<p> Recent advances regarding commensals in the gastrointestinal tract point to an intimate &ldquo;accessory&rdquo; organ status. To study the cross-talk that an accessory organ must have, the Piletz laboratory began in 2014 developing a three-dimensional (3D) <i>in vitro</i> co-culture model system, whereby two differentiated cell lines are juxtaposed along with &ldquo;luminal&rdquo; contents. The model uses differentiated C2BBe1 cell line enterocytes grown to confluency on polycarbonate filters with 0.4 &micro;m pores over-layered atop SH-SY5Y cell line neurons to study cross-talk from either the lumen-side or the neuron-side. The focus is on an endogenous molecule, agmatine (1-amino-4-guanidobutane), made by gut bacteria at millimolar concentrations in the mucosa of the small intestine&mdash;yet in the brain known to be a neurotransmitter. Starting with each individual cell line in standard mono-cultures, agmatine was added at varying doses and varying times to replicate what is essentially dogma to the agmatine field, that of being anti-proliferative to all mammalian cells. Above 1 mM agmatine, the predicted anti-proliferative response was realized as a non-toxic, non-divisional state sustained for at least 4 days from single dosing. Moving to the 3D co-culture system, wherein the C2BBe1 cells were differentiated as per high transepithelial electrical resistance (TEER) over a 24-hour equilibration period, it was expected that agmatine would again be <i>anti-proliferative</i>. Yet, apical agmatine appeared to exert a <i>pro-proliferative</i> effect starting as low as 0.002 mM. A parallel decline in metabolism per SH-SY5Y cell was found using the color dye reaction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). It was therefore hypothesized that apical agmatine had caused the C2BBe1 cells to secrete a growth signal(s) impacting the underlying SH-SY5Y cells; and to test this, conditioned basal media collected from just C2BBe1 cells grown 4 days in the presence of apical 2 mM agmatine was taken to replace the media of na&iuml;ve SH-SY5Y cells growing in log phase mono-cultures. The expectation was that growth factors would be carried over, but to the contrary, an anti-proliferative response emerged from the conditioned media, mirroring the earlier studies with agmatine in mono-cultures. Cellular lysates were also prepared from treated cells exposed for 24 h to 2 mM agmatine, and these were probed on immune-blots to assess if any of 32 common receptor tyrosine kinases had phosphorylated /activated post-addition of apical mM agmatine. No evidence was obtained that agmatine (mM apical) had elicited such flags of cell activation. Next, the 3D co-culture condition was re-run for longer periods and with more controls, and from this came the realization that the model had hidden the existence of an anti-proliferative response from the C2BBe1 cells before agmatine was even added. In short, the starting hypothesis was disproven, but in doing so it was realized that micromolar apical agmatine is able to rejuvenate a cytostasis rendered by the C2BBe1 co-culturing. Two fundamentally different mechanisms must be invoked by agmatine, because the concentrations of agmatine at which these two processes occurred were 500-fold different (0.002 mM for the reversal of cytostasis vs. 1 mM for anti-proliferative, respectively). In summary, any microbial dysbiosis involving agmatine-producing bacteria is likely to act through two molecular signaling mechanisms from the &ldquo;accessory&rdquo; organ bacteria to enteric nervous system.</p><p>

<p> The human brain is an ongoing dynamic system not activated by experience but nudged from intrinsic activity into new network configurations during perception and learning. Ongoing neural activity during rest is assumed to reflect these intrinsic dynamics in a relatively closed system state. Traditionally, inter-regional connectivity in this system is measured by obtaining time-locked correlations in BOLD activity using fMRI. It is well documented, however, that neural activity unfolds across time and is not isolatent to some reference point. </p><p> This exploratory study is a theoretical analysis of how a lagged analysis of resting state dynamics in fMRI could represent persistent representations of knowledge in the neocortex. A novel procedure using both surface based maps and independent component analysis (ICA) is applied to a small group of 54 adolescents. The ICA methods appear to reveal lagged structures with different information than traditional resting state analysis. The group level results are symmetrical between hemispheres and may represent high level perceptual systems.&nbsp;</p><p> The components obtained from this exploration are then used to attempt understand how these knowledge systems in neocortex frame mind-wandering frequency when reading aloud in a subset of 38 individuals. The results did not correlate with any known neural systems related to mind wandering, but the methods here are unique. One of the identified components shows significant difference in the lag structure of the occipital cortex as a function of mind wandering frequency during oral reading. This demonstrates that it may be worth exploring the timing in visual system to understand why individuals mind wander when reading aloud. Reverse inference is used to interpret results and suggest future approaches.</p><p>

