Language production is characterized by an unlimited expressive capacity and creative flexibility that allows speakers to rapidly generate novel and complex utterances. In turn, listeners interpret language "on-line", incrementally integrating diverse representations to create meaning in real-time. A challenge for theories of language has been to understand how speakers generate, integrate, and maintain representations in service of language use and processing and how this is accomplished in the brain. Much of this work has focused prefrontal cortex mechanisms such as "working memory". The goal of this dissertation is to understand the role of the hippocampal declarative memory system (HDMS) in language use and processing, specifically in referential processing and narrative construction. To test the role of the hippocampus in referential processing, healthy comparisons, brain damaged comparisons (BDC), individuals with bilateral hippocampal damage participated in an eyetracking experiment in which individuals viewed scenes and listened to short stories. The amount of time participants spent looking at the characters after a pronoun reference was recorded. Healthy comparisons and BDC participants preferentially targeted the first mentioned character while participants with hippocampal damage did not, suggesting that the hippocampus plays a role in maintaining and integrating information, even in short discourse history. In a second experiment, participants with bilateral hippocampal damage and healthy comparisons told narratives multiple times over the course of a month. The narratives were analyzed for the number of words, the number of episodic details, the number of semantic details, the number of editorials and the consistency of details over the multiple tellings. The patients with hippocampal damage told stories that were significantly shorter, more semanticized and less consistent from telling to telling than healthy comparisons. The final goal of this study was to understand the effects of unilateral hippocampal damage on language processing. Individuals with unilateral hippocampal damage participated in all of the previous experiments. It was predicted that individuals with left hippocampal damage would perform worse than individuals with right hippocampal damage, and their performance was significantly impaired across measures. This suggests that the left hippocampus may be particularly important for processing linguistic material outside of even verbal memory.

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Maternal behavior in rodents is dependent, at least in part, on prolactin acting in the brain. Pheromones carried by male mouse major urinary proteins lower serum prolactin levels in female mice. Therefore, we hypothesized that virgin female C57BL/6J mice housed in split cages, where they had pheromonal but not physical contact with a male, would show suppressed maternal behavior. Contrary to our hypothesis, we found split-cage housed females were significantly faster to retrieve 3 foster pups on the first and second day of maternal behavior testing compared to mice housed in individual cages. The advancement in maternal behavior was replicated when virgin females were simply exposed to male mouse urine-soaked bedding. Ovariectomising the mice, to remove the influence of steroid hormones, prior to placement in the split cages, prevented the pheromonal advancement of maternal behavior. The data infer that an ovarian steroid-dependent action of male mouse pheromones primes virgin female mice to express maternal behavior more rapidly when mouse pups are introduced. This effect required greater than 14 days exposure to male pheromones. Male mouse pheromones are reported to suppress prolactin secretion. However, serum prolactin levels in split-caged housed females, where they had pheromonal but not physical contact with a male, were only briefly lowered and became significantly elevated from 24 hours until 72 hours of pheromonal contact. Despite the early increases in prolactin after pheromone exposure, levels were significantly lower in the pheromone-exposed females when maternal behavior was tested after 21 days. It has been previously reported that prolactin is important in the onset of maternal behavior, but is not required for the ongoing maintenance of maternal behavior. We hypothesised that the hyperprolactinemia observed in the first 24-72 hours of pheromonai exposure had subsequently led to the enhanced maternal behavior. To test this we injected a group of individually-housed mice with slow release prolactin for 48 hours to simulate the period of hyperprolactinemia, and blocked prolactin secretion in a group of split-caged housed females with bromocriptine, and tested their maternal behavior 18 days later. The mice injected with prolactin had enhanced maternal behavior, compared to controls injected with a placebo. By contrast, bromocriptine inhibition of prolactin secretion completely prevented the pheromonal enhancement of maternal behavior. This suggests that the pheromonal advancement of maternal behavior is specifically mediated by a 48-hour period of sustained hyperprolactinemia. It has been previously shown that pregnancy increases neurogenesis in the subventricular zone in a prolactin-dependent manner. Therefore, as the male pheromone-induced advancement of maternal behavior is prolactin-dependent and takes some time to occur, we hypothesized that long-term pheromonal contact initiates mitogenesis in the subventricular zone. Split-caged housed mice showed