The anteroventral periventricular nucleus (AVPV) of the preoptic area mediates the positive feedback effects of estradiol (E2) on LH surge in rats. Consistent with their role in female reproduction, the neurons in this region are more numerous in adult females than males. This sex difference is established due to E2-mediated cell death in the developing male AVPV. Loss of neurons in the AVPV permanently abolishes the ability of E2 to trigger LH surge release. However, the identity of the neurons lost during AVPV masculinization and the mechanism underlying E2-triggered cell death have not been clearly defined to date. This dissertation shows that, developmental exposure to E2 permanently reduces the number of dual-phenotype GABAergic/Glutamatergic (GABA/Glu) neurons, supporting the role of these neurons in female-specific LH surge release. My results identified a key role for TNFα-activated NFκB-mediated cell survival in establishing sex differences in the GABA/Glu population in the AVPV. GABAergic neurons in males had higher levels of TRAF Interacting Protein (TRIP), an inhibitor of the NFκB pathway. Thus, the male AVPV had lower levels of nuclear NFκB and its downstream target, pro-survival Bcl-2 mRNA than females. I also showed that E2 produces these sex differences by upregulating TRIP gene expression and thus reduces the number of GABA/Glu neurons in the male AVPV. Using the N42 GABA/Glu cell line as an in vitro model for the AVPV, I verified that E2 reversed TNFα-mediated effects on NFκB activation, Bcl-2 mRNA, and caspase activity. Moreover, E2 could directly upregulate TRIP mRNA levels, only in the presence of TNFα. To understand the nature of this cooperativity, I cloned the proximal TRIP promoter, identified an ERE half-site and a novel TNFRE in a region 1000 base pairs upstream of the transcription start site. Mutation of either of these sites abolished the stimulatory effect of E2 on promoter activity, suggesting that cooperative action of E2 and TNFα is required for TRIP promoter activation. To summarize, these studies provide a novel mechanism for sexual differentiation of the AVPV in which E2 acts cooperatively with TNFα to inhibit a neuroprotective pathway in GABA/Glu neurons of the male AVPV.

High doses of 3,4-methylenedioxymethamphetamine (MDMA; "Ecstasy") are known to reduce levels of various serotonergic markers outside of the raphe nuclei. To test the hypothesis that these deficits reflect a degeneration of distal axons/terminals, we investigated the effects of an MDMA binge (10mg/kg x 4) on the relative protein and genetic expression of several serotonergic markers in rats, as well as the effects of this compound on the quantity of serotonergic terminals in these animals. In experiment I, we examined whether MDMA alters serotonin transporter (SERT) levels as determined by lysate binding and immunoblotting analyses. Both methods of analysis revealed MDMA-induced reductions in regional SERT content. Experiment II investigated MDMA-induced changes in terminal-specific levels of SERT and the vesicular monoamine transporter 2 (VMAT-2) in the hippocampus, a region with sparse dopaminergic innervation, after lesioning noradrenergic input with N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP-4). Animals were administered 100 mg/kg DSP-4 or saline 1 week prior to MDMA (or saline). As determined by immunoblotting of synaptosomal tissue, the DSP-4/MDMA group showed little change in hippocampal VMAT-2 protein expression compared to DSP-4/Saline controls, despite large reductions in SERT levels in all regions examined in the MDMA-treated animals. Experiment III examined whether MDMA alters genetic expression of SERT and VMAT-2. When compared to saline-treated controls, animals given MDMA showed a striking decrease in SERT gene expression (and a lesser effect on VMAT-2) in dorsal/median raphe as assessed by quantitative RT-PCR. Experiment IV(a) investigated the effects of MDMA on gene and protein expression of tryptophan hydroxylase (TPH) in the hippocampus. Levels of TPH protein were unchanged between treatment groups, while transcript levels were decreased 15-fold in the dorsal/median raphe. In experiment IV(b), flow cytometry was used to measure whether MDMA alters the quantity of serotonergic terminals in the hippocampus. MDMA-treated animals showed an increase in the number of serotonergic synaptosomes identified by co-labeling for synaptosome-associated protein of 25 kDa (SNAP-25) and TPH. These results demonstrate that MDMA causes substantial regulatory changes in the expression of serotonergic markers with no evidence for synaptic loss, questioning the need to invoke distal axotomy as an explanation of MDMA-related serotonergic deficits. Keywords: MDMA, neurotoxicity, neurodegeneration, serotonin transporter, vesicular monoamine transporter 2, tryptophan hydroxylase, serotonin, immunoblotting, gene expression, flow cytometry.