<p> Basal forebrain corticopetal cholinergic neurons are critical for contextual and cued fear memory in the conditioned suppression paradigm, but neural mechanisms that alter these neurons in fear memory remain unknown. Interestingly, basal forebrain cholinergic lesions have no effect on behavioral performance in commonly-studied fear conditioning paradigms like Pavlovian conditioned freezing or fear-potentiated startle, yet impair fear memory in the conditioned suppression paradigm. Many studies conducted have experimented with lesions of cell bodies of corticopetal cholinergic neurons in the nucleus basalis magnocellularis (NBM), but there is a void in the literature defining which specific projections may be responsible for their discrepant role in different fear memory paradigms. The basal forebrain projects to the anterior cingulate cortex (ACC), a subregion of the medial prefrontal cortex. The ACC is a well-established portion of the fear circuit across all fear conditioning paradigms and has a clear role in decision-making in the conditioned suppression paradigm. Given the role in choice conflict that the ACC plays in operant tasks involved in the conditioned suppression paradigm, it is plausible that it may be a region that allows basal forebrain cholinergic neurons to alter a fear memory in the conditioned suppression paradigm. The goal of this study is to examine the specific roles that basal forebrain cholinergic projections to the ACC play in fear memory, specifically in the conditioned suppression paradigm. These lesions may target specific cholinergic input to the ACC from the NBM in the basal forebrain and this may isolate a specific fear circuit involved in fear memory in the conditioned suppression paradigm. Data have suggested that ACC lesioned animals demonstrate less fear-conditioned suppression over sham animals, but further experiments and cohorts of animals are required. If ACC cholinergic lesions are shown to produce deficits in fear memory in the conditioned suppression paradigm, it may suggest that the presence of the appetitive task, which only occurs in the conditioned suppression paradigm and not in any of the other commonly studied fear paradigms, may be able to elicit changes in functional connectivity to incorporate this projection from the NBM to the ACC to the fear circuit. Discrepancies in fear memory between fear conditioning paradigms demand to be addressed because assumptions about functional connectivity across different paradigms are assumed to be similar in the literature. If the notion of paradigmdependent functional connectivity presented here is true, deductions about this functional connectivity may only be made in the context of one fear paradigm and may not necessarily be applicable across paradigms. In other words, to say that Pavlovian fear conditioning and fear-potentiated startle are indicative of the broad neurobiology of fear memory would only be looking at a fraction of the reality behind how fear circuitry operates. In order to further the literature to propose holistic circuits, molecular processes and constructs that apply to all fear memory regardless of protocol or paradigm, it is necessary to investigate neural involvement across alternative fear paradigms, like conditioned suppression. This study supports the novel idea that neural circuitry that supports fear can expand with new learning tasks or events and therefore, may be more susceptible to change than previously considered, but future studies are required</p><p>

<p> When someone hears regular, periodic sounds, such as drum beats, footsteps, or stressed syllables in speech, these individual stimuli tend to be grouped into a perceived rhythm. One of the hallmarks of rhythm perception is that the listener generates expectations for the timing of upcoming stimuli, which theorists have described as endogenous periodic modulations of attention around the time of anticipated sounds. By constructing an internal representation of a rhythm, perceptual processes can be augmented by proactively deploying attention at the expected moment of an upcoming stressed syllable, the next step in an observed stride, or during the stroke of a co-speech hand gesture. A hypothetical benefit of this anticipatory allocation of attention is that it might facilitate temporal integration across the senses, binding multisensory aspects of our experiences into a unified &ldquo;now,&rdquo; anchored by temporally-precise auditory expectations. The current dissertation examines this hypothesis, exploring the effects of auditory singletons, and auditory rhythms, on electrophysiological indices of perception and attention to a visual stimulus, using the flash-lag paradigm. An electroencephalography study was conducted, where sounds, either isolated or presented rhythmically, occurred in alignment with a task-relevant visual flash. Results suggest a novel dissociation between the multisensory effects of discrete and rhythmic sounds on visual event perception, as assessed by the N1 component of the event-related potential, and by oscillatory power in the beta (15&ndash;20 Hz) frequency range. This dissociation is discussed in the context of classic and contemporary research on rhythm perception, temporal orienting, and temporal binding across the senses, and contributes to a more refined understanding of rhythmically-deployed attention. </p><p>