a significant increase in BrdU-labeled cells in the subventricular zone after 7 days of contact which reduced to baseline levels by 14 days of contact. The mice injected with BrdU on day 7 of contact and killed 21 days later showed a significant increase in labeled cells in the accessory olfactory bulb compared to controls. The data suggest that male mouse pheromones initiate mitogenesis in the subventricular zone of virgin C57B6 mice, in an exposure-dependent manner, and that these cells travel via the rostral migratory stream to the accessory olfactory bulb. As with the effect on maternal behavior, the pheromone-induced increase in neurogenesis was steroid- and prolactin-dependent. During pregnancy and lactation in rodents, prolactin receptor expression is increased in the MPOA, an adaptive change, which could lead to an increased neuronal response to serum prolactin levels, which are high just prior to parturition, and consequently could underlie the enhanced maternal responses seen in late pregnancy and after parturition. It is known that systemic prolactin can access the brain, but it is also possible that there could be local synthesis of brain prolactin acting in an autocrine or paracrine manner. Therefore we hypothesized that the pheromonal-induced changes in maternal behavior are being mediated by altered prolactin receptor expression/sensitivity and/or increased production of brain prolactin. Using RT-PCR to measure levels of prolactin receptor and prolactin mRNA, we found changed expression of the 3 short forms and the long form of prolactin receptor mRNA in the arcuate nucleus, paraventricular nucleus, bed nucleus of the stria terminalis, and MPOA with either exposure to male pheromones or pups. We also found changes in prolactin mRNA in the MPOA and paraventricular nucleus after exposure to pups or male pheromones. The data suggest that altered levels of expression of the receptor, coupled with local production of brain prolactin acting in an autocrine or paracrine manner, may cause a net change in prolactin cell signaling, which leads to adaptive responses which ensure reproductive success. There is extensive evidence that dopamine is a key neurotransmitter mediating maternal behavior. In addition, there is some evidence that serotonin may also be involved in regulating maternal behavior. Therefore, we hypothesised that the pheromonal-induced changes in maternal behavior would be associated with increased dopaminergic and/or serotonergic neuronal activity in the MPOA and other areas of the brain implicated in maternal behavior expression. Using HPLC to measure levels of dopamine and serotonin and their respective metabolites, we found a significant increase in serotonergic and dopaminergic neuronal activity in the MPOA of virgin female C57BL/6J mice after 24 hours of pheromonal contact. The neuronal activity returned to basal levels after exposure to pups. The data suggest that male mouse pheromones increase serotonergic and dopaminergic neuronal activity in the MPOA, but that dopamine and serotonin levels are tightly regulated within strict parameters dependent on what physical stimuli the female is receiving. Changes in prolactin levels are associated with altered responses to anxiety. There is an increased risk of anxiety and depression with sustained periods of hyperprolactinemia, and in the postpartum period, where there are fluctuations in prolactin levels, there is an increased risk of mood disorders. As pheromones change both serum and brain prolactin levels and prolactin modulates anxiety, we hypothesised that female mice exposed to pheromones would show altered behavioral responses to a standardized test of anxiety. We found that male pheromone-exposed mice showed decreased levels of anxiety on an elevated plus maze compared to individually housed controls. Female mice exposed to female pheromones displayed 2 disparate responses to the plus maze. One female from each cage showed increased anxiety, while her cage-mate showed decreased anxiety, yet both groups of female mice showed impaired maternal behavior. We infer, that in this model, male pheromones decrease anxiety, but anxiety and expression of maternal behavior are not directly correlated. The major signal transduction pathway activated by prolactin binding to its receptors in the brain is the JAK/STAT signalling pathway, and in some neurons, in particular, the STAT5B pathway. The expression of prolactin and its receptor affect maternal behavior in mice. Therefore, we hypothesised that if the JAK/STAT STAT5B pathway is involved in maternal behavior, then STAT5B-deficient mice would have altered maternal behavior. We found that there were no significant differences in expression of full maternal behavior between the STAT5B-deficient mice and wild-type controls. The data suggest that STAT5B is not required for normal expression of maternal behavior. We propose that the prolactin-mediated pheromonal increase in neurogenesis, alteration in monoamine synthesis, and alteration of prolactin and prolactin receptor mRNA levels facilitate expression of enhanced maternal behavior. We further propose that the pheromonal decrease in anxiety does not mediate enhanced maternal behavior. In addition, we propose that prolactin does not mediate maternal behavior through STAT5B. While pheromones have previously been reported to exert powerful actions on the reproductive system, these results demonstrate for the first time that male pheromones potentially complement the prolactin-mediated establishment of maternal behavior.