Circadian rhythms are ∼24 hour cycles in behavioral and physiological responses observed in a wide range of organisms. The role of this central clock lies in its ability to recognize different environmental stimuli and adapt the behavior of organisms accordingly. This response is critical for an organism's survival and evolution as it allows for the anticipation of environmental cues. The circadian pacemaker of mammalian organisms is located in the brain region of the suprachiasmatic nucleus (SCN) of the hypothalamus. The SCN produces self-sustained oscillations and further controls a number of metabolic processes across distinct organs. This thesis has focused on the mathematical modeling of the SCN to investigate mechanisms responsible for the generation and synchronization of daily signals across the circadian network. For this purpose, we developed several multicellular models of the mammalian circadian clock characterized by a high degree of heterogeneity with respect to single cell periodicity and behavior (intrinsic and driven oscillators), neurotransmitter release (vasoactive intestinal peptide (VIP), γ-aminobutyric acid (GABA) and glutamate synthesis) and spatial organization (long range versus short range connectivity). A firing rate code model was further developed to incorporate known electrophysiological properties of SCN pacemakers that give rise to the electrical firing of individual neurons. In this model, daily changes in ion conductances, ion concentrations and membrane properties (such as membrane resistance) were integrated to produce circadian changes in the action potential frequency of SCN neurons. Intracellular signaling pathways, initiated by cytosolic calcium and VIP, were also included as the putative link between electrical firing and gene expression. The developed model predicted a direct relationship between firing frequency and gene expression amplitudes, demonstrated the importance of intracellular pathways for circadian behavior and synchrony and provided a novel multiscale framework which captured characteristics of the SCN at both the electrophysiological and gene regulatory levels. We further attempted to mimic the structural organization of the SCN and utilize experimentally derived connectivity schemes to simulate the SCN’s ventrolateral and dorsomedial subdivisions. The model predicted that sufficient connectivity between the two separate regions, associated with distinct circadian functions, was responsible for the expression of sustained circadian behavior.

Progesterone regulates female reproduction in many ways, yet it is still unclear how signals are conveyed through nuclear and extranuclear receptors. The traditional notion was that progesterone binds classical progesterone receptors to alter gene transcription. This view has been challenged by the discovery of additional progesterone signaling molecules important for progesterone actions in non-neural cells. In granulosa cells, the progesterone receptor membrane component 1 (Pgrmc1) mediates progesterone effects by forming a receptor complex with binding partner, Serpine mRNA binding protein 1, but it is unknown whether these molecules function similarly in the brain. To begin to address these issues, I investigated the neural role of Pgrmc1 in female mouse brain, rat brain and in neural cells. By examining the neuroanatomical localization, hormonal regulation, and colocalization of Pgrmc1 within key neurons in the neural control of ovulation, Pgrmc1 emerged as a candidate signaling molecule likely to mediate progesterone functions. Furthermore, Pgrmc1 levels regulate the expression of several diverse genes and signaling pathways in neural cells. Taken together, these results demonstrate that Pgrmc1 function is likely to impact diverse neural functions.

Temporally selective attention allows for the preferential processing of stimuli presented at particular times, and is reasoned to be important for processing rapidly presented information such as speech. Recent event-related potential (ERP) evidence demonstrates that listeners direct temporally selective attention to times that contain word onsets in speech. This may be an effective listening strategy since these moments provide critical information to the listener, but the mechanism that underlies this process remains unexplored. In three experiments, putative attention cues including word recognition and predictability were manipulated in both artificial and natural speech and ERP responses at various times were compared to determine how listeners selectively process word onsets in speech. The results demonstrate that listeners allocate attention to word-initial segments because they are less predictable than other times in the speech stream. Attending to unpredictable moments may improve spoken language comprehension by allowing listeners to glean the most relevant information from an otherwise overwhelming speech signal.