<p> Mitochondrial metabolism of reactive oxygen species (ROS) is tightly regulated during brain development. Imbalance has been correlated to neuropsychiatric disorders. Nevertheless, the contribution of ROS accumulation to aberrant cortical circuit organization and function remains unknown. Individuals with 22q11 deletion syndrome (22q11DS) are highly susceptible to psychiatric disorders; therefore, 22q11DS has been suggested as a model for studying the neurodevelopmental origins of these disorders. Six genes &ndash;<i>Mrpl40, Tango2, Prodh, Zdhhc8, Txnrd2</i> and <i>Scl25a1</i>&ndash; located in the 22q11DS commonly deleted region encode proteins that localize to mitochondria. This project aimed to characterize the effects of altered mitochondrial function, due to diminished dosage of these genes, on cortical projection neuron development, using the <i>LgDel</i> mouse model of 22q11DS. I found growth deficits in <i>LgDel</i> neurons that are due to increased mitochondrial ROS and are <i>Txnrd2</i>-dependent. Antioxidant treatment, by n-acetyl cysteine (NAC), rescues neuronal morphogenesis in <i>LgDel</i> and <i> Txnrd2</i>-depleted neurons <i>in vitro</i> and <i>in vivo.</i> Electroporation of <i>Txnrd2</i> restores ROS levels and normal dendritic and axonal growth. <i>Txnrd2</i>-dependent redox regulation underlies a key aspect of cortical circuit differentiation in a mouse model of 22q11DS. These studies define the effects of mitochondrial accumulation of ROS on neuronal integrity, and establish the role of altered pyramidal neuron differentiation in the formation of circuits in 22q11DS. These data provide novel insight into the role of redox imbalance in aberrant development of cortical circuits.</p><p>

<p>Fibroblast growth factors (FGFs) and their receptors (FGFRs) play a vital part in the proper development and maintenance of the brain. FGFR1, which is one of four FGFRs total and one of three found in the brain (FGFR1-3), has been shown to be important in cellular proliferation, cellular migration, synaptogenesis, cellular morphology, and has also been implicated in multiple neuropsychiatric disorders. Understating the role FGFR1plays in these and other cellular processes is vital to our understanding of the human body and in the prevention and treatment of some neuropsychiatric and developmental disorders. Although previous studies have produced groundbreaking findings in the field, they have fallen short in the accurate identification of which cell type express Fgfr1. Therefore, to validate the use of a transgenic mouse line in the accurate and efficient study of Fgfr1 expression during experimental manipulations and cellular localization, we utilized the tgFGFR1- EGFPGP338Gsat BAC mouse line (tgFgfr1-EGFP+) obtained from the GENSAT project. By utilizing the tgFgfr1-EGFP+ mouse line, we were able to accurately identify which cell types in the embryonic and perinatal mouse brain express Fgfr1. Furthermore, we were able to measure relative changes in Fgfr1 expression via GFP fluorescence as a proxy after both exposure to chronic stress and the chemical demyelinator, cuprizone. The combination of our results lead us to conclude that the tgFgfr1-EGFP+ mouse line is a very useful tool in the study of FGFR1 and may aid in the identification of potential targets for therapeutic treatment of the many disorders associated with FGFR1 signaling.

Neural communication is imperative for physical and mental health. Dysfunction in either ionic signaling or chemical neurotransmission can cause debilitating disorders. Thus, study of neurotransmission is critical not only to answer important fundamental questions regarding learning, decision making, and behavior but also to gain information that can provide insight into the neurochemistry of neurological disorders and lead to improved treatments. The work presented herein describes the development of techniques and instrumentation to enable advancements in neuroscientific inquiry. The effect of different temporal patterns and durations of simulation of the prefrontal cortex on dopamine release in the nucleus accumbens was examined and revealed a complex interaction that can help improve deep brain stimulation therapies. A measurement platform that combines electrophysiological and electrochemical techniques is described. The instrumentation is capable of concurrent monitoring of neural activity and dopamine release in vivo and in freely moving rodents. Analysis techniques to allow absolute quantification of tonic dopamine concentrations in vivo are detailed and the temporal resolution of the technique was vastly improved from ten minutes to forty seconds. An instrument that can simultaneously probe both dopamine and serotonin dynamics in either of their two temporal modes of signaling (tonic and phasic) using either fast-scan cyclic voltammetry or fast-scan controlled-adsorption voltammetry at two individually addressable microelectrodes is described. Together these new tools represent a significant step forward in the field of neuroanalytical chemistry by enable multiple brain regions, signaling modes (ionic flux in addition to both tonic and phasic neurotransmission), neurochemicals, and to be measured together.

