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Neurons in the arcuate nucleus of the hypothalamus (ARH) play a central role in the regulation of body weight and energy homeostasis. ARH neurons directly sense nutrient and hormonal signals of energy availability from the periphery and relay this information to secondary nuclei targets, where signals of energy status are integrated to regulate behaviors related to food intake and energy expenditure. Transduction of signals related to energy status by Pro-opiomelanocortin (POMC) and Neuropeptide-Y/agouti-related protein (NPY/AgRP) neurons in the ARH exert opposing influences on secondary neurons in central circuits regulating energy balance. My thesis research focused on the developmental events regulating the differentiation and specification of cell fates in the ARH. My first project was designed to characterize the ontogeny of Pomc- and Npy-expressing neurons in the developing mediobasal hypothalamus (Chapter 2). These experiments led to the unexpected finding that during mid-gestation, Pomc is broadly expressed in the majority of newly-born ARH neurons, but is subsequently down-regulated during later stages of development as cells acquire a terminal cell identity. Moreover, these studies demonstrated that most immature Pomc-expressing progenitors subsequently differentiate into non-POMC neurons, including a subset of functionally distinct NPY/AgRP neurons. The second aspect of my work focused on characterizing Pomc-expressing precursors throughout the brain (Chapter 3). Similar to findings in Chapter 2, Pomc was broadly expressed in many aspects of the developing brain and subsequently down-regulated as cells further differentiated into non-Pomc terminal identities. In the CNS, the percentage of POMC neurons derived from Pomc-expressing precursors are marginal. These findings are of general importance to the field of energy homeostasis research, because many genetically targeted functional manipulations of POMC cells have been induced during early development, a time in which Pomc is expressed in many cells that will not persist into a POMC terminal identity. The off target consequences of these manipulations have yet to be considered. Our work will provide the foundational evidence for potential confounds of targeted genetic POMC manipulations and may help to explain some of the unexpected phenotypes that have arisin using a BAC transgenic Pomc-Cre reagent. My current research efforts are focused on elucidating the molecular mechanism regulating differentiation and cell fate specification in the ARH during gestation and the early postnatal period (Chapter 4). Consistent with the identification of Pomc transcriptional activity during embryogenesis, functional POMC-processing products (β-endorphin and α-melanocyte-stimulating hormone) are also detected in embryonic hypothalamic extracts (at embryonic day (E) 13.5 and E15.5, respectively). The presence of POMC processing poducts in the embryo, prior to the establishment of circuits regulating food intake, may be involved in the local differentiation events. Preliminary loss-of-function studies are underway to determine the role of POMC processing products in differentiation of hypothalamic terminal fates. Early results from this work indicate that both β-endorphin and α-melanocyte-stimulating hormone are critical to the differentiation of the Pomc lineage. The goal of this work is to define the developmental origins of critical components of neuronal circuits that determine body weight. Evidence in the literature supports that maternal signals influence these events, our studies may provide a means to the design of effective strategies to reduce transmission of signals that increase susceptibility to obesity in offspring.