Most recreational users of 3,4-methylenedioxymethamphetamine (MDMA; "ecstasy") also take cannabis, in part because cannabis can reduce the dysphoric symptoms of the ecstasy come-down, such as agitation and insomnia. Although previous animal studies have explored the acute effects of co-administering MDMA and Δ9-tetrahydrocannabinol (THC), the major psychoactive ingredient in cannabis, research on chronic exposure to this drug combination is lacking. The four experiments included in the current dissertation were designed to provide a wide breadth of information on the physiological, behavioral, and neurochemical effects of intermittent MDMA administration combined with daily THC exposure using a dosing regimen designed to reflect a clinically-relevant pattern of human ecstasy and cannabis co-usage. Because ecstasy and cannabis abuse usually starts during human adolescence, drug treatment was administered from postnatal day (PD) 35 to 60 in order to target the period of rat development lasting from approximately mid-adolescence to early adulthood. In addition, the dosing regimen in rats was also chosen to best correlate to patterns of human ecstasy and marijuana use. Drug-treated rats received two subcutaneous (s.c.) injections of 10 mg/kg of (±) MDMA-HCL every fifth day and/or a single daily intraperitoneal (i.p.) injection of 5 mg/kg of THC every day. The twice every fifth day MDMA dosing regimen was designed to simulate the intermittent weekend usage of ecstasy at "rave" parties and the "boosting" behavior (taking additional doses of MDMA in the same session to maintain desired effects) that has been noted in human users. THC was administered daily to simulate heavy cannabis usage in humans, which has been defined to mean using cannabis more than seven times per week. While THC helped to alleviate MDMA-induced anxiety-like, impulsivity-like, and exploratory behavior, co-administration of MDMA and THC additively produced depressive-like behavior and deficits in spatial memory. Furthermore, our experiments provide physiological and neurochemical evidence that helps to explain the behavioral outcomes, specifically as THC failed to protect against MDMA-induced neurotoxicity in the hippocampus, the brain region that is responsible for processing of spatial memory information, in both the SERT binding assay and in SERT autoradiography. Finally, our data suggested that male rats are more susceptible to MDMA-induced damages than females—which has significant implications for assessing the risks of recreational ecstasy and cannabis co-usage in humans.

Removal of protein activity by genetic mutation or pharmacological inhibition has been used extensively to understand the normal function of a protein. However, null mutations eliminate gene function in all cells and pharmacological agents can diffuse through tissues to have similar global effects that can obscure the physiological function of a protein. This is a particular problem when studying proteins that function in many cell types or that have different cell-specific activities. The most direct strategy to study the function of a protein is to reduce or eliminate its activity only in specific cell types, rather than in all cells of an organism. The idea of targeting gene knockdown to specific cell types or to individual cells is not new and many strategies aim to do just this. However, these strategies result in variable knockdown efficiencies and can have silencing effects in neighboring cells and therefore knockdown is never cell-specific. We developed a novel method to knock down the expression of any gene and to restrict this knockdown to specific cell types in C. elegans. In this method we replaced endogenous genes with single copy integrated transgenes containing an engineered sequence tag that introduces premature stop codons (PTCs) into transgene mRNA. This tag causes the natural stop codon to be recognized as a PTC by the host's nonsense-mediated decay (NMD) machinery and does not disrupt gene function. In NMD-competent animals, a PTC-containing transgene is degraded and in NMD-defective animals, a PTC-containing transgene is expressed. Therefore, the expression of PTC-containing transgenes can be controlled by cell-specific activation of NMD. Using this technique, we replaced two endogenous genes with PTC-containing transgenes and directed degradation of their mRNA to specific cell types by restoring NMD activity in these cells. The single copy transgenes were expressed at levels comparable to the endogenous genes and were knocked down to ∼10% of endogenous by NMD, resulting in both global and cell-specific null-like phenotypes. This knockdown strategy can be used to cell-specifically knock down essentially any gene in the C. elegans genome and should provide new insights into understanding protein function.

Cocaine abuse and addiction are widespread problems with profound medical and socioeconomic consequences. At present, the neurobiological adaptations associated with short- and long-term cocaine abuse are not well understood, which contributes to the lack of availability of broadly effective treatments for this type of addiction. Recently, some studies have implicated GABAA receptor subtypes in the neuroadaptations underlying addiction. To explore the contributions of GABAA receptors to the neurobiological basis of cocaine abuse, we utilized a non-human primate model of cocaine self-administration and examined changes in species typical behaviors, and corresponding alterations in three GABAA receptor subtypes within five reward-related areas of the brain. Sixteen rhesus monkeys either self-administered cocaine intravenously (1-hr/day, 0.03 mg/kg/injection of cocaine) or received passive infusions of saline yoked to the cocaine injections (yoked control). Monkeys either self-administered cocaine for ~10 days (short-term group) or ~100 days (long-term group). Twenty-four hours after the last session, animals were sacrificed and brains were removed. We examined alterations in &agr;1, &agr;2, and &agr;3 subunit-containing GABAA receptors (&agr;1, &agr;2, and &agr;3GABAA receptors) using immunohistochemistry (IHC), in situ hybridization (ISH), and real-time PCR experiments (RTPCR) within reward-related areas of the brain including the nucleus accumbens, ventral tegmental area, caudate, putamen, and anterior cingulate cortex. Long-term cocaine taking animals self-administered cocaine in a cyclical pattern, and increased number of cocaine injections taken within the initial portion of daily self-administration sessions. We observed behavioral alterations in behaviors including locomotor, stereotypic, scratching and affiliative behaviors. IHC results demonstrated alterations in &agr;1GABAA receptors within all regions of interest after long-term self-administration. After short-term cocaine self-administration decreases in &agr;3GABAA receptors were observed in all regions examined. When examining transcript levels using ISH and RTPCR, we found relatively few changes in comparison to protein alterations. The notable change was a decrease of all three receptor mRNAs within the anterior cingulate cortex after short-term cocaine exposure. The present model of drug may expand our understanding of addiction-related behaviors and the role of GABA in addiction. Furthermore, our findings suggest GABAA receptors may serve as viable targets for pharmacotherapeutic approaches to treat addiction.