Diabetic neuropathy is believed to arise due to oxidative stress following hyperglycemic situations. Uncoupling proteins (UCPs) constitute a subgroup of mitochondrial transporter proteins with putative antioxidant properties. By dissipating the proton gradient over the mitochondrial inner membrane, these proteins reduce the mitochondrial inner membrane potential (MMP), and thereby, the mitochondrial production of reactive oxygen species (ROS) is decreased. In this thesis I have examined the regulation of UCP2, UCP3, and UCP4 by the neuroprotective hormone insulin-like growth factor type 1 (IGF-1). I have also investigated the possible involvement of UCP3 in IGF-1-mediated neuroprotection following high glucose treatments. All studies were performed using human neuroblastoma SH-SY5Y cells as an in vitro cell model. The major findings were as follows: i. Native SH-SY5Y cells expressed UCP2, UCP3, and UCP4. ii. UCP3 was upregulated by IGF-1 via activation of the IGF-1 receptor. IGF-1 increased UCP3 mRNA and protein levels primarily via activation of the “classical” anti-apoptotic phosphatidyl inositol 3 (PI3)-kinase signaling pathway, as shown by incubation with specific inhibitors of the PI3-kinase and mitogen activated protein (MAP) kinase signaling pathways. iii. UCP2 and UCP4 protein levels were only marginally or not at all regulated by IGF-1. These UCPs are probably not involved in IGF-1-mediated neuroprotection. iv. High glucose concentrations reduced the UCP3 protein levels in highly differentiated SH-SY5Y cells. Concomitantly, the MMP and the levels of ROS and glutathione increased, whereas the number of neurites per cell was reduced. This supports an antioxidant and neuroprotective role of UCP3 v. IGF-1 prevented the glucose-induced reduction in UCP3 protein levels. In parallel, the effects on MMP, levels of ROS and glutathione, and number of neurites per cell were abolished or significantly reduced. These data suggest that UCP3 is involved in IGF-1-mediated neuroprotection.

During neuronal development, the axonal and dendritic projections are polarized and oriented along specific body axis. To further explore the molecular basis of neuritogenesis in vivo, we used the nematode Caenorhabditis elegans as a developmental model and performed a forward genetic screen to identify genes that specify the polarity of neurite outgrowth. We examined the VC4 and VC5 neurons, members of the six VC motor neurons using the Pcat-1::gfp transgene cyIs4. The VC motor neurons are ventrally located neurons that extend two processes. VC1, VC2, VC3, and VC6 extend axons along the anterior-posterior (A/P) axis; VC4 and VC5 extend axons around the vulva along a mediolateral left-right (L/R) axis perpendicular to the A/P axis. We identified and showed that vang-1/Van Gogh, a core component of planar cell polarity (PCP) signalling pathway, acts cell-autonomously in VC4 and VC5 neurons and non-autonomously from the epithelial cells to restrict neurite formation along the A/P axis. vang-1 mutant animals display ectopic neurites along the A/P axis. Using a candidate gene approach, we further identified and revealed two additional core members of PCP signalling, Prickle (PRKL-1) and Dishevelled (DSH-1), to play a role in A/P-directed neurite suppression. We also showed prkl-1 and dsh-1 genetically interact with vang-1 and VANG-1 is required to suppress A/P-directed neurite outgrowth from larval stage 4 to adulthood. Overexpression of VANG-1 results in a loss-of-function (lof) phenotype, suggesting that an appropriate level of VANG-1 activity is important. Additionally, vang-1/prkl-1, and dsh-1 may interact in parallel pathways. Our findings implicate PCP genes to play a previously unidentified role in maintaining polarized neuronal morphology by inhibiting neuronal outgrowth responses to environmental cues.