Part I: Consider the n-dimensional vector y = Xβ + ǫ where β ∈ Rp has only k nonzero entries and ǫ ∈ Rn is a Gaussian noise. This can be viewed as a linear system with sparsity constraints corrupted by noise, where the objective is to estimate the sparsity pattern of β given the observation vector y and the measurement matrix X. First, we derive a non-asymptotic upper bound on the probability that a specific wrong sparsity pattern is identified by the maximum-likelihood estimator. We find that this probability depends (inversely) exponentially on the difference of kXβk2 and the ℓ2-norm of Xβ projected onto the range of columns of X indexed by the wrong sparsity pattern. Second, when X is randomly drawn from a Gaussian ensemble, we calculate a non-asymptotic upper bound on the probability of the maximum-likelihood decoder not declaring (partially) the true sparsity pattern. Consequently, we obtain sufficient conditions on the sample size n that guarantee almost surely the recovery of the true sparsity pattern. We find that the required growth rate of sample size n matches the growth rate of previously established necessary conditions. Part II: Estimating two-dimensional firing rate maps is a common problem, arising in a number of contexts: the estimation of place fields in hippocampus, the analysis of temporally nonstationary tuning curves in sensory and motor areas, the estimation of firing rates following spike-triggered covariance analyses, etc. Here we introduce methods based on Gaussian process nonparametric Bayesian techniques for estimating these two-dimensional rate maps. These techniques offer a number of advantages: the estimates may be computed efficiently, come equipped with natural errorbars, adapt their smoothness automatically to the local density and informativeness of the observed data, and permit direct fitting of the model hyperparameters (e.g., the prior smoothness of the rate map) via maximum marginal likelihood. We illustrate the flexibility and performance of the new techniques on a variety of simulated and real data. Part III: Many fundamental questions in theoretical neuroscience involve optimal decoding and the computation of Shannon information rates in populations of spiking neurons. In this paper, we apply methods from the asymptotic theory of statistical inference to obtain a clearer analytical understanding of these quantities. We find that for large neural populations carrying a finite total amount of information, the full spiking population response is asymptotically as informative as a single observation from a Gaussian process whose mean and covariance can be characterized explicitly in terms of network and single neuron properties. The Gaussian form of this asymptotic sufficient statistic allows us in certain cases to perform optimal Bayesian decoding by simple linear transformations, and to obtain closed-form expressions of the Shannon information carried by the network. One technical advantage of the theory is that it may be applied easily even to non-Poisson point process network models; for example, we find that under some conditions, neural populations with strong history-dependent (non-Poisson) effects carry exactly the same information as do simpler equivalent populations of non-interacting Poisson neurons with matched firing rates. We argue that our findings help to clarify some results from the recent literature on neural decoding and neuroprosthetic design. Part IV: A model of distributed parameter estimation in networks is introduced, where agents have access to partially informative measurements over time. Each agent faces a local identification problem, in the sense that it cannot consistently estimate the parameter in isolation. We prove that, despite local identification problems, if agents update their estimates recursively as a function of their neighbors’ beliefs, they can consistently estimate the true parameter provided that the communication network is strongly connected; that is, there exists an information path between any two agents in the network. We also show that the estimates of all agents are asymptotically normally distributed. Finally, we compute the asymptotic variance of the agents’ estimates in terms of their observation models and the network topology, and provide conditions under which the distributed estimators are as efficient as any centralized estimator.

The ability to transform physical energies from internal and external environments into neuronal signals underlies the senses of hearing and touch as well as many aspects of body self-awareness. The small soil dwelling nematode Caenorhabditis elegans has proven to be a useful system in which to study neuronal mechanosensation and the development of specialized neuronal subtypes. Response to gentle body touch in C. elegans is mediated by the six Touch Receptor Neurons (TRNs). TRN cell fate is specified by the LIM-Homeodomain transcription factor MEC-3. Gene expression profiling has revealed a set of putatively MEC-3 dependant transcripts, which may represent the set of genes necessary to establish a TRN fate. I characterized this set of putatively MEC-3 regulated transcripts confirming the MEC-3 dependant expression of several previously unrecognized MEC-3 targets providing insight into TRN development and function and identifying the CCT chaperonin complex as being needed for TRN function. LIM-HD transcription factors play important developmental roles across phyla particularly in neurons. The data presented in this thesis provide insight into LIM-HD function in general and provide targets for further research in C. elegans TRNs as well as neurons from higher organisms. Microtubules (MTs) play crucial roles in the majority of eukaryotic cells where they are required for cell division, intracellular transport, morphological stability and a variety of other functions. The TRNs of C. elegans are characterized by unique large-diameter, heavily acetylated microtubules (MTs), which are required for mechanosensation. The TRNs thus present a valuable model for the study of neuronal MTs and MT acetylation, which is a poorly understood but widespread MT modification particularly in post-mitotic neurons. I employed TRN-specific RNAi to identify MT-associated proteins not previously known to be involved with mechanosensation including the CCPP-1 tubulin deglutamylase, the C. elegans homolog of the MT severing enzyme katanin, and several other proteins. I also shed light on the significance of á-tubulin acetylation by investigating the acetyltransferases responsible. MEC-17 and ATAT-2 are á-tubulin acetyltransferases found in the TRNs, which illustrate mechanosensory defects when mutated. I found that mec-17 mutants exhibit a drastic decrease in TRN MT number as well as an elimination of the unknown electron-dense material found in the lumen of TRN MTs and other MTs across phyla. I also found that eliminating MEC-17 activity changed TRN morphology, resulting in ectopic sprouting and process growth. These results suggest that acetyltransferase activity is required for TRN MT integrity, mechanosensory function, and the maintenance of TRN morphology.