Dynamic Attending Theory predicts that attention is allocated hierarchically across time during processing of hierarchically structured rhythms. Event-related potential (ERP) research demonstrates that attention to a moment in time modulates early auditory processing as evidenced by the amplitude of the first negative peak (N1) approximately 100 ms after sound onset. Four experiments were designed to test the hypothesis that hierarchically structured rhythms result in a hierarchical allocation of attention across time by comparing behavioral responses and N1 amplitudes for sounds presented at times of varying hierarchical strength. Specifically, ERPs elicited by tones presented at times of high and low strength were compared in short melodies (Experiment 2) of salient metric structure (Experiment 1), and in subjective metric hierarchies (Experiments 3 and 4). Experiment 4 also added a level of medium strength in a subjective metric hierarchy. A more negative N1 was observed for metrically strong beats compared to metrically weak beats under nearly all conditions in Experiments 2, 3 and 4, providing strong evidence that attention is allocated preferentially to hierarchically strong times and supporting the central hypothesis. This effect was evident for both stimulus-inherent and listener-imposed metric structure, suggesting it represents ongoing direction of attention to metrically strong times rather than establishment of a metric percept. A patterned distribution of N1 amplitude was evident among metrically weaker times, demonstrating that attention is not allocated to the strongest times in an all-or-none manner. However, this pattern was not fully hierarchical, suggesting that hierarchical rhythmic structure does not modulate early auditory processing in a one-to-one manner. Additionally, a late negativity and late positivity were associated with metric strength under some conditions, indicating that multiple cognitive processes are associated with metric perception. Interestingly, the primary finding of a more negative N1 for sounds presented at hierarchically strong times in musical and pseudomusical stimuli was not modulated by musical expertise, suggesting that it indexes the use of a more general cognitive process that may also be employed to efficiently process other complex auditory streams including speech.

The adage practice makes perfect makes for sound advice when learning a novel motor skill. Be it typing a new password or hitting a forehand in tennis, proficiency increases with experience. Behavioral changes associated with motor skill acquisition can be broken down into three broad categories: (1) movements are executed faster and become more coordinated, (2) they come to rely on sensory information gained while executing the task, rather than just sensory information used during initial stages of learning the task, and (3) they seem to be executed with less conscious thought and attention. In addition, neural activity changes: many imaging and neural recording studies suggest that with experience, control is transferred from cortical planning areas to the basal ganglia. The two areas are thought to employ different learning and control schemes. In general, planning can quickly take new information into account to make reasonable decisions, but its control mechanisms have large computational requirements. The basal ganglia use a simpler and less computationally expensive control scheme, but they require much experience before they can produce reasonable behavior. In this thesis, I contribute to answering the question, "what goes on during practice?" More formally, I am interested in the mechanisms by which motor skills are acquired. I take a theoretical approach in that I hypothesize a multiple controller scheme, based on the learning and control mechanisms of cortical planning areas and the basal ganglia, and test it with simulations designed emulate generic motor skill tasks. Because skill proficiency increases with experience, I am particularly interested in the role of the experience-dependent mechanisms of the basal ganglia in motor skill acquisition. Thus, learning mechanisms attributed to cortical areas are artificially restricted so that any change in model behavior is attributed to the learning mechanisms of the basal ganglia. Model behaviors exhibit characteristics indicative of motor skills, supporting the plausibility of the multiple controller scheme as one used by our nervous system and suggesting that the learning mechanisms of the basal ganglia can contribute to developing most characteristics. In addition, I show how the strategies developed by the models are functionally advantageous, providing a reason why such a scheme may be used.