Thesis (Ph.D.) - University of Queensland, 2005. / Includes bibliography.

Thesis (PH.D.) -- Syracuse University, 2006. / "Publication number AAT 3241849."

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In rodents, there exists a strong interaction between the reproductive and circadian systems. For this thesis the female hamster was used as a model for the study of this interaction. Studies described in chapter II investigated whether the circadian regulation of reproductive processes may be through direct input of the suprachiasmatic nucleus (SCN) to neurons containing estrogen receptor (ER) and/or to neurons containing luteinizing hormone releasing hormone (LHRH). The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was applied to the SCN and double label immunocytochemistry for PHA-L and either ER or LHRH was carried out. Both ER- and LHRH-immunoreactive cells show appositions with SCN efferents or with efferents of the subparaventricular nucleus and the retrochiasmatic area. Results suggest that the circadian system can regulate reproductive processes via input to LHRH- and/or ER-containing neurons. Studies described in chapter 111 investigated whether effects of estrogen on circadian rhythms may be exerted through estrogen-binding systems afferent to the SCN. Immunocytochemistry for ER and the retrograde tracer cholera toxin B subunit, after its application to the SCN, demonstrated that some areas contain relatively high percentages of SCN afferent neurons which show ER immunoreactivity. Retrograde tracing results were compared with results of anterograde tracing from some of the sites containing SCN afferents. Furthermore, using a combined retrograde and anterograde tracing technique, SCN input to some SCN afferent neurons was demonstrated. However, no evidence of reciprocity between single ER-immunoreactive cells and the SCN was found. Results indicate the existence of estrogen binding systems afferent to the SCN which might mediate the effects of gonadal steroid hormones on circadian rhythms. Studies in chapter IV analyze the effects of blockade of SCN axonal output by local unilateral application of tetrodotoxin (TTX) on the LH surge. Injections of TTX on either the morning or the afternoon of proestrus were unable to block the LH surge. Results favor the interpretation that the SCN output signal responsible of the circadian gating of the LH surge occurs before the onset of the light period on the day of proestrus.

Ovarian steroid hormones influence both behavior and physiology in a variety of species by binding to intracellular steroid receptors. Recent studies suggest that steroid receptors (e.g., estrogen and progestin receptors) may also be activated in the absence of steroid. Infusion of dopamine agonists into the third ventricle of estradiol-primed female rats can increase estrous behavior, and this increase can be blocked by prior treatment with progestin antagonists even in the absence of progesterone. However, it is not known if progestin receptors in rat brain are activated in the absence of circulating progesterone under physiological conditions affecting neuronal responses and behavior. This dissertation attempts to determine if somatosensory cues that are normally experienced by females, such as stimuli associated with sexual contact with males, activate progestin receptors to influence both neuronal response and estrous behavior in the absence of circulating progesterone. Using immunocytochemistry, it is possible to determine the expression of immediate early gene products that suggest neuronal response to particular stimuli. It was found that either progesterone or stimuli associated with mating can increase immunostaining of the immediate early gene product, Fos, within cells that contain progestin receptors. Thus, some neurons in female rat brain are capable of integrating somatosensory information provided by the male and information relating to serum progesterone levels. In addition, increases in Fos expression in female rat brain following stimuli associated with mating can be blocked by prior treatment with progestin antagonists even in the absence of circulating progesterone. This suggests that the response of some neurons to mating-related stimuli are mediated via progestin receptors. It was also found that a component of estrous behavior (e.g., lordosis) in female rats can be facilitated by repeated exposure to males in the absence of progesterone, and this facilitation can be blocked by prior treatment with progestin antagonists. The present results suggest that progestin receptors in some neurons in rat brain are activated by mating related stimuli in a progesterone-independent manner. These data suggest a pathway by which mating-related stimuli or other environmental influences could activate steroid receptors influencing neuronal response and behavior in the absence of circulating progesterone.