Psychological models have proposed that valence, how pleasant or unpleasant a stimulus is perceived, and intensity, the strength with which a stimulus is perceived, constitute two primary dimensions that describe affective experience. However, the inherent relationship between valence and intensity has limited imaging studies of these models and the neural substrates are poorly understood. For example, it is not known if the neural representations for each dimension are discrete or shared. To overcome these limitations, we applied properties of alliesthesia, the phenomenon where the valence of a stimulus is dependent upon the physiological state of the body, using thermal stimuli to the hand in combination with whole-body warming and cooling. In this way, we were able to manipulate the hedonic aspect of our thermal stimuli independent of their perceived intensity. Brain regions correlating with stimulus valence included the medial orbitofrontal cortex, subgenual anterior cingulate cortex and amygdala, whereas stimulus intensity was correlated with activity in the insula, thalamus and striatum, among others. Our results suggest segregated patterns of neural activity underlying perceptions of valence and intensity, consistent with dimensional models of emotion.

In this paper, we studied neural networks of both excitatory and inhibitory populations with inhibition stabilized network (ISN) models. In ISN models, the recurrent excitatory connections are so strong that the excitatory sub-network is unstable if the inhibitory firing rate is fixed; however, the entire network is stable due to inhibitory connections. In such networks, external input to inhibitory neurons reduced their responses due to the withdrawal of network excitation (Tsodyks et al., 1997). This paradoxical effect of the ISN was observed in recent surround suppression experiments in the primary visual cortex with direct membrane conductance measurements (Ozeki et al., 2009). In our work, we used a linearized rate model of both excitatory and inhibitory populations with weight matrices dependent on the locations of the neurons. We applied this model to study surround suppression effects and searched for networks with appropriated parameters. The same model was also applied in the study of spontaneous activities in awake ferrets. Both studies led to network solutions in the ISN regime, suggesting that ISN mechanisms might play an important role in the neural circuitry in the primary visual cortex.

Dopamine receptors mediate various important neurophysiological functions. At a molecular level, G protein coupling is considered the main activation mechanism for most of the receptor-mediated cellular processes. A number of studies using native tissue have supported the idea that receptors can interact to form dimers or higher order oligomers. Particularly in medium spiny neurons of the striatum, dopamine receptor subtypes are reported to form dimers with themselves or other receptors (e.g. adenosine receptor A2A). Although a functional relevance for these dimers has been proposed, current assay systems are not capable of teasing out dimer-specific signaling events from those from other receptor populations. We have developed an assay that allows investigation of receptor-effector coupling specifically with defined dimer pairs. Using this assay, we investigated putative dopamine D1-D2 and A2A-D2 receptor dimer functions and studied the issue of a purported G protein coupling switch in the D1-D2 receptor dimer in which the heteromer was proposed to activate Gq, unlike D1 or D2 receptor when expressed alone. We were unable, however, to find evidence for Gq activation by the D1-D2 heteromer, as the protomers in the heteromer maintained fidelity of signaling to their cognate G proteins. We also developed and optimized a series of novel Gs biosensors to elucidate differences in heterotrimeric G protein conformational changes triggered by dopamine D1 and A2A receptors, two of the prominent pharmacological targets in the striatum. In addition to G protein signaling, intracellular calcium is also involved in many important cellular functions in all cell types. In neurons, intracellular calcium is implicated in learning and memory (synaptic plasticity) as well as neurodegeneration (apoptosis). In medium spiny neurons, dopamine-mediated calcium release from internal stores has been reported to result from activation of phospholipase C (PLC). However, different subtypes of dopamine receptors and intermediary proteins have been proposed to play a role in this dopamine-mediated PLC activation, and the underlying mechanisms are unclear. We found that activation of D1 and D2 receptors expressed individually can mobilize calcium in a PLC-dependent manner. In parallel, we also examined D1 and D2 receptor colocalization in striatal brain slices as well as in cultured medium spiny neurons. Although we found evidence using bacterial artificial chromosome-D1 and D2 reporter mice that D1 and D2 receptors are co-expressed in a small number of brain regions, we failed to observe D1-D2 receptor colocalization, suggesting the possibility that in neurons the receptors are somehow segregated.