Excitation and inhibition in the nervous system must be carefully balanced. I utilized the zebrafish (Danio rerio) in order to study this balance in the vertebrate motor system. Zebrafish (Danio rerio) can be used effectively for both forward and reverse genetics. My primary interest lies in genes involved in neural network development and function. Forward genetic mutagenesis screens are particularly powerful as they are unbiased. Using this approach we characterized a mutant based on its abnormal motor behavior. However, mutants identified with abnormal motor behavior may have mutations in muscle proteins. We identified a semi-dominant mutation in the atp2a1 gene, which encodes a protein vital for normal muscle function, from a previously completed mutagenesis screen. In order to investigate genes directly involved in neuronal signaling I used a reverse genetics approach to study GABAA receptors. γ-Aminobutyric acid (GABA) is a major source of inhibition in the motor system. In order to test the effect of blockade of GABAA receptors, I injected gabazine, a GABAA receptor antagonist, into zebrafish embryos. Injected embryos display an abnormal escape response later in development. To determine the GABAA receptor subunits responsible for the observed phenotype, I utilized RNA in situ hybridization to examine the expression of the &agr; class of GABAA receptor subunits. I found that the gabra5 gene, which encodes the GABAA &agr;5 subunit, was expressed in the hindbrain of larvae. To examine the role of the &agr;5 subunit I designed anti-sense morpholinos to target the start codon of the gabra5 gene. Knockdown of the gabra5 gene caused abnormal behavior in larvae similar to that seen in gabazine injected larvae. I have characterized a novel semi-dominant atp2a1 mutant in zebrafish. This mutant completes a zebrafish model system of the human disease Brody's disease. Turning toward a reverse genetic approach I investigated the expression of several GABAA receptor &agr; subunits. I have characterized the behavior of GABAA &agr;5 subunit knockdown embryos and larvae and begun work to generate a stable knockout line. This line will be useful in exploring the function of the &agr;5 subunit and compounds that interact with it.

Thyroid hormone (TH) plays an important role in fetal brain development. Therefore, exogenous factors that interfere with TH signaling may exert potentially important adverse effects on brain development. However the specific roles of TH in brain development are poorly understood. The goal of this research is to delineate the mechanisms of TH and potential mechanisms of endocrine disruption at the TH receptor in the developing brain. The dissertation showed that PCBs can reduce the circulating levels of TH, but simultaneously exerted TH-like effects on TH-responsive genes in fetal brain. To determine whether a specific PCB metabolite, 4-OH-PCB106, could exert a direct agonistic effect on the TRβ1, we employed chromatin immunoprecipitation (ChIP). These studies demonstrate that 4-OH-PCB106 acts as an agonist in GH3 cells, and does not alter the ability of TRβ1 to physically interact with the TRE in the growth hormone (GH) promoter, or with SRC1/NCoR. Interestingly, 4-OH-PCB106 appears to exert actions on gene expression in GH3 cells predominantly through TR, as evidenced by a focused study using differential mRNA display in GH3 cells. A significant impediment in identifying the ability of PCBs to interact with TRs in vivo is that few direct gene targets of TH are known. Therefore, we employed ChIP-on-chip in combination with whole transcriptome expression analysis. We identified 526 direct TH gene targets and these revealed major signaling networks regulated by maternal TH during fetal brain development, including cell-fate specification, cell migration and synaptogenesis. This combination of approaches provides a new look at the role of TH in fetal brain development. In a summary, despite the great deal of research focused on the mechanism of TH action, we do not have comprehensive understanding of the role of TH and its modulators in the brain. Therefore it is important to identify genes that may be direct targets of TH action. This is the first large in vivo database for native TREs in the fetal brain before the onset of fetal thyroid function. Therefore the result will provide profound impact in study of mechanisms of TH as well as endocrine disruptors at the TR in developing brain.