In a set of 7 experiments, the author examined if cue-competition effects such as blocking and overshadowing reflect deficits in learning (e.g., Rescorla & Wagner, 1972) or deficits in performance (Miller & Schachtman, 1985). To this end, the author tested if the ability of a blocked and/or overshadowed stimulus was weakened in its ability to serve as a blocker or second-order reinforcer for a novel stimulus. It was assumed that the ability of a stimulus to serve as a blocker or second-order reinforcer depended on its associative status, and not on the performance it evoked. CS-evoked suppression of appetitively-motivated barpressing served as the dependent measure of conditioned performance. Experiment 1 found that an overshadowed CS was weakened in its ability to serve as a blocker. Experiment 2 replicated Experiment 1 and further showed that a blocked stimulus was also weakened in its ability to serve as a blocker. Experiment 3 showed that a blocked and overshadowed stimulus was weakened in its ability to serve as a second-order reinforcer. The results of Experiments 1, 2, and 3 were construed as supporting a learning-deficit as opposed to a performance-deficit interpretation of cue-competition effects. Performance-deficit theorists, however, might claim that the weakened ability of a blocked and/or overshadowed CS to serve as a blocker or second-order reinforcer was due to the presence of an intact A-US association (the association produced by the blocking and/or overshadowing CS). Experiments 4 to 7 addressed this issue, using various techniques, which might weaken the allegedly interfering A-US association. Experiments 4 and 5 showed that extinguishing the blocking and/or overshadowing stimulus did not facilitate performance to and blocking ability of a blocked and/or overshadowed stimulus (Experiment 4) or overshadowed stimulus (Experiment 5). Experiment 6 further showed that subjecting the blocking and/or overshadowing cue to a Pavlovian conditioned inhibition procedure also did not enhance performance to the blocked and/or overshadowed stimulus. Finally, Experiment 7 showed that extinguishing the overshadowing stimulus weakened performance to the overshadowed stimulus. Collectively, the results of Experiments 1 to 7 are consistent with learning-deficit interpretations of cue-competition effects (e.g., Rescorla & Wagner, 1972; Mackintosh, 1975).

The factors controlling desensitization to ATP stimulation were investigated in PC12 cells. Reducing the concentration of ATP to produce half maximal response reduced the degree to which cells desensitize to ATP. Increasing external Me concentration, which produced a comparable decrease in the secretory response of the cells, had no effect on the degree of desensitization. Neither did decreasing external Ca2+ concentration, which produced a similar decrease in secretory response. Desensitizing cells in 0.5 mM Ca2+ did not result in a corresponding decrease in response when cells were subsequently tested in 2.2 mM Ca2+. PC12 cells desensitized in 2.2 mM Ca2+ were found to show the same degree of desensitization when tested in 0.5 mM Ca2+. A similar pattern was found when desensitization to 30 and 300 uM ATP was examined. The role of the ATP receptor subtypes, P2x and P2y, was studied using the ATP agonists 2-MeS ATP and UTP, respectively. 60 uM 2-MeS ATP was found to cause desensitization to the same degree as 30 uM ATP. As with ATP, the initial response to 2-MeS ATP was found to be sensitive to changes in external Mg2+. Unlike ATP, the desensitization to 2-MeS ATP was sensitive to changes in external Mg2+. When cells were co-stimulated with 2-MeS ATP and UTP, the sensitivity of 2-MeS ATP desensitization to Mg2+ remained. UTP in the background solution, however, increased desensitization to ATP and 2-MeS ATP. The effect of voltage-operated Ca2+ channel (VOCC) blockers on the response and desensitization to ATP and 2-MeS ATP was examined. Cd2+ produced a significant increase in the secretory response to both stimulants. Both nicardipine and Cd2+ increased the rate of desensitization to ATP. Only nicardipine was able to increase the rate of desensitization to 2-MeS ATP. These results were integrated to provide insights into the relationship between ATP receptors and VOCCs.

Polychlorinated biphenyls are one type of organic pollutant. Although they are no longer produced due to their resistance to degradation they have persisted in the environment and have become globally distributed. They have been found within the tissues of nearly every organism that has been tested for their presence. Because they are capable of interacting with biological systems their effect on environmental and human health have become a topic of interest and research. It is known that PCBs alter the function and levels of thyroid hormone. This is significant because of the importance of properly regulated thyroid hormone levels during development. To gain a better understanding of the specific effects of PCBs on thyroid hormone function and to better assess the risks associated with PCB exposure I set out to identify mechanisms by which PCBs alter thyroid hormone-directed brain development. I used a number of approaches to identify impacts of PCBs on thyroid hormone-directed brain development. I first looked at gene expression and identified new targets of thyroid hormone, the cofactors N-CoR and SRC-1. I next looked at the effects of PCB exposure on these genes. I found that PCBs did not affect the expression of these genes. I next went on to look at cell and death and proliferation in the cerebellum and found that PCBs did not alter these processes. When I tried an in vitro approach to look at the impact of PCB exposure on receptor-independent effects of thyroid hormone on actin polymerization I found that my cultures did not respond to thyroid hormone as reported. In the last experiment I used a differential display screen to identify targets of PCB exposure and identified number of genes whose expression is putatively affected by PCB exposure. These genes included Stathmin, BESH, and Zic-1 all of which are associated with the cytoskeleton. It has also been shown that the effects of thyroid hormone can alter neuronal migration. These data suggest that PCBs may be altering neuronal migration and that the genes identified in this screen are downstream consequences of the thyroid hormone receptor -independent effects of thyroid hormone.