MicroRNAs (miRNAs) are evolutionarily-conserved small non-coding RNAs that are important posttranscriptional regulators of gene expression. Genetic Variants may cause microRNA dysregulation and the concomitant aberrant target expression. The dysregulation of one or a few targets may in turn lead to functional consequences ranging from phenotypic variations to disease conditions. In this thesis, I present our studies of mouse models of two human genetic variants - a rare copy number variant (CNV), 22q11.2 microdeletions, and a common single nucleotide polymorphism (SNP), BDNF Val66Met. 22q11.2 microdeletions result in specific cognitive deficits and high risk to develop schizophrenia. Analysis of Df(16)A+/- mice, which model this microdeletion, revealed abnormalities in the formation of neuronal dendrites and spines as well as microRNA dysregulation in brain. We show a drastic reduction of miR- 185, which resides within the 22q11.2 locus, to levels more than expected by a hemizygous deletion and demonstrate that this reduction impairs dendritic and spine development. miR-185 targets and represses, through an evolutionary conserved target site, a previously unknown inhibitor of these processes that resides in the Golgi apparatus. Sustained derepression of this inhibitor after birth represents the most robust transcriptional disturbance in the brains of Df(16)A+/- mice and could affect the formation and maintenance of neural circuits. Reduction of miR-185 also has milder effects on the expression of a group of Golgi-related genes. One the other hand, BNDF Val66Met results in impaired activity-dependent secretion of BDNF from neuronal terminals and affects episodic memory and affective behaviors. We found a modest reduction of miR-146b which causes derepression of mRNA and/or protein levels of a few targets. Our findings add to the growing evidence of the pivotal involvement of miRNAs in the development of neuropsychiatric disorders and cognitive dysfunction. In addition, the identification of key players in miRNA dysregulation has implications for both basic and translational research in psychiatric disorders and cognitive dysfunction.

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder characterized by the dysfunction and death of motor neurons. Patients afflicted with this condition commonly experiences muscle weakness that progresses to generalized paralysis. Although most ALS cases are sporadic, mutations in several human genes of divergent molecular function have been linked to the development of ALS. Recently, two new ALS genes, TDP-43 and FUS, were identified that have structural similarities suggesting that they may function in a common process. TDP-43 and FUS have both been implicated in a number of cellular functions, including the regulation of splicing, transcription, and microRNA processing. The work in this thesis includes studies that identified roles for TDP-43 and FUS in the health and function of the nervous system of Drosophila melanogaster. Genetic and biochemical studies were used to define the molecular relationships between TDP-43 and FUS, placing TDP-43 upstream of FUS in a common pathway that is important for the function and health of the nervous system. The finding that TDP-43 and FUS interact suggests that they regulate a common disease pathway that is disrupted in ALS and that therapeutic intervention in this pathway may ameliorate many of the symptoms of the disease.

