Genetic screens which assay behavior have been successfully used to identify genes required for neural function. This thesis is the analysis of midline uncoordinated (muc), a mutation identified for its effect on grooming behavior. This mutation was caused by a single P (lac W) insertion at position 28A. A number of additional muc alleles have been generated by excision of the P element. Using markers for two types of femoral chordotonal neurons we have been able to show that muc disrupts the axon trajectories of these cells. In addition to grooming behavior and neuroanatomy, many muc alleles also affect midline parting of the thoracic microchaetae, flightlessness, lethality and male sterility. Genetic analysis of the various muc mutations suggest that they form a unique complementation group. Three transcripts were found near the area of the muc mutation. The most likely gene affected by muc is the Drosophila homolog of dihydrolipoamide acetyltransferase, component E$\sb2$ of the mitochondrial pyruvate dehydrogenase complex. The P (lac W) element sits in an intron of this gene. We have found that the most severe grooming alleles retain all or almost of all of the P element used to cause the original mutation. In addition to severe grooming behavior, these alleles also have severe axon projection defects. Revertant alleles which have cleanly excised the P element have wild type grooming behavior and normal axon projection patterns.

Fetal alcohol exposure is the leading known cause of mental retardation in the western world. However, the mechanisms underlying alcohol-induced damage in fetal brain are largely unknown. The goal of this dissertation is to identify ethanol-responsive genes during brain development to provide more insights into the mechanisms. I chose a well-established animal model for all the studies in this dissertation. First, I demonstrated that this ethanol paradigm increased the mRNA of cellular retinol binding protein I (CRBP-1) in gestational day 13 (G13) brain and the incidence of apoptosis in G16 brain. Second, I identified 12 putative ethanol-responsive genes using mRNA differential display. After the quantitative analysis by Northern blot, in situ hyridization, western blot and relative quantitative RT-PCR, ribosomal protein S6 (rpS6), neuroendocrine-specific protein-A (NSP-A) and a novel gene were verified as ethanol-responsive genes. Third, I isolated 32 putative ethanol-responsive genes using cDNA microarray analysis. They encode proteins engaged in cell signaling, cell cycle regulation, metabolism, stress response and cell structure. Among all the putative genes, alcohol dehydrogenase 3 (Adh3) and glutathione S transferase pi 2 (GST pi 2) are previously known ethanol-responsive genes. Additionally, bone morphogenetic protein receptor type IA (BMPR-IA) showed the largest change induced by ethanol, 2.1-fold, and the ethanol effect on its expression was confirmed by relative quantitative RT-PCR. Fourth, because NSP-A is also a thyroid hormone-regulated gene, I analyzed the expression of two other thyroid hormone-regulated genes, Oct-1 and Hes-1, in my ethanol-treated animal model. However, ethanol did not affect thyroid hormone regulation of these two genes in G16 cerebral cortex. Fifth, I examined the effects of ethanol on protein expression and phosphoralytion using two-dimensional (2D) electrophoresis and western blotting. Ethanol was shown not to robustly change the abundance of individual proteins, but may change the post translation modifications of some proteins during brain development, such as glycosylation. In conclusion, these studies systematically and thoroughly examined the effects of fetal alcohol exposure on gene expression during brain development. They provide useful insights for analyzing the complex pathways leading to CNS damage in the children born to mothers who drank heavily during pregnancy.

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.

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.

The flow of information through neuronal circuits relies on the ability of neurons to form synaptic connections with specific temporal and spatial properties. These properties are not static but have the ability to change, allowing synaptic connections to be strengthened or weakened. It is this plastic nature of synapses that is central to higher order processes such as learning and memory. However, major gaps remain in our understanding of this process. Throughout my dissertation work I have examined mechanisms of a form of structural synaptic plasticity by analyzing the roles of a variety of proteins that we have found serve to regulate the formation and maintenance of glutamatergic synapses at the Drosophila NMJ. These proteins include APPL, the Drosophila homolog of Alzheimer's disease-associated β-Amyloid Precursor Protein (APP), the tumor suppressor protein Scribble (Scrib), the secreted signaling molecule Wingless (Wg), and the cell adhesion molecule Fasciclin II (FasII). In this work, in collaboration with members of the labs of Dr. Vivian Budnik, Dr. Kalpana White, and Dr. Susan Cumberledge, I have demonstrated that Wingless (Wg) provides a secreted signal that is required to initiate the formation of pre- and postsynaptic structures. Further, I have also demonstrated that once synapse formation is initiated, presumably by Wg signaling, APPL regulates synaptic bouton proliferation. This process also involves signaling by FasII, a protein required for synapse maintenance, and growth. Moreover, I have also demonstrated that Scrib is a scaffolding protein that plays a key role at these synapses in influencing neurotransmitter release.

These studies examined the pharmacokinetics of cocaine following its chronic subcutaneous (s.c.) administration to pregnant rats and the effects of this treatment on neurotensin/neuromedin (NT/N) mRNA expression in the brains of their offspring. First, I examined the distribution of cocaine and its metabolites benzoylecgonine (BE) and norcocaine in pregnant rats following twice-daily s.c. injections of 20 mg/kg cocaine from gestational day (GD) 8–GD 21. Following a single injection on GD 21, maternal and fetal trunk blood, fetal brains, and amniotic fluid (AF) were collected at 8 separate time points from 5 min to 12 h. Cocaine peaked in maternal plasma at 1 h and at 2 h in fetal plasma, fetal brain and AF. Peak BE levels were detected at 4 h in maternal plasma, fetal plasma, and fetal brain, and at 8 h in the AF. An additional group of dams given both injections on GD 21 and sacrificed 2 h later showed increased concentrations of BE in both fetal compartments and in the AF. Previously undetectable, norcocaine was now measurable in the AF. Chronic cocaine administration increases NT/N mRNA in the nucleus accumbens. To further understand the mechanisms involved, I conducted a dose response study evaluating the role of the D3 receptor on the expression of NT/N mRNA in the nucleus accumbens shell using in situ hybridization. Animals were sacrificed 3 h following an acute challenge with either the D3 agonist PD 128904 or the antagonist nafadotride. As neither compound significantly altered NT/N mRNA levels, no further work was performed with these drugs. To examine the effects of prenatal cocaine exposure on neurotensin expression, adult male offspring from either cocaine (40 mg/kg daily, GD 8–21) or saline-injected dams were treated with a single daily i.p. injection of cocaine or saline for 10 days and sacrificed 1 h after the last injection. This treatment resulted in increased NT/N mRNA in the nucleus accumbens, fundus, striati, and dorsomedial striatum regardless of prenatal treatment, and significantly greater NT/N mRNA expression within the medial preoptic nucleus (MPN) of offspring from pair-fed saline dams. Thus, prenatal cocaine exposure alters the NT/N response in the MPN to postnatal cocaine challenge.