In predator-prey interactions, prey species have complex defensive behaviors to protect themselves from predators. Chemical defenses are one tool that is employed to protect against predators, especially for slow-moving or otherwise susceptible prey. Many of these chemical defenses have been studied and the effective compounds identified, but few studies were performed on their mechanisms of detection. In my research, I used the sea hare, Aplysia californica, as chemically defended prey. This slow moving mollusk is soft-bodied with no external shell, but it has adapted a number of defenses including chemical defenses. Ink is a sticky mixture of the products of the ink gland and the opaline gland which are mixed in the mantle cavity and released toward an attacker. I show that this ink secretion protects the sea hare from predation by a fish predator. Because many deterrent compounds taste bitter, bitter taste receptors are thought to protect predators from ingesting harmful compounds in prey. Studies of deterrent taste detection have commonly utilized bitter compounds from human hedonics to study the responses in animals, such as fruit flies, fishes, rats, and monkeys. In my dissertation, I argue that the study of chemical defenses allows us to ask more questions about detection of relevant deterrents and interactions between predators and prey at the individual and population levels. My results show that diet-derived pigments in Aplysia ink, aplysioviolin and phycoerythrobilin, are strongly deterrent to fish predators. Electrophysiological analyses of the gustatory system show that these compounds are equipotent and cross-adapt each others’ responses completely. Aplysioviolin and phycoerythrobilin produced incomplete reciprocal cross-adaptation with amino acids and adapted bile salt responses but were not significantly adapted by these latter stimuli. These results showed multiple pathways that are sensitive to aplysioviolin and phycoerythrobilin, which may have different effects on the physiology and behavior of the predatory fish. My findings demonstrate the value to the fields of chemical ecology and chemosensory biology of studying sensory processing of relevant deterrent compounds. This work lays the foundation for how a diet-derived photopigment is adapted by a species to protect itself from predators by stimulating their chemosensory systems.

Adolescence in humans is a vulnerable period for illicit drug use, and teenage onset of drug use is associated with long-term addiction. Adolescent sensitivity to drug reinforcement, relapse, and withdrawal has not been explored thoroughly in animal models, especially considering opiate drugs such as morphine and heroin. The present series of studies profiles adolescent sensitivity to opiates using adolescent and adult male rats to test for age differences in opiate self-administration, reinstatement, withdrawal signs, locomotor sensitization, and even brain activation during drug-seeking. To test for acute sensitivity to the reinforcing effects of morphine or heroin, we compared patterns of self-administration by adolescent vs. adult male rats on various schedules of reinforcement, drug doses, and daily access conditions. Using fixed ratio schedules and short daily access, adolescents self-administered less morphine than adults, an effect commonly interpreted as higher drug sensitivity. In contrast, escalation of morphine intake under long access conditions was similar across ages, as was heroin intake using fixed or progressive ratio schedules of reinforcement. To test for enduring effects of opiates, we compared opiate-seeking in the absence of the drug in tests of extinction responding and cue-induced reinstatement. Regardless of the acute effects of morphine or heroin, all adolescent treatment groups showed attenuated opiate-seeking compared to adults. Next we considered behavioral correlates of reinforcement, drug withdrawal and locomotor sensitization, during and after escalating doses of experimenter-administered heroin. Consistent with attenuated opiate-seeking, adolescents exhibited attenuated somatic and locomotor signs of withdrawal compared with adults, although locomotor sensitization was similar across ages. Finally, the medial prefrontal cortex (mPFC) is a brain region heavily implicated in drug reinforcement, so we used tissue levels of Fos-like immunoreactivity to compare activation of this region by heroin-seeking. Indeed mPFC activation was absent in rats that self-administered heroin as adolescents, but robust in adults. Together these behavioral and neuroanatomical results surprisingly suggest that adolescent male rats are less sensitive than adults to some acute and enduring effects of opiates, and may predict better response profiles among younger human addicts. Through future studies, adolescent rats may provide a new model to help identify treatments for drug abuse.