In male hamsters mating behavior is dependent on sufficient androgens and chemosensory input from the main olfactory and vomeronasal systems, whose central pathways contain cell bodies and fibers of gonadotropin-releasing hormone (GnRH) neurons. Regions of the medial amygdala (vomeronasal amygdala) contain androgen receptors and differentially process chemosignals with different social implications. According to published reports of "categorical" patterns of response, conspecific chemosensory stimuli activate the anterior (MeA) and posterior (MeP) medial amygdala, while heterospecific stimuli only activate MeA, in male hamsters (and male mice). Furthermore, chemosignals with distinct social implications differentially activate the dorsal and ventral subregions of MeA and MeP (MeAd/v, MePd/v). In sexually-naïve male hamsters, lesions of the vomeronasal organ (VNX), but not the main olfactory bulb, impair mating behavior. Intracerebroventricular (icv)-GnRH restores mating in sexually-naïve VNX males and enhances medial amygdala (Me) activation by chemosensory stimulation. In sexually-experienced males, VNX does not impair mating and icv-GnRH suppresses Me activation. Thus, main olfactory input is sufficient for mating in experienced- but not naïve-VNX males. I tested whether GnRH enhances access of main olfactory input to the amygdala using icv-GnRH and either electrical or pharmacological stimulation of the main olfactory bulb (MOB), and then examined immediate early gene (IEG) expression there. Electrical stimulation of the MOB did not significantly activate the ipsilateral main olfactory cortex or amygdala in intact or VNX animals. When the IEG counts from both sides of the brain were averaged together, GnRH appeared to enhance activation in the medial amygdala in naïve-intact males, but appeared to decrease activation in naïve-VNX males. I concluded that electrical stimulation was not a sufficient means of driving main olfactory input to downstream brain regions, possibly due to activation of intra-bulbar inhibitory circuits. To alleviate this possible confound, I pharmacologically stimulated the MOB with a mixture of bicuculline methiodide and d,l Homocysteic acid. In sexually-naïve intact-males, MOB stimulation produced significant activation in MeAv and MePv. MePv activation is also characteristic of chemosensory stimuli from potential competitors and predators. In sexually-naïve VNX-males, in which GnRH facilitates mating, GnRH enhanced activation by MOB stimulation in posterodorsal medial amygdala (MePd), a region known to be rich in androgen resceptors and activated by conspecific reproductive chemosignals. Conversely, in sexually-experienced VNX-males, animals that do not require exogenous GnRH to mate normally after VNX, there is a depression in activation in MePd due to GnRH and stimulation in MePd, similar to its response to natural chemosensory stimulation. There also appeared to be a possible effect of VNX due to the difference in selective activation of GnRH in naïve-intact vs. naïve-VNX animals. MeP is also rich in steroid receptors and many chemosensory behaviors are steroid dependent. Therefore, I also tested the activation of androgen receptor (AR)-containing cells in Me after conspecific or heterospecific chemosensory stimulation. Conspecific and heterospecific chemosensory stimuli significantly activated AR-containing cells in Me and significantly increased the number of AR-positive cells in Me above control. The increase in the number of AR-ir cells produced by conspecific stimuli was also significantly above the numbers of AR-ir cells produced by the heterospecific stimulus. These effects may be due to increases of testosterone in response to chemosignals or circuit activity dependent on steroid levels. Future studies on castrated testosterone-replaced males will test these possibilities. The studies of this dissertation provide important information about the neurohormonal regulation of chemosensory and olfactory input to the medial amygdala. The integration of hormonal and chemosensory factors is vital to mating and other social behaviors, and thus species survival. The amygdala is crucial to this process in many vertebrate species, including the hamsters, which use chemicals to communicate with one another. This dissertation suggests, and provides some evidence for a part of the mechanism by which the amygdala accomplishes this integration. / A Dissertation submitted to the Department of Biological Science in partial fulfillment of the requirements for the degree of Doctor of Philosophy. / Spring Semester, 2009. / December 9, 2008. / Olfactory Bulb, GnRH, Androgen Receptor, Vomeronasal, Hamster, Amygdala / Includes bibliographical references. / Michael Meredith, Professor Directing Dissertation; Jon Maner, Outside Committee Member; P. Bryant Chase, Committee Member; Richard Hyson, Committee Member; Zuoxin Wang, Committee Member.