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Estradiol (E) and progesterone (P) induce the expression of sexual behavior in female rats by binding to their intracellular receptors (ER and PR, respectively) in specific brain regions. Nuclear receptor coactivators, including Steroid Receptor Coactivator-1 (SRC-1) and CREB Binding Protein (CBP), have been shown to enhance ligand-dependent steroid receptor transcriptional activity in vitro. One mechanism by which nuclear receptor coactivators promote gene expression is by bridging the receptor complex with the basal transcription machinery and inducing chromatin remodeling. Although knockout mice have provided some insight into the general function of SRC-1 and CBP in vivo, it is difficult to interpret these data because both SRC-1 and CBP interface with a large family of nuclear receptors. Consequently, the data do not provide information specific to ER and/or PR, or provide information specific to brain region. Therefore, to test the hypothesis that SRC-1 and CBP are important mediators of E and P action on sexual behavior, I decreased coactivator proteins in the ventromedial nucleus of the hypothalamus (VMN) using an antisense technique. I found that reduction of SRC-1 and CBP in the VMN decreased (1) ER-mediated induction of PR expression in the VMN, (2) ER-dependent sexual receptivity as characterized by the lordosis posture, and (3) PR-dependent proceptive behaviors including hopping and darting and ear wiggling. Using pull-down assays to test whether SRC-1 from hypothalamic or hippocampal/cortical extracts interacts with ER and PR, I found that SRC-1 from brain associates with ER and PR when receptors were bound to agonists, but not when bound to antagonists, or in the absence of ligand. Furthermore, the efficiency of the interactions between SRC-1 and ER or PR was dependent upon receptor subtype. Taken together, the present studies suggest that coactivators interact with ER and PR in the brain as they modulate hormone actions in the VMN that regulate sexual behavior.

The present study employed electrophysiological and behavioral approaches to investigate conditioned response (CR) timing and integration when rabbits were trained with a temporal uncertainty paradigm. The temporal uncertainty paradigm involved training with a mixture of two interstimulus intervals (ISIs) of 300 and 700-ms. Previous work reported that when rabbits were trained using two CS-US, the resulting CRs showed bimodal amplitude peaks (Moore & Choi, 1997). Principle activities investigated in this study included a recordings experiment designed to investigate the expression of CR integration in cerebellar Purkinje cells and experiments designed to provide normative data on CR integration at the behavioral level. The electrophysiological recording experiment investigated the topography of conditioned eyeblink responses in rabbits trained under a mixed ISI procedure is predicted by the conditioned stimulus-triggered firing of cerebellar Purkinje cells. Recordings from Purkinje cells showed that the activity of individual neurons located in the cerebellum fired in a way that reflects the topography of conditioned eyeblink responses. Single neurons fired in anticipation of both amplitude peaks of bimodal CRs, suggesting that integration of learning may be expressed by individual Purkinje cells. Additionally, on short ISI trials the response to the second peak was suppressed suggesting that the US becomes a conditioned inhibitor during training. The behavioral experiments examined compound conditioning under temporal uncertainty and how it presents challenges to various models of learning, such as Sutton and Barto's TD (CSC) model (1990), which are designed to predict the amplitude and timing of complex CR waveforms in eyeblink conditioning. These behavioral experiments specifically examined rules for reconstructing complex CR waveforms from component waveforms that arise for mixed-ISI protocols. Decomposition of compound waveforms preserved the bimodality of compound waveforms, but the overall waveforms were shifted in time resulting in longer latencies than observed compound waveforms. Summation of trained component waveforms, each trained at different ISIs, resulted in unimodal compound waveforms that were also shifted so that the peaks of the compound waveforms were between those of the component waveforms. Combination rules would have fit better if not for the shift in latency that occurred.

Hormonally regulated cell death is thought to underlie many sex differences in the nervous system, but little is known about the molecular mechanisms involved. This question was investigated in a sexually dimorphic neuromuscular system in mice. Motoneurons in the spinal nucleus of the bulbocavernosus (SNB) innervate striated perineal muscles including the bulbocavernosus (BC) and levator ani (LA). In rats, SNB cell number and BC/LA muscle size are similar in males and females prenatally, but the postnatal persistence of this neuromuscular system is androgen dependent. Androgens reduce cell death of SNB motoneurons during a perinatal critical period and, as a result, males have more SNB motoneurons than do females in adulthood. The Bcl-2 family of proteins, which includes anti-apoptotic (e.g., Bcl-2) and pro-apoptotic (e.g., Bax, Bak) family members, regulates cell death in neurons and other tissues. I found that Bax is essential for the normal sex difference in SNB motoneuron number in adult mice. Experiments to characterize the time course of developmental cell death in the SNB of mice proved inconclusive based on counts of retrogradely-labeled SNB motoneurons and pyknotic cells in the SNB region, but suggest that developmental cell death may overlap with a period of late secondary SNB migration. Further, SNB motoneuron location differs between perinatal male and female mice. It's controversial whether the sex difference in perineal muscle size is due to sexually dimorphic cell death or to androgen dependent growth, especially of the LA. We find that Bax deletion slightly increased LA fiber number in adult females, but did not eliminate the gross sex difference in perineal muscle size. Modest effects of Bax deletion on the perineal muscles in adulthood may result from functional redundancy between Bax and Bak. In confirmation, I found that Bax/Bak DKO females have a more than 10-fold increase in LA fiber number over that in wild type females. In addition, wild type females have a higher density of TUNEL-positive cells than do males in both the BC and the LA during perinatal life. Thus, cell death makes an important contribution to the sexually dimorphic development of the BC and the LA.

