to pattern the organization of retinal neurons.

Obsessive-compulsive disorder (OCD) is a devastating illness that afflicts around 2% of the world's population with recurrent distressing thoughts (obsessions) and repetitive ritualistic behaviors (compulsions). While dysfunction at excitatory glutaminergic excitatory synapses leading to hyperactivity of the orbitofrontal cortex and head of the caudate - brain regions involved in reinforcement learning - are implicated in the pathology of OCD, clinical studies involving patients are unable to dissect the molecular mechanisms underlying this cortico-striatal circuitry defect. Since OCD is highly heritable, recent studies using mutant mouse models have shed light on the cellular pathology mediating OCD symptoms. These studies point toward a crucial role for deltaFosB, a persistent transcription factor that accumulates with chronic neuronal activity and is involved in various diseases of the striatum. Furthermore, elevated deltaFosB levels results in the transcriptional upregulation of Grin2b, which codes GluN2B, an N-methyl-D-aspartate glutamate receptor (NMDAR) subunit required for the formation and maintenance of silent synapses. Taken together, the current evidence indicates that deltaFosB-mediated expression of aberrant silent synapses in caudate medium spiny neurons (MSNs), in particular D1 dopamine-receptor expressing MSNs (D1 MSNs), mediates the defective cortico-striatal synaptic transmission that underlies compulsive behavior in OCD.

Schizophrenia is marked by significant disruptions to dopaminergic signaling across the mesolimbic and mesocortical circuits. Antipsychotic drugs have been largely unsuccessfully treating cognitive symptoms that debilitate the schizophrenia patient population. Dopamine 2 Receptor (D2R)- βeta arrestin 2 (βarr2) biased signaling, independent of the canonical G protein signaling, has emerged as a potential mechanism for antipsychotic drugs to restore dopaminergic signaling and improve treatment resistant cognitive symptoms. In the following experiments, I described gene editing tools to systematically investigate D2R signaling in a region or cell specific manner. Next, I evaluated the behavioral effects of two functionally selective D2-like βarr2 biased ligands against psychotomimetic challenge from phencyclidine or amphetamine. Then I employed chemogenetics to perform synthetic pharmacology experiments e.g. studying the signaling cascade of a drug without using the drug, to discover how D2- R βarr2 signaling produces antipsychotic effects in the prefrontal cortex. Lastly, I characterized the neurophysiological changes induced by phencyclidine and a D2R βarr2 biased ligand within relevant brain regions in the meso -limbic and -cortical circuits. Our results determined antipsychotic like activity is 1) regulated by excitation-inhibitory balance maintained by cortical GABA interneurons 2) dependent on βarr2.

Organisms are presented with a wide variety of environmental stimuli and must interpret and respond to these cues in to perform a wide variety of behaviors, such as foraging, mating, fleeing, and fighting. The ability of an organism to recognize various stimuli, such as pheromones, to identify mates or competitors through the activation of various circuits and molecular components in the brain is tightly regulated. In order to delineate how molecular changes occur in the brain during stimuli response we used Drosophila melanogaster as it has a well-defined nervous system. We focus in on the circuit which regulates sex-specific mating behaviors in male D. melanogaster. Sex-specific splicing regulates the expression of two genes known as fruitless (fruM) and doublesex (dsxM) in the courtship circuit. Here we demonstrate using in the fly olfactory system that Olfactory receptor 47b (Or47b) and Olfactory receptor 67d (Or67d) activity, through sensory experience, regulates the expression patterns of male-specific fruM through coincident activity of hormone binding transcription factors Gce and Met and histone acetyltransferase P300 activity. We also identify various genes which changes in various mutant and social contexts, including exon specific changes in fruitless transcripts as well as changes in the expression of hormone metabolism genes, and neuromodulators in antennae. Given these changes in neuromodulators and the known structure of the FruM and DsxM central circuits, we looked at changes in the chromatin state and expression levels and find changes in peripheral sensory neurons have downstream effects on higher order circuits. We identify that FruM regulates the chromatin structure of both itself and dsxM in whole brain lysates and that changes in chromatin structure depend on pheromone receptor and neurotransmitter activity across processing centers in the brain. Taken together, we identify potential candidates for future study, as well as lay the framework for understanding how sensory changes in the periphery have effects on various neuronal clusters in the brain.

Pain is defined by the international association of pain as an "unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage". Most people have experienced one form of pain or another and although such experiences can be unsavory, pain serves the basic need for the detection of dangerous stimuli that can cause bodily harm. Because pain serves such an essential need, it is important to understand how the nervous system processes and encodes noxious or potentially tissue damaging stimuli. This neural processing is called nociception.

In this study, I use Drosophila larvae as a genetic model organism to study nociception. In response to noxious thermal and mechanical stimuli, Drosophila larvae perform a nociceptive defensive behavior (termed nocifensive) where larvae rotate in a corkscrew like fashion along the long axis causing them to move in a lateral direction. Using this behavior and genetic tools which can manipulate neuronal output, we have identified the sensory neurons which serve as larval nociceptors as class IV multidendritic sensory neurons. Further characterization of these larval nociceptors, has also shown that they are both cholinergic and peptidergic.

After the identifying the larval nociceptors, I next identified several molecular components which are required for larval mechanical nociception. I have found that the degenerin epithelial sodium channel (DEG/ENaC) called pickpocket is required for larval mechanical nociception by using genetic mutants and RNAi knockdwon. In addition, after performing a screen using RNAi to knockdown ion channel transcripts in larval nociceptors, I have identified two other DEG/ENaC channels which are required for larval mechanical nociception. DEG/ENaCs are particularly interesting because they have been identified as candidate mechanotransducers in C. elegans for the gentle touch behavior. I propose that DEG/ENaCs may serve as candidate mechanotransducers in larval mechanical nociception because they are not generally required for neuronal excitability. However, future research will be required to establish their true role in mechanical nociceptive signaling.

In addition to DEG/ENaCs, transient receptor potential (TRP) channels also play a role in nociception. painless, a channel that was first identified in a thermal nociception screen on Drosophila larvae, is required for both thermal and mechanical nociception. The last section shows that multiple isoforms of painless exist and that these different isoforms may play different roles in thermal and mechanical nociception.

Taken together, these results have begun to establish Drosophila larva as a model for studying nociception. I have identified the sensory neurons used as larval nociceptors and shown that DEG/ENaC channels play an important role in larval mechanical nociception.