Most neuropsychiatric disorders have developmental origins and an emerging model postulates that such developmental vulnerability is often restricted to sensitive periods. The concept of sensitive developmental periods for the indelible modulation of complex behaviors is similar to that described for sensory systems (e.g. visual cortex, ocular dominance plasticity), but effected behaviors, modulating factors, and underlying mechanisms are much less well understood. Furthering our knowledge of sensitive periods that determine the developmental trajectory of complex behaviors is a necessary step towards improving diagnosis, prevention and treatment approaches for neuropsychiatric disorders. To fulfill this mission, I here investigate how genetic and environmental risk factors act during sensitive periods of brain development to alter adult behavior and thereby confer vulnerability to neuropsychiatric disorders. My thesis is divided into four chapters. Chapter I provides general background and significance information relevant to chapters II-IV. Chapter II focuses on elucidating and comparing the consequences of developmental serotonin (5-HT) transporter (5-HTT) and monoamine oxidase A (MAOA) blockade. Pharmacologic MAOA or 5-HTT blockade in adulthood has antidepressant and anxiolytic efficacy. Yet, genetically conferred MAOA or 5-HTT hypo-activity is associated with altered aggression and increased anxiety/depression. Here I test the hypothesis that increased monoamine signaling during development causes these paradoxical aggressive and affective phenotypes. I find that pharmacologic MAOA blockade during early postnatal development (P2-P21) increases anxiety- and depression-like behavior in mice, mimicking the effect of P2-21 5-HTT inhibition. Moreover, MAOA or dopamine transporter (but not norepinephrine transporter) blockade during peri-adolescence (P22-P41) increases adult aggressive behavior. 5-HTT blockade from P2-P21 or P22-P41 reduces adult aggressive behavior. Altered aggression correlates positively with locomotor response to amphetamine challenge in adulthood and striatal dopamine and DOPAC content is increased while brainstem 5-HIAA content is decreased in high aggression. Taken together, these data suggest that genetic and pharmacologic factors impacting dopamine and serotonin signaling during sensitive developmental periods confer risk for aggressive and emotional dysfunction in humans. Chapter III focuses on refining the 5-HT sensitive period affecting anxiety and depression-like behavior. Specifically, I hypothesized that the identified P2-21 period, which encompasses many developmental processes, contains a narrower critical period, affecting fewer developmental processes but having the same impact on adult behavior. This experiment serves two purposes: First, I seek to gain insight into the neural substrates and possible developmental processes underlying developmental programing of anxiety- and depression-like behaviors through 5-HT signaling. Second, I aim at providing translationally relevant data, informing clinical and epidemiological studies as to which developmental window might be sensitive to 5-HT altering factors in humans. This thesis research shows that postnatal fluoxetine (PN-FLX) treatment from P2-11 leads to increased adult anxiety- and depressive-like behavior in mice, while PN-FLX treatment from P12-21 or P22-41 has no effect in adult anxiety- and depressive-like behavior. In addition, adult chronic FLX treatment could not rescue the behavioral phenotype produced by P2-11 5-HTT blockade. Chapter IV focuses on the role of 5-HT2A receptor signaling in mediating the effect of P2-11 5-HTT blockade on adult behavior. Htr2a-/- mice display reduced conflict anxiety. Because 5-HT2A receptor antagonists do not reduce conflict anxiety in adulthood, I hypothesized that the behavioral htr2a-/- phenotype is at least partially of developmental origin, which would further indicate that increased 5-HT2A receptor signaling during development could increase conflict anxiety. To investigate this hypothesis, I analyzed the effect of P2-11 5-HTT blockade on anxiety and depression-like behaviors in htr2a+/+, +/-, and -/- mice. Supporting my hypothesis, I find that absence of htr2a improved performance of PN-FLX treated mice in the novelty suppressed feeding task, by decreasing the latency to feed to control levels. Absence of htr2a, however, did not have ameliorative effects on PN-FLX phenotypes in the open field and shock escape tests. In summary, these data demonstrate that 5-HT2A receptor signaling mediates some but not all consequences of increased P2-11 5-HT signaling. Taken together, in my thesis work I identified and characterized two sensitive developmental periods whereupon early-life perturbation of monoamine signaling alters adult behavior: an early postnatal (P2-P11) 5-HT-sensitive period that affects anxiety and depression-related behaviors and a later peri-adolescent (P22-P41) DA- and 5-HT-sensitive period altering aggression and behavioral sensitivity amphetamine. These data give insight into the etiology of neuropsychiatric disorders and should ultimately help improving diagnosis, prevention and treatment approaches.