The intracellular environment of eukaryotic cells has been found to have membrane-less organelles, which form through liquid-liquid phase separation.1,2 The membrane-less organelles within the cell can be modeled with multiphase complex coacervates that are formed through the combination of two liquid polyelectrolyte complex coacervates, which are individually made up of a pair of one positively charged polymer and one negatively charged polymer.3,4 Membrane-less organelles in a eukaryotic cell have been found to aggregate from liquid to solid in neurological disorders and during the aging process5, and investigation into the factors affecting aggregation of multiphase complex coacervates may shed light on how membrane-less organelles aggregate in the eukaryotic cell. This project specifically aims to investigate how hydrophobicity differences between the polyelectrolyte complex coacervates affect the secondary structure of the multiphase complex coacervate, particularly the beta-sheet structure of the multiphase complex coacervate, which indicates a more solid structure.6 Positively and negatively charged heterochiral polypeptides with varying hydrophobicity were synthesized with solid-phase peptide synthesis and combined to form polyelectrolyte complex coacervates and multiphase complex coacervates; these were then characterized with optical microscopy, Fourier transform infrared spectroscopy, and critical salt concentration measurements. Results indicate that a larger hydrophobicity difference between the two polyelectrolyte complex coacervates of a multiphase complex correlates to a more solid character of the multiphase complex, although the character of the individual polyelectrolyte complex coacervates also affects the character of the multiphase complex.

Huntington Disease (HD) is a neurodegenerative disorder that is caused by a CAG trinucleotide repeat expansion in the huntingtin gene. The onset of the disease is defined by the presence of motor deficits, such as chorea. However, cognitive and psychiatric symptoms often develop before motor onset and typically have a larger impact on patient quality of life. Psychiatric symptoms include depression, anxiety, and OCD, but also aggression and irritability, which have been comparatively understudied due to stigma. Currently, treatments to modify these behaviors in premanifest HD patients are not consistently effective and often have side effects, creating a need for research into these psychiatric disturbances. Our lab has observed increased-aggression in our humanized HD mouse model (Hu97/18) during routine handling that is not present in our knock-in HD mouse model (Q175FDN). In this study, we seek to quantify the aggressive behavior exhibited by these two mouse models and determine the neurological basis for these observed behavioral differences. From this analysis, we seek to identify potential therapeutic targets for modulating aggressive behavior in mice, which could lead to the development of therapies that reduce the aggressive behavioral symptoms experienced by HD patients.

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<p>In the current investigation several biochemical techniques, including solubilization, affinity chromatography and photoaffinity labelling, were used to purify the D-1 dopamine agonist ADTN (2-amino-5, 6-dihydroxy-tetrahydornaphthealene) to be used as affinity and photoaffinity probes was an integral part of this investigation.</p> <p>Solubilization of the D-1 receptor was achieved with several detergents although cholic acid proved to be the most effective for receptor solubilization prior to affinity chromatography. Using this procedure, yields of solubilized receptors of greater than 30% were consistently obtained.</p> <p>An affinity chromatography protocol utilizing an ADTN analogue covalently coupled to an affinity matrix was established for cholate-solubilized D-1 receptor. The affinity protocol developed during this investigation purified the D-1 receptor approximately 50-fold, while an average of 8% of the receptors were recovered. These results were superior to any previous literature reports of D-1 receptor purification.</p> <p>Several photoactive compounds were synthesized and used to crosslink D-1 receptors. One compound in particular, a photoactive derivative of the dopamine against ADRN, proved to be a useful ligand for this purpose. A tritiated derivative of this compound was covalently and specifically incorporated into a protein of M.W. =79kDa. This was the first report that the D-1 receptor had a M.W. of greater than 70kDa as determined by affinity crosslinking. Several other investigators have subsequently confirmed this observation.</p> <p>Other photoactive compounds radiolablled with 125I were synthesized and examined for activity as D-1 and D-2 receptor probes. These compounds were not as useful as photoaffinity labels for dopamine receptors as had been originally proposed. The compounds did label a 50kDa protein which was determined to be neither the D-1 nor D-2 receptor. This protein (originally designated Apo-50 and later CatNAP) has very interesting properties as it possesses binding activities with several catecholamines which are without precedent in the literature.</p> / Doctor of Philosophy